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macrophages belong to the mononuclear phagocytic system ( mps ) [ 1 , 2 ] and comprise a heterogeneous population of cells that have the capacity to perform a wide range of critical functions . they play an important role in tissue homeostasis and immune responses in normal and diseased kidneys [ 2 , 4 ] . macrophages are present in all tissues and originate from common myeloid progenitor cells in the bone marrow [ 1 , 5 ] under the influence of colony - stimulating factor 1 ( csf-1 ) . monocyte development sequentially gives rise to monoblasts , pro - monocytes and finally monocytes , which are released from the bone marrow into the bloodstream . monocytes then migrate from the blood to the injured tissue and replenish tissue macrophage numbers , especially during inflammation . macrophages are crucial components of innate immunity ; their main function is to clear the interstitial environment of extraneous cellular material and also to generate an adaptive immune response by serving as antigen - presenting cells ( apcs ) and by recruiting other immune cells such as lymphocytes [ 5 , 8 ] . macrophages are divided into different subpopulations based on their functionality and anatomical location , e.g. kupffer cells , langerhans cells and microglial cells . they are defined as tissue - resident phagocytic cells that contribute to critical roles in homeostasis , surveillance and tissue injury and repair [ 5 , 10 ] . in the tissue injury scenario , blood monocytes are recruited to the site of damage and undergo differentiation in response to microenvironment signals to which they are exposed . therefore , macrophage infiltration in the kidney is a common feature of chronic kidney disease ( ckd ) in humans , and the correlation between the degree of macrophage infiltration and the severity of renal injury suggests an effector function for macrophages . hence , during ckd , resident and infiltrating macrophages undergo a range of activation responses such as phagocytosis and production of pro - inflammatory cytokines and toxic metabolites . ckd is becoming one of the most important health problems worldwide and greatly affects patients ' quality of life . there is broad agreement that all primary causes of ckd share a common pathogenic pathway of progressive injury resulting from the destructive consequences of scarring ( fibrosis ) . the development of ckd is characterized by an accumulation of extracellular matrix ( ecm ) proteins in the glomerulus and interstitium , which is thought to be promoted by an aberrant wound healing response involving tubular epithelial cells ( tecs ) , myofibroblasts , fibrocytes and immune cells , among others , thus leading to progressive fibrosis in the kidney and loss of viable nephrons . following recruitment to the damaged kidney , macrophages can be broadly classified into two different subtypes , depending on their local microenvironment : classically activated ( m1 ) and alternatively activated ( m2 ) macrophages [ 10 , 14 , 16 ] . although they have been strongly associated with tissue injury , they also have a critical role in both host defence and tissue repair [ 18 , 19 ] . pro - inflammatory m1 macrophages are produced by exposure to interferon ( ifn)- or lipopolysaccharide ( lps ) and are considered pro - inflammatory due to their capacity to release certain cytokines such as interleukin ( il)-1 , il-6 and tumour necrosis factor ( tnf)- . in contrast , m2 macrophages have anti - inflammatory functions and express arginase , mannose receptor and il-10 , among others ( table 1 ) [ 10 , 20 ] . alternatively activated m2 macrophages can be further categorized into three subgroups : m2a induced by il-4 and/or il-13 , which display a wound - healing ( tissue repair ) role ; m2b induced by immune complexes and m2c , with anti - inflammatory effects and induced by il-10 , transforming growth factor ( tgf)- or glucocorticoids [ 7 , 21 ] . thus , macrophages are highly heterogeneous cells that exhibit distinct phenotypes and functionality in response to microenvironment stimuli , leading to the previously described classification system [ 5 , 10 ] . although extensive in vitro studies have supported the phenotype classification , this does not necessarily reflect their true phenotypes in vivo . table 1.distinct macrophage populationsmacrophage phenotypestimulationeffectmarker expressionm1ifn- , tnf- , lps , gm - gsfpro - inflammatorycd86 , cd80 , mhc ii , ly6c , tlr2 , tlr4m2ail-4 and/or il-13profibroticmr / cd206 , mhc ii , arg-1m2bic + lpsimmunoregulationcd86 , mhc iim2cil-10 , tgf- , apoptotic cells , glucocorticoidsanti - inflammatorymr / cd206 , b7-h4 , tlr1 , tlr8adapted from anders and ryu , cao et al . and martinez et al . .depending on the microenvironment , macrophages can differentiate into specific populations with distinct functions.gm-csf , granulocyte macrophage colony - stimulating factor ; ic , immune complexes ; il , interleukin ; ifn , interferon ; lps , lipopolysaccharide ; mhc ii , major histocompatibility complex class ii ; tlr , toll - like receptor ; tnf , tumour necrosis factor . distinct macrophage populations adapted from anders and ryu , cao et al . and martinez et al . . gm - csf , granulocyte macrophage colony - stimulating factor ; ic , immune complexes ; il , interleukin ; ifn , interferon ; lps , lipopolysaccharide ; mhc ii , major histocompatibility complex class ii ; tlr , toll - like receptor ; tnf , tumour necrosis factor . early phases of ckd trigger a remarkable infiltrate of immune cells , firstly neutrophils , natural killer ( nk ) cells and t helper ( th)1/17 cells , followed soon after by m1 macrophages . at sites of tissue injury , the interstitial microenvironment is dominated by pathogen - associated molecular patterns ( pamps ) such as adenosine triphosphate ( atp ) , high mobility group box 1 ( hmgb-1 ) and uric acid , derived from damage - associated molecular patterns ( damps ) released by necrotic cells [ 5 , 2124 ] . during toxic , infectious or traumatic injuries , pamps activate resident macrophages as well as parenchymal cells via innate pattern recognition receptors ( prrs ) [ 25 , 26 ] , thus leading to the secretion of pro - inflammatory cytokines and providing defence against pathogens and also a functional barrier to prevent further pathogen entries [ 5 , 27 , 28 ] . inflammation kills host cells at the site of infection , which causes some non - specific collateral tissue damage [ 26 , 29 ] . in sterile kidney injury , pamps are often absent , and damps mostly drive the infiltration of inflammatory macrophages into the sterile kidney [ 5 , 30 , 31 ] . macrophages detect endogenous danger signals via toll - like receptors ( tlrs ) [ 21 , 22 , 30 , 32 ] , intracellular prrs and the il-1 receptor ( il-1r ) through the adaptor molecule myeloid differentiation primary response gene 88 ( myd88 ) . thus , a tlr ligand acting in an myd88-dependent manner will induce tnf transcription , which can act in conjunction with ifn- in an autocrine manner to activate the macrophage population . ifn- is released by nk cells in response to stress and infections , which can prime macrophages to secrete pro - inflammatory cytokines . the combination of these two signals results in a macrophage population with enhanced microbicidal effects as well as in increased production of pro - inflammatory cytokines ( il-6 , il-1 and tnf- ) , superoxide anions and oxygen and nitrogen radicals . the two signals together also promote cytotoxic adaptive immunity by upregulating major histocompatibility complex class ii ( mhc ii ) in conjunction with co - stimulatory molecules ( cd40 , cd80 and cd86 ) [ 35 , 36 ] . support a theory in which renal injury and inflammation are reciprocally enhanced in an autoamplification loop , referred to as necroinflammation . cell necrosis releases damps and alarmins that activate infiltrating monocytes via tlrs towards a pro - inflammatory phenotype . thus , infiltrating macrophages , in turn , further contribute to necroinflammation due to the secretion of numerous pro - inflammatory cytokines ; therefore , inhibiting activated pro - inflammatory macrophages would prevent immunopathology in injured kidneys . these m1 macrophages promote th1 and th17 responses and are therefore involved in initiating and sustaining inflammatory processes [ 7 , 37 ] . in inflammation in mice , ly6c monocytes are recruited and differentiated into m1 macrophages that express cc - chemokine receptor 2 ( ccr-2 ) , thus responding to cc - chemokine ligand 2 ( ccl-2 ) , an important chemokine required for monocyte / macrophage recruitment to damaged sites [ 1 , 38 ] . pro - inflammatory macrophages also release matrix metalloproteinases ( mmps ) to enable their migration through basement membranes and interstitial ecm networks [ 5 , 39 ] . lps / ifn--activated m1 macrophages induce renal fibrosis by secretion of mmp-9 , which increase tubular cell ecm transition via the -catenin pathway . the transcription factor irf5 also seems to play a key role in m1 macrophage polarization , suggesting that inhibiting irf5 might be useful for chronic macrophage - induced inflammation . the pathogenic role of macrophages has been demonstrated by depletion of kidney - resident macrophages with liposomal clodronate ( lc ) in different types of experimental kidney disease [ 4143 ] . however , increasing evidence from extensive studies shows that macrophages also play a reparative role during disease progression . macrophages that secrete anti - inflammatory cytokines and promote wound healing and tissue remodelling have been referred to as alternatively activated macrophages ( aams ) [ 5 , 10 ] , also called m2 . the mechanisms by which kidney - resident m1 macrophages switch to an anti - inflammatory m2 phenotype are still not well understood . detailed studies in which renal injury resolves have identified that macrophages undergo a phenotypic change during recovery and this confers a protective and reparative role [ 4446 ] . moreover , depletion of macrophages during this phase delays recovery , indicating a functional role for macrophages in renal repair . macrophages modulated ex vivo with il-4 and/or il-13 ( m2a ) express high levels of mannose receptor ( cd206 ) , produce anti - inflammatory il-10 and have immunoregulatory functions [ 8 , 47 ] . these cells secrete components of the ecm and therefore their main functions seem to be associated with wound healing and tissue remodelling and repair [ 7 , 10 ] . martinez et al . demonstrated that when ecm clearance is deficient due to defects in the engulfment of dying cells , functional markers of renal injury , such as serum creatinine , blood urea nitrogen and proteinuria , progressively increase . another category of aams is the m2b macrophages , which represents crosstalk with b cells . this category induces il-10 secretion in addition to upregulating antigen presentation and promoting th2 responses . both m2a and m2b macrophages have an immunoregulatory role through downregulation of il-12 , il-6 and tnf . moreover , m2c macrophages are induced by il-10 , tgf- and glucocorticoids ; this subset is known to exhibit anti - inflammatory cytokine production and suppressive functions in vitro , and they are often referred to as deactivated . similarly to classically activated macrophages , wound healing macrophages can develop in response to innate and adaptive signals . il-4 is one of the first signals released during tissue injury by basophils and mast cells , among others , and this early il-4 production converts resident macrophages into a population reprogrammed to promote wound healing . to date , the diverse roles of macrophages in in vivo studies are still not fully understood , although it is generally known that macrophages eagerly participate in the clearance of apoptotic and necrotic cells in injury resolution and tissue remodelling [ 5 , 51 , 52 ] . macrophage depletion via anti - macrophage serum or lc nearly always reduced persistent inflammation and also the subsequent development of fibrosis [ 17 , 21 ] . nevertheless , renal fibrosis may not only be triggered by pro - inflammatory m1 macrophages , but instead by insufficient epithelial healing or by profibrotic m2 macrophages and fibrocytes . in the face of ongoing damage , sustained m2 macrophage infiltration may result in constant production of several wound healing growth factors , and what initially begins as a reparative mechanism may subsequently become harmful . in fact , persistence of the wound healing process could be pathological , resulting in irreversible fibrosis and progressive kidney tissue destruction . on the other hand , m2 macrophages may help to resolve inflammation through high endocytic clearance capacities and the production of trophic factors that promote angiogenesis and mediate wound healing producing ecm [ 53 , 54 ] . for instance , in vitro cytokines such as il-4 and il-13 further promote the m2 phenotype , which predominantly releases fibronectin 1 ( fn-1 ) and other ecm molecules that could contribute directly to renal fibrosis [ 55 , 56 ] . likewise , kim et al . recently demonstrated that m2 macrophages play a more important role than m1 macrophages in the development of fibrosis in an in vitro cisplatin - treated culture . it is important to note that these studies were performed in vitro , and the role of macrophages as a contributing factor in the development of renal fibrosis in vivo remains under discussion . members of the tgf- superfamily are the most extensively studied growth factors derived from macrophages , which are mainly associated with an m2-like phenotype , among other cell types such as tecs and myofibroblasts . within the kidney , macrophage - derived tgf- may promote fibrosis by paracrine activation of matrix - producing myofibroblasts . unilateral ureteral obstruction ( uuo ) is a well - characterized model to investigate the factors that contribute to renal fibrosis . for instance , braga et al . demonstrated that m2-phenotype macrophages contribute to renal fibrosis in an myd88-dependent manner and through tlr signalling pathways . moreover , galectin-3 , a nuclear m2 marker , has been shown to be produced by kidney - resident macrophages and to enhance renal fibrosis in uuo . . also demonstrated that macrophages expressing the marker mannose receptor-2 ( mrc2 ) displayed a fibrosis - attenuating role in uuo , as mice deficient in mrc2 exhibited worsened renal fibrosis . indeed , to study the macrophage - specific role of tgf-1 in the development of renal fibrosis , huen et al . developed mice with a homozygous deletion of tgf-1 in myeloid lineage cells and demonstrated that despite tgf-1 mrna reduction and the prevention of downstream smad activation , interstitial fibrosis and tubular injury were not significantly different after uuo compared with the control uuo group . thus , they suggested that specifically targeting myeloid tgf-1 may not be sufficient to combat the progression of renal fibrosis . several studies targeting tgf-1 have highlighted the complex role of cytokine in both injury and wound repair processes , showing that further research is necessary to clarify the functional impact of this complex . taken together , renal fibrosis may not only be triggered by pro - inflammatory m1 macrophages , but m2 macrophages could also contribute somehow to the development of fibrosis and progressive fibrotic scarring . further studies in experimental animal models of ckd , such as diabetic nephropathy , undergo suggested that , at later disease stages , macrophages confer a shift towards chronic activation of the m2 phenotype [ 64 , 65 ] , which leads to glomerulosclerosis , tubular interstitial fibrosis and eventually organ failure [ 65 , 66 ] . thus , distinct macrophage subsets can coexist in kidney tissue and certain subsets can predominate at different disease stages , from the beginning of kidney damage to the recovery phase . shen et al . demonstrated that phagocytosis by m1 macrophages to remove dead cells may only function in the early stages of uuo , and m2 macrophages appeared to be the major cell type in advanced stages . these results are in agreement with those reported by cao et al . , who described how , in ckd , due to progressive and persistent injury and inflammation , m1 macrophages persistently remained at sites of tissue injury and consequently reduced numbers of m2-phenotype macrophages were recruited into the kidney . therefore , m2 macrophages either coexist in small numbers or are absent because of permanent inflammation . taken together , the mechanisms regulating the distinct functions of m2 macrophages during tissue repair or fibrosis remain largely unclear ( figure 1 ) . renal function varies over time , depending on the type of injury , the persistence and severity of the damage and the reparative ability of the kidney . in the early stages of ckd , pro - inflammatory macrophages ( m1 ) infiltrate the injury site and release pro - inflammatory cytokines , which promote an inflammatory state . if the injury resolves , renal function as well as renal mass ameliorate , depending on damage severity and duration . macrophages also switch to an anti - inflammatory ( m2 ) phenotype , leading to a wound healing phase that may involve tissue fibrosis . however , if there is no injury resolution , m1 macrophages persist at injured sites and there is a decrease in the number of m2 macrophages , which could also subsequently undergo a phenotypic switch to m1 . the continuous release of profibrotic and inflammatory factors promote renal fibrosis , leading to renal failure . m1/m2 renal function varies over time , depending on the type of injury , the persistence and severity of the damage and the reparative ability of the kidney . in the early stages of ckd , pro - inflammatory macrophages ( m1 ) infiltrate the injury site and release pro - inflammatory cytokines , which promote an inflammatory state . if the injury resolves , renal function as well as renal mass ameliorate , depending on damage severity and duration . macrophages also switch to an anti - inflammatory ( m2 ) phenotype , leading to a wound healing phase that may involve tissue fibrosis . however , if there is no injury resolution , m1 macrophages persist at injured sites and there is a decrease in the number of m2 macrophages , which could also subsequently undergo a phenotypic switch to m1 . the continuous release of profibrotic and inflammatory factors promote renal fibrosis , leading to renal failure . therefore , interventions such as gene and cell - based therapies are being extensively developed as an alternative treatment modality for the prevention of progression to end - stage renal failure . for instance , flaquer et al . demonstrated that hepatocyte growth factor ( hgf ) gene therapy was able to enhance the amount of bone marrow derived cells in the diabetic kidney and showed that these cells located around the glomeruli were mainly m2 macrophages . thus , rodent models are valuable for studying macrophage phenotypes in the context of ckd . among potential strategies , macrophage cell - based therapy provides a contrast of promising and discordant results at the same time [ 54 , 72 , 73 ] . macrophages modulated ex vivo and displaying an anti - inflammatory or reparatory activity have been used as cell - based therapy in a number of mouse models of ckd ( table 2 ) . m2 macrophages must meet at least two conditions if they are to be used as a therapeutic tool in vivo : first , the ability to reach the injured tissue or organ , and second , a stable phenotype . these two conditions are particularly important , since infused macrophages may be harmful if they switch from an anti - inflammatory to a pro - inflammatory phenotype . table 2.effects of macrophage cell - based therapy in different mouse models of ckdckd mouse modelcharacteristicsmacrophage cell - based therapyresultslimitationsunilateral ureteral obstruction ( uuo ) [ 56 , 59 , 73 ] well - established model with rapid interstitial fibrosisnon - reversible bone marrow - derived macrophages ( bm - m0)raw246.7 m2 macrophages controversial results : transition into collagen - producing myofibroblasts . at late stage , renal fibrosis is reduced no renal function dataaggressive fibrosisadriamycin - induced nephropathy ( an ) [ 18 , 47 , 62]gradual development of proteinuria , podocyte injury followed by glomerulosclerosis , tubulointerstitial inflammation and fibrosis splenocytes ( m2a and m2c)bone marrow - derived macrophages ( bm - m2a ) failed renoprotection of bm - m2a , whereas splenocytes prevented renal injuryreduction of histological and functional injurythe pathophysiology has not been clarified in full detail by the sequence of events , with proteinuria preceding the development of renal fibrotic lesionsdiabetic kidney disease stz - induced diabetesdestruction of -cells and induction of the hyperglycaemic state associated with inflammatory infiltratessplenocytes ( m0 and m2a)amelioration of tubular atrophy , glomerular hypertrophy and interstitial expansion . degree of interstitial fibrosis , but no effects on renal function regeneration of pancreatic islets can occur after stz treatmentdose - dependent strainsearch criteria : macrophage infusion kidney , m2 macrophage fibrosis , transfused macrophages and alternatively activated macrophages kidney. effects of macrophage cell - based therapy in different mouse models of ckd well - established model with rapid interstitial fibrosis bone marrow - derived macrophages ( bm - m0 ) controversial results : transition into collagen - producing myofibroblasts . at late stage , renal fibrosis is reduced no renal function data splenocytes ( m2a and m2c ) bone marrow - derived macrophages ( bm - m2a ) failed renoprotection of bm - m2a , whereas splenocytes prevented renal injury reduction of histological and functional injury destruction of -cells and induction of the hyperglycaemic state associated with inflammatory infiltrates regeneration of pancreatic islets can occur after stz treatment dose - dependent strain search criteria : macrophage infusion kidney , m2 macrophage fibrosis , transfused macrophages and alternatively activated macrophages kidney. professor david harris 's group has extensively investigated the effects of infusing in vitro generated m2 macrophages in rodent models of both acute kidney failure and ckd [ 18 , 54 , 72 ] . using a mouse model of adriamycin nephropathy ( an ) , they demonstrated that a single intravenous injection of 1 10 macrophages generated by splenic cd11b cells and exposed to il-10 and tgf- ( m2c ) provided increased protection against renal structural and functional injury compared with il-4/il-13-exposed macrophages ( m2a ) . these il-10/tgf--modulated m2 macrophages ( m2c ) expressed high levels of b7-h4 , whereas il-4/il-13-modulated macrophages ( m2a ) did not . thus , these authors attributed the greater potency of m2c macrophages to the expression of the co - stimulation molecule b7-h4 , which suppresses t cell proliferation and induces regulatory t cells both in vitro and in vivo . however , they also demonstrated that infused m2c macrophages did change their phenotype during the disease course , although not towards a distinct m1 phenotype [ 72 , 74 ] . in a severe combined immunodeficient ( scid ) mouse model of an , the infusion of m2 macrophages isolated from the spleen and modulated ex vivo by il-4 and il-13 was associated with an amelioration of renal injury . in contrast , a report by cao et al . showed that using the same mouse model but with m2 macrophages isolated from the bone marrow ( bm - m2 macrophages ) failed to reduce proteinuria and to preserve renal function , owing to a change in macrophage phenotype . therefore , these studies demonstrated that transfused bm - m2 macrophages lost their suppressive function in vivo due to their proliferation , whereas splenic m2 macrophages ( sp - m2 ) were protective because they did not proliferate . the enhanced proliferation of bm - m2 macrophages can be explained by their increased expression of macrophage colony - stimulating factor ( m - csf ) receptor , in comparison with sp - m2 macrophages , which could be partly prevented by blocking csf-1-mediated signalling . transfer of sp - m2 macrophages seems to protect against renal injury , whereas transfer of bm - m2 macrophages appears to promote renal fibrosis [ 18 , 75 ] . however , the bone marrow , rather than the spleen , represents an accessible source of macrophage precursors for aam therapy . therefore , the source of origin of macrophages is another critical issue to bear in mind due to the contradictory outcomes that have been published recently . nowadays , the use of anti - inflammatory and regenerative macrophage - derived molecules is increasing . heme - oxygenase-1 ( ho-1 ) , for example , is a protective and anti - inflammatory enzyme upregulated in response to renal injury , whereas its downregulation is associated with susceptibility to damage [ 76 , 77 ] . chen et al . demonstrated that sustained overexpression of ho-1 counteracted multiple detrimental renal fibrosis - associated pathological processes in a uuo mouse model . on the other hand , accumulating evidence suggests that tnf- is involved in diabetic nephropathy progression [ 79 , 80 ] . furthermore , awad et al . demonstrated that blockade of tnf- conferred kidney protection by reducing albuminuria , plasma creatinine , kidney macrophage recruitment and plasma cytokine levels . therefore , their results suggest that inhibition of tnf- may be a viable strategy to treat diabetic nephropathy in humans . moreover , lin et al . found that the wnt pathway may play an important role in tissue regeneration . they showed that wnt7b was produced by macrophages and was required to stimulate renal repair and regeneration by acting on injured tecs to promote regeneration of the tubule basement membrane , thereby re - establishing renal function and reducing renal fibrosis . therefore , macrophages have been shown to be important in renal repair , wound healing and regeneration processes . however , it is still unknown whether they can promote renal repair processes directly by fusing with other cells or transforming into new ones or indirectly by providing help to other cell types [ 82 , 83 ] . nevertheless , it is important to note that many intermediate phenotypes and many subpopulations are likely to coexist in the same tissue . moreover , macrophages do not remain in a specific phenotype due to their cell plasticity ; they may revert to a resting state and can be subsequently reactivated , depending on the microenvironment . macrophage dynamics during the different phases of ckd progression are not fully known , and assessment of the predominant macrophage phenotype may be relevant in terms of defining the type of therapy . aams have been demonstrated to be protective in reducing renal injury due to their anti - inflammatory role . however , whether these macrophages could become fibrolytic to reduce renal fibrosis still remains unknown . anders and ryu have proposed to classify tissue macrophages according to their predominant roles in different phases of kidney disease : pro - inflammatory , anti - inflammatory , profibrotic and fibrolytic macrophages . however , there is a lack of information regarding macrophage types and their dynamics , plasticity and function in human ckd . hence , more studies are needed before testing macrophage cell - based therapy in humans , since macrophages represent a spectrum of activated phenotypes rather than discrete stable subpopulations [ 5 , 84 ] . therefore , better strategies to induce truly regenerative and reparative macrophages in vivo need to be developed .

chronic kidney disease ( ckd ) has become a major health problem worldwide . this review describes the role of macrophages in ckd and highlights the importance of anti - inflammatory m2 macrophage activation in both renal fibrosis and wound healing processes . furthermore , the mechanisms by which m2 macrophages induce renal repair and regeneration are still under debate and currently demand more attention . the m1/m2 macrophage balance is related to the renal microenvironment and could influence ckd progression . in fact , an inflammatory renal environment and m2 plasticity can be the major hurdles to establishing macrophage cell - based therapies in ckd . m2 macrophage cell - based therapy is promising if the m2 phenotype remains stable and is fixed by in vitro manipulation . however , a greater understanding of phenotype polarization is still required . moreover , better strategies and targets to induce reparative macrophages in vivo should guide future investigations in order to abate kidney diseases .

Macrophage origin and heterogeneity Pro-inflammatory M1 macrophages in CKD Anti-inflammatory M2 macrophages in CKD Persistent M2 macrophages are associated with fibrosis M2 macrophage cell-based therapy for renal repair and regeneration Macrophages: future challenges Conflict of interest statement

monocytes then migrate from the blood to the injured tissue and replenish tissue macrophage numbers , especially during inflammation . therefore , macrophage infiltration in the kidney is a common feature of chronic kidney disease ( ckd ) in humans , and the correlation between the degree of macrophage infiltration and the severity of renal injury suggests an effector function for macrophages . the development of ckd is characterized by an accumulation of extracellular matrix ( ecm ) proteins in the glomerulus and interstitium , which is thought to be promoted by an aberrant wound healing response involving tubular epithelial cells ( tecs ) , myofibroblasts , fibrocytes and immune cells , among others , thus leading to progressive fibrosis in the kidney and loss of viable nephrons . following recruitment to the damaged kidney , macrophages can be broadly classified into two different subtypes , depending on their local microenvironment : classically activated ( m1 ) and alternatively activated ( m2 ) macrophages [ 10 , 14 , 16 ] . pro - inflammatory m1 macrophages are produced by exposure to interferon ( ifn)- or lipopolysaccharide ( lps ) and are considered pro - inflammatory due to their capacity to release certain cytokines such as interleukin ( il)-1 , il-6 and tumour necrosis factor ( tnf)- . in contrast , m2 macrophages have anti - inflammatory functions and express arginase , mannose receptor and il-10 , among others ( table 1 ) [ 10 , 20 ] . alternatively activated m2 macrophages can be further categorized into three subgroups : m2a induced by il-4 and/or il-13 , which display a wound - healing ( tissue repair ) role ; m2b induced by immune complexes and m2c , with anti - inflammatory effects and induced by il-10 , transforming growth factor ( tgf)- or glucocorticoids [ 7 , 21 ] . thus , macrophages are highly heterogeneous cells that exhibit distinct phenotypes and functionality in response to microenvironment stimuli , leading to the previously described classification system [ 5 , 10 ] . although extensive in vitro studies have supported the phenotype classification , this does not necessarily reflect their true phenotypes in vivo . at sites of tissue injury , the interstitial microenvironment is dominated by pathogen - associated molecular patterns ( pamps ) such as adenosine triphosphate ( atp ) , high mobility group box 1 ( hmgb-1 ) and uric acid , derived from damage - associated molecular patterns ( damps ) released by necrotic cells [ 5 , 2124 ] . during toxic , infectious or traumatic injuries , pamps activate resident macrophages as well as parenchymal cells via innate pattern recognition receptors ( prrs ) [ 25 , 26 ] , thus leading to the secretion of pro - inflammatory cytokines and providing defence against pathogens and also a functional barrier to prevent further pathogen entries [ 5 , 27 , 28 ] . thus , a tlr ligand acting in an myd88-dependent manner will induce tnf transcription , which can act in conjunction with ifn- in an autocrine manner to activate the macrophage population . the combination of these two signals results in a macrophage population with enhanced microbicidal effects as well as in increased production of pro - inflammatory cytokines ( il-6 , il-1 and tnf- ) , superoxide anions and oxygen and nitrogen radicals . cell necrosis releases damps and alarmins that activate infiltrating monocytes via tlrs towards a pro - inflammatory phenotype . thus , infiltrating macrophages , in turn , further contribute to necroinflammation due to the secretion of numerous pro - inflammatory cytokines ; therefore , inhibiting activated pro - inflammatory macrophages would prevent immunopathology in injured kidneys . in inflammation in mice , ly6c monocytes are recruited and differentiated into m1 macrophages that express cc - chemokine receptor 2 ( ccr-2 ) , thus responding to cc - chemokine ligand 2 ( ccl-2 ) , an important chemokine required for monocyte / macrophage recruitment to damaged sites [ 1 , 38 ] . lps / ifn--activated m1 macrophages induce renal fibrosis by secretion of mmp-9 , which increase tubular cell ecm transition via the -catenin pathway . the pathogenic role of macrophages has been demonstrated by depletion of kidney - resident macrophages with liposomal clodronate ( lc ) in different types of experimental kidney disease [ 4143 ] . macrophages that secrete anti - inflammatory cytokines and promote wound healing and tissue remodelling have been referred to as alternatively activated macrophages ( aams ) [ 5 , 10 ] , also called m2 . the mechanisms by which kidney - resident m1 macrophages switch to an anti - inflammatory m2 phenotype are still not well understood . moreover , depletion of macrophages during this phase delays recovery , indicating a functional role for macrophages in renal repair . macrophages modulated ex vivo with il-4 and/or il-13 ( m2a ) express high levels of mannose receptor ( cd206 ) , produce anti - inflammatory il-10 and have immunoregulatory functions [ 8 , 47 ] . moreover , m2c macrophages are induced by il-10 , tgf- and glucocorticoids ; this subset is known to exhibit anti - inflammatory cytokine production and suppressive functions in vitro , and they are often referred to as deactivated . similarly to classically activated macrophages , wound healing macrophages can develop in response to innate and adaptive signals . il-4 is one of the first signals released during tissue injury by basophils and mast cells , among others , and this early il-4 production converts resident macrophages into a population reprogrammed to promote wound healing . to date , the diverse roles of macrophages in in vivo studies are still not fully understood , although it is generally known that macrophages eagerly participate in the clearance of apoptotic and necrotic cells in injury resolution and tissue remodelling [ 5 , 51 , 52 ] . nevertheless , renal fibrosis may not only be triggered by pro - inflammatory m1 macrophages , but instead by insufficient epithelial healing or by profibrotic m2 macrophages and fibrocytes . in the face of ongoing damage , sustained m2 macrophage infiltration may result in constant production of several wound healing growth factors , and what initially begins as a reparative mechanism may subsequently become harmful . in fact , persistence of the wound healing process could be pathological , resulting in irreversible fibrosis and progressive kidney tissue destruction . on the other hand , m2 macrophages may help to resolve inflammation through high endocytic clearance capacities and the production of trophic factors that promote angiogenesis and mediate wound healing producing ecm [ 53 , 54 ] . for instance , in vitro cytokines such as il-4 and il-13 further promote the m2 phenotype , which predominantly releases fibronectin 1 ( fn-1 ) and other ecm molecules that could contribute directly to renal fibrosis [ 55 , 56 ] . recently demonstrated that m2 macrophages play a more important role than m1 macrophages in the development of fibrosis in an in vitro cisplatin - treated culture . it is important to note that these studies were performed in vitro , and the role of macrophages as a contributing factor in the development of renal fibrosis in vivo remains under discussion . demonstrated that m2-phenotype macrophages contribute to renal fibrosis in an myd88-dependent manner and through tlr signalling pathways . moreover , galectin-3 , a nuclear m2 marker , has been shown to be produced by kidney - resident macrophages and to enhance renal fibrosis in uuo . also demonstrated that macrophages expressing the marker mannose receptor-2 ( mrc2 ) displayed a fibrosis - attenuating role in uuo , as mice deficient in mrc2 exhibited worsened renal fibrosis . indeed , to study the macrophage - specific role of tgf-1 in the development of renal fibrosis , huen et al . developed mice with a homozygous deletion of tgf-1 in myeloid lineage cells and demonstrated that despite tgf-1 mrna reduction and the prevention of downstream smad activation , interstitial fibrosis and tubular injury were not significantly different after uuo compared with the control uuo group . thus , they suggested that specifically targeting myeloid tgf-1 may not be sufficient to combat the progression of renal fibrosis . several studies targeting tgf-1 have highlighted the complex role of cytokine in both injury and wound repair processes , showing that further research is necessary to clarify the functional impact of this complex . taken together , renal fibrosis may not only be triggered by pro - inflammatory m1 macrophages , but m2 macrophages could also contribute somehow to the development of fibrosis and progressive fibrotic scarring . further studies in experimental animal models of ckd , such as diabetic nephropathy , undergo suggested that , at later disease stages , macrophages confer a shift towards chronic activation of the m2 phenotype [ 64 , 65 ] , which leads to glomerulosclerosis , tubular interstitial fibrosis and eventually organ failure [ 65 , 66 ] . demonstrated that phagocytosis by m1 macrophages to remove dead cells may only function in the early stages of uuo , and m2 macrophages appeared to be the major cell type in advanced stages . therefore , m2 macrophages either coexist in small numbers or are absent because of permanent inflammation . taken together , the mechanisms regulating the distinct functions of m2 macrophages during tissue repair or fibrosis remain largely unclear ( figure 1 ) . renal function varies over time , depending on the type of injury , the persistence and severity of the damage and the reparative ability of the kidney . in the early stages of ckd , pro - inflammatory macrophages ( m1 ) infiltrate the injury site and release pro - inflammatory cytokines , which promote an inflammatory state . macrophages also switch to an anti - inflammatory ( m2 ) phenotype , leading to a wound healing phase that may involve tissue fibrosis . however , if there is no injury resolution , m1 macrophages persist at injured sites and there is a decrease in the number of m2 macrophages , which could also subsequently undergo a phenotypic switch to m1 . the continuous release of profibrotic and inflammatory factors promote renal fibrosis , leading to renal failure . m1/m2 renal function varies over time , depending on the type of injury , the persistence and severity of the damage and the reparative ability of the kidney . in the early stages of ckd , pro - inflammatory macrophages ( m1 ) infiltrate the injury site and release pro - inflammatory cytokines , which promote an inflammatory state . if the injury resolves , renal function as well as renal mass ameliorate , depending on damage severity and duration . macrophages also switch to an anti - inflammatory ( m2 ) phenotype , leading to a wound healing phase that may involve tissue fibrosis . however , if there is no injury resolution , m1 macrophages persist at injured sites and there is a decrease in the number of m2 macrophages , which could also subsequently undergo a phenotypic switch to m1 . therefore , interventions such as gene and cell - based therapies are being extensively developed as an alternative treatment modality for the prevention of progression to end - stage renal failure . among potential strategies , macrophage cell - based therapy provides a contrast of promising and discordant results at the same time [ 54 , 72 , 73 ] . macrophages modulated ex vivo and displaying an anti - inflammatory or reparatory activity have been used as cell - based therapy in a number of mouse models of ckd ( table 2 ) . m2 macrophages must meet at least two conditions if they are to be used as a therapeutic tool in vivo : first , the ability to reach the injured tissue or organ , and second , a stable phenotype . these two conditions are particularly important , since infused macrophages may be harmful if they switch from an anti - inflammatory to a pro - inflammatory phenotype . table 2.effects of macrophage cell - based therapy in different mouse models of ckdckd mouse modelcharacteristicsmacrophage cell - based therapyresultslimitationsunilateral ureteral obstruction ( uuo ) [ 56 , 59 , 73 ] well - established model with rapid interstitial fibrosisnon - reversible bone marrow - derived macrophages ( bm - m0)raw246.7 m2 macrophages controversial results : transition into collagen - producing myofibroblasts . at late stage , renal fibrosis is reduced no renal function dataaggressive fibrosisadriamycin - induced nephropathy ( an ) [ 18 , 47 , 62]gradual development of proteinuria , podocyte injury followed by glomerulosclerosis , tubulointerstitial inflammation and fibrosis splenocytes ( m2a and m2c)bone marrow - derived macrophages ( bm - m2a ) failed renoprotection of bm - m2a , whereas splenocytes prevented renal injuryreduction of histological and functional injurythe pathophysiology has not been clarified in full detail by the sequence of events , with proteinuria preceding the development of renal fibrotic lesionsdiabetic kidney disease stz - induced diabetesdestruction of -cells and induction of the hyperglycaemic state associated with inflammatory infiltratessplenocytes ( m0 and m2a)amelioration of tubular atrophy , glomerular hypertrophy and interstitial expansion . degree of interstitial fibrosis , but no effects on renal function regeneration of pancreatic islets can occur after stz treatmentdose - dependent strainsearch criteria : macrophage infusion kidney , m2 macrophage fibrosis , transfused macrophages and alternatively activated macrophages kidney. effects of macrophage cell - based therapy in different mouse models of ckd well - established model with rapid interstitial fibrosis bone marrow - derived macrophages ( bm - m0 ) controversial results : transition into collagen - producing myofibroblasts . at late stage , renal fibrosis is reduced no renal function data splenocytes ( m2a and m2c ) bone marrow - derived macrophages ( bm - m2a ) failed renoprotection of bm - m2a , whereas splenocytes prevented renal injury reduction of histological and functional injury destruction of -cells and induction of the hyperglycaemic state associated with inflammatory infiltrates regeneration of pancreatic islets can occur after stz treatment dose - dependent strain search criteria : macrophage infusion kidney , m2 macrophage fibrosis , transfused macrophages and alternatively activated macrophages kidney. professor david harris 's group has extensively investigated the effects of infusing in vitro generated m2 macrophages in rodent models of both acute kidney failure and ckd [ 18 , 54 , 72 ] . these il-10/tgf--modulated m2 macrophages ( m2c ) expressed high levels of b7-h4 , whereas il-4/il-13-modulated macrophages ( m2a ) did not . thus , these authors attributed the greater potency of m2c macrophages to the expression of the co - stimulation molecule b7-h4 , which suppresses t cell proliferation and induces regulatory t cells both in vitro and in vivo . however , they also demonstrated that infused m2c macrophages did change their phenotype during the disease course , although not towards a distinct m1 phenotype [ 72 , 74 ] . in a severe combined immunodeficient ( scid ) mouse model of an , the infusion of m2 macrophages isolated from the spleen and modulated ex vivo by il-4 and il-13 was associated with an amelioration of renal injury . in contrast , a report by cao et al . showed that using the same mouse model but with m2 macrophages isolated from the bone marrow ( bm - m2 macrophages ) failed to reduce proteinuria and to preserve renal function , owing to a change in macrophage phenotype . therefore , these studies demonstrated that transfused bm - m2 macrophages lost their suppressive function in vivo due to their proliferation , whereas splenic m2 macrophages ( sp - m2 ) were protective because they did not proliferate . the enhanced proliferation of bm - m2 macrophages can be explained by their increased expression of macrophage colony - stimulating factor ( m - csf ) receptor , in comparison with sp - m2 macrophages , which could be partly prevented by blocking csf-1-mediated signalling . transfer of sp - m2 macrophages seems to protect against renal injury , whereas transfer of bm - m2 macrophages appears to promote renal fibrosis [ 18 , 75 ] . however , the bone marrow , rather than the spleen , represents an accessible source of macrophage precursors for aam therapy . therefore , the source of origin of macrophages is another critical issue to bear in mind due to the contradictory outcomes that have been published recently . nowadays , the use of anti - inflammatory and regenerative macrophage - derived molecules is increasing . heme - oxygenase-1 ( ho-1 ) , for example , is a protective and anti - inflammatory enzyme upregulated in response to renal injury , whereas its downregulation is associated with susceptibility to damage [ 76 , 77 ] . demonstrated that sustained overexpression of ho-1 counteracted multiple detrimental renal fibrosis - associated pathological processes in a uuo mouse model . moreover , lin et al . they showed that wnt7b was produced by macrophages and was required to stimulate renal repair and regeneration by acting on injured tecs to promote regeneration of the tubule basement membrane , thereby re - establishing renal function and reducing renal fibrosis . therefore , macrophages have been shown to be important in renal repair , wound healing and regeneration processes . however , it is still unknown whether they can promote renal repair processes directly by fusing with other cells or transforming into new ones or indirectly by providing help to other cell types [ 82 , 83 ] . moreover , macrophages do not remain in a specific phenotype due to their cell plasticity ; they may revert to a resting state and can be subsequently reactivated , depending on the microenvironment . aams have been demonstrated to be protective in reducing renal injury due to their anti - inflammatory role . however , whether these macrophages could become fibrolytic to reduce renal fibrosis still remains unknown . anders and ryu have proposed to classify tissue macrophages according to their predominant roles in different phases of kidney disease : pro - inflammatory , anti - inflammatory , profibrotic and fibrolytic macrophages . hence , more studies are needed before testing macrophage cell - based therapy in humans , since macrophages represent a spectrum of activated phenotypes rather than discrete stable subpopulations [ 5 , 84 ] . therefore , better strategies to induce truly regenerative and reparative macrophages in vivo need to be developed .

retinal angiomatous proliferation ( rap ) has recently been described as a variant of exudative - type age - related macular degeneration ( armd ) , characterized by the initial presence of new intraretinal capillaries that grow toward the subretinal space and choroid.13 three stages of the disease are distinguished : stage i , characterized by the presence of intraretinal neovascularization ; stage ii , in which new capillaries are formed in the subretinal space ( subretinal neovascularization ) ; and stage iii , characterized by choroid neovascularization ( cnv ) with retinochoroidal anastomosis.1 approximately 10%15% of all eyes with exudative armd present rap.1,4,5 the diagnosis of rap is complex , since in most cases , fluorescein angiography ( fa ) is used , revealing a blurred area of exudate within the intra- or subretinal space that is usually classified as occult cnv . in this context , angiography with indocyanine green ( icg ) is recommended for clear identification . given the difficulty of its detection , it is estimated that rap could represent almost a quarter of all cases of occult or minimally classical cnv.1,6 the natural course of rap differs from that in typical exudative armd , and its prognosis in relation to treatment response is poorer.7,8 different therapeutic approaches have been evaluated in rap treatment,9,10 such as surgery,11,12 laser photocoagulation,8 transpupillary thermotherapy,13 photodynamic therapy ( pdt ) with verteporfin,1416 and the intravitreous injection of triamcinolone,12,17 among others . more recently , several studies have been published which analyze the efficacy of antiangiogenic drugs in monotherapy1821 or in combination with other treatments,2224 revealing visual improvement in certain cases . nowadays , there is no evidence to support higher efficacy with one treatment strategy over the rest , since no randomized , controlled clinical trials assessing long - term efficacy have been conducted.9 ranibizumab , which is administered as an intravitreal injection , is an antiangiogenic drug that has shown efficacy and safety for the treatment of wet armd.9,18,2224 ranibizumab is a humanized monoclonal antibody fragment designed to inhibit vegf - a , a key protein in angiogenesis , preventing the growth of new blood capillaries and leakage.25,26 ranibizumab has been used for the treatment of retinal angiomatous proliferation ( rap ) lesions as well , exhibiting a similar profile in comparison with regular armd lesions.18,19,24 in the case of pdt , following luminous activation after the intravenous injection of a photosensitive drug , endothelial damage is induced that favors localized platelet adhesion , resulting in occlusion of the anomalous vessels with selective sealing of the neovascularization without affecting the proximal neurosensory retinal layer . pdt with verteporfin has been shown to be effective in the treatment of cnv associated with exudative armd.16 the existing data suggest that combined administration of pdt with verteporfin and an antiangiogenic or anti - inflammatory drug could offer advantages over monotherapy , slowing or completely arresting the neovascularization process found in rap.22,24 the purpose of the present study was to obtain efficacy and safety data on ranibizumab in monotherapy and in combination with pdt with verteporfin for the treatment of rap during 1 year of follow - up . a randomized , open - label , multicenter , parallel - group study was designed , considering the inclusion of a total of 30 patients with rap ( 15 in each treatment group ) . the following groups were established : group a ( ranibizumab [ lucentis ; novartis , basel , switzerland ] 0.5 mg in monotherapy , administered as an intravitreal injection ) and group b ( combination of ranibizumab 0.5 mg and pdt with verteporfin [ visudyne ; novartis , basel , switzerland ] ) . the patients were randomized in 1:1 proportion to treatment on a stratified basis according to the stage of rap . in group b , the study included patients aged 50 years , diagnosed with rap in stages i iii , with best - corrected visual acuity ( bcva ) in the study eye between 73 and 24 letters , measured by early treatment diabetic retinopathy study ( etdrs ) charts at a distance of 4 m or snellen equivalent . patients receiving topical or systemic steroids or pdt with verteporfin in the study eye during the three previous months or antiangiogenic treatment during the six previous months were excluded from the study . subjects with cataracts in the study eye that were likely to require surgery during the study period , patients with a history of glaucoma in the study eye or with an intraocular pressure 23 mmhg or with uncontrolled arterial hypertension ( systolic blood pressure > 180 mmhg and/or diastolic blood pressure > 100 mmhg ) , women of childbearing potential not using effective contraceptive methods , as well as pregnant and nursing women or women with suspected pregnancy were also excluded . the study was approved by the ethics committees of all participating centers and authorized by the spanish agency for medicines and medical devices . group a received ranibizumab 0.5 mg on days 1 , 30 , and 60 of the trial ( loading phase ) . in addition , group b also received pdt with verteporfin on day 1 . when needed , patients in both groups received retreatment in case of leakage detected on fa or icga , loss of over five letters in visual acuity ( va ) , or a mean increase in macular thickness of 100 m as measured by optical coherence tomography ( oct ) . all retreatments in group b consisted of combined therapy of a single intravitreal injection of ranibizumab and pdt with verteporfin . in addition , in group b , ranibizumab 0.5 mg could be administered in monotherapy as rescue therapy , if necessary . the efficacy of treatment was determined through monthly etdrs chart evaluation of va and oct . all reported adverse events ( aes ) occurring during the clinical trial , as well as concomitant medication were recorded . given the exploratory nature of the study , the efficacy analysis was carried out with an intent - to - treat sample including the randomized patients who had received at least one treatment , with baseline and one posttreatment assessment of the principal study variable , bcva the last observation carried forward method was applied to estimate the missing values . the safety sample , in turn , comprised all patients included in the trial who had received at least one dose of the study drug . the categorical variables were described as absolute and relative frequencies , while the continuous variables were reported as mean , median , standard deviation , and range . comparison of the quantitative variables was based on the use of parametric ( student s t - test or analysis of variance [ anova ] ) or nonparametric tests ( mann whitney u - test ) , depending on the characteristics of the study variables . all statistical calculations were performed using the statistical analysis software version 9.1.3 ( sas institute inc . , cary , nc , usa ) , applying two - tailed tests , with a level of significance of 0.05 . a randomized , open - label , multicenter , parallel - group study was designed , considering the inclusion of a total of 30 patients with rap ( 15 in each treatment group ) . the following groups were established : group a ( ranibizumab [ lucentis ; novartis , basel , switzerland ] 0.5 mg in monotherapy , administered as an intravitreal injection ) and group b ( combination of ranibizumab 0.5 mg and pdt with verteporfin [ visudyne ; novartis , basel , switzerland ] ) . the patients were randomized in 1:1 proportion to treatment on a stratified basis according to the stage of rap . in group b , the study included patients aged 50 years , diagnosed with rap in stages i iii , with best - corrected visual acuity ( bcva ) in the study eye between 73 and 24 letters , measured by early treatment diabetic retinopathy study ( etdrs ) charts at a distance of 4 m or snellen equivalent . patients receiving topical or systemic steroids or pdt with verteporfin in the study eye during the three previous months or antiangiogenic treatment during the six previous months were excluded from the study . subjects with cataracts in the study eye that were likely to require surgery during the study period , patients with a history of glaucoma in the study eye or with an intraocular pressure 23 mmhg or with uncontrolled arterial hypertension ( systolic blood pressure > 180 mmhg and/or diastolic blood pressure > 100 mmhg ) , women of childbearing potential not using effective contraceptive methods , as well as pregnant and nursing women or women with suspected pregnancy were also excluded . the study was approved by the ethics committees of all participating centers and authorized by the spanish agency for medicines and medical devices . group a received ranibizumab 0.5 mg on days 1 , 30 , and 60 of the trial ( loading phase ) . in addition , group b also received pdt with verteporfin on day 1 . when needed , patients in both groups received retreatment in case of leakage detected on fa or icga , loss of over five letters in visual acuity ( va ) , or a mean increase in macular thickness of 100 m as measured by optical coherence tomography ( oct ) . all retreatments in group b consisted of combined therapy of a single intravitreal injection of ranibizumab and pdt with verteporfin . in addition , in group b , ranibizumab 0.5 mg could be administered in monotherapy as rescue therapy , if necessary . the efficacy of treatment was determined through monthly etdrs chart evaluation of va and oct . all reported adverse events ( aes ) occurring during the clinical trial , as well as concomitant medication were recorded . given the exploratory nature of the study , the efficacy analysis was carried out with an intent - to - treat sample including the randomized patients who had received at least one treatment , with baseline and one posttreatment assessment of the principal study variable , bcva the last observation carried forward method was applied to estimate the missing values . the safety sample , in turn , comprised all patients included in the trial who had received at least one dose of the study drug . the categorical variables were described as absolute and relative frequencies , while the continuous variables were reported as mean , median , standard deviation , and range . comparison of the quantitative variables was based on the use of parametric ( student s t - test or analysis of variance [ anova ] ) or nonparametric tests ( mann whitney u - test ) , depending on the characteristics of the study variables . all statistical calculations were performed using the statistical analysis software version 9.1.3 ( sas institute inc . , cary , nc , usa ) , applying two - tailed tests , with a level of significance of 0.05 . a total of 20 patients ( 20 eyes under study ) were included in the trial , and all were evaluable for efficacy ( intent - to - treat sample ) and safety analysis . table 2 shows the demographic and clinical characteristics related to the study disease at baseline , according to treatment groups . seven patients ( 70% ) in group a were females and six patients ( 60% ) in group b were males . the mean age was 79.4 years ( standard deviation [ sd ] = 6.1 ) . at the time of inclusion , the most common rap stage in both groups was stage ii , which was present in six patients ( 60% ) in group a and five patients ( 50% ) in group b. a total of six patients ( three patients in group a and three patients in group b ) had received some previous treatment for cnv in the study eye . glaucoma was the most common previous ophthalmological condition in both groups in the non - study eye . prior cataract surgery on the study eye was recorded in five patients ( 50% ) and two patients ( 20% ) in groups a and b , respectively . the most frequent signs were the presence of epithelial pigment alterations ( 70% and 40% , respectively ) and macular drusen ( 70% and 60% , respectively ) . the most common angiographic pattern in the study eye was minimally classic cnv in five patients of group a ( 50% ) and occult cnv in five patients of group b ( 50% ) . oct of the study eye at the time of inclusion showed five patients ( 50% ) in group a and three patients ( 30% ) in group b to have exudative retinal detachment , while eight patients ( 80% ) and six patients ( 60% ) , respectively , presented cystoid macular edema . no statistically significant differences were observed between the two groups for any of the characteristics analyzed both being homogeneous in terms of the baseline clinical and biodemographic parameters . only two patients from group a were prematurely withdrawn from the study due to informed consent withdrawal by a patient and the onset of a severe concomitant disease which affected the participation of another patient in the clinical trial . the principal efficacy variable or endpoint of the study was the mean change in bcva after 6 and 12 months , calculated with respect to baseline va values for the patients in both treatment groups . the results obtained for groups a and b were : 1.30 ( 95% confidence interval [ ci ] : 5.90 to 8.50 ) and 7.60 ( 95% ci : 1.2014.00 ) after 6 months of follow - up , and 1.50 ( 95% ci : 8.44 to 11.44 ) and 5.60 ( 95% ci : 2.30 to 13.50 ) after 12 months of follow - up , with no statistically significant differences observed according to treatment group ( anova ; p>0.05 ) ( figure 1 ) . the percentage of patients showing improved bcva , defined as a va improvement of over five , ten , and 15 letters after 6 and 12 months of follow - up , versus baseline was determined . after 6 months of follow - up , 40% of the patients ( four patients ) in both groups presented a va increase of over five letters , 10% ( one patient ) in group a and 20% ( two patients ) in group b showed an improvement of over ten letters , and 10% ( one patient ) in group b showed a va gain of over 15 letters . after 12 months of follow - up , 50% of the patients ( five patients ) in group a and 40% ( four patients ) in group b showed a va increase of over five letters , 30% ( three patients ) in group a and 20% ( two patients ) in group b showed a gain of over ten letters , and 20% ( two patients ) in both groups showed an improvement of over 15 letters . no statistically significant differences were found between the two treatment groups or between the visits analyzed for each group ( chi - square test ; p>0.05 ) ( figure 2 ) . regarding the evolution of va , both groups showed statistically significant differences in the mean bcva value versus baseline at visits 4 and 5 ( months 4 and 5 of the trial ) in group a , and at visits 4 , 6 , 7 , and 11 ( months 4 , 6 , 7 , and 11 of the trial ) in group b ( student s t - test ; p<0.05 ) . the percentage of patients presenting a slight decrease in va after 6 and 12 months of follow - up ( defined as a loss of < 15 letters vs baseline ) was 10% ( one patient ) in both groups at 6 months , while a slight decrease was only present in 10% of the patients ( one patient ) in group b after 12 months . no significant differences were observed according to treatment group or between the visits analyzed for each group ( chi - square test ; p>0.05 ) . there were no statistically significant differences between the treatment groups regarding the presence of exudative retinal detachment determined by oct during the study with respect to baseline ( chi - square test ; p>0.05 ) . mean change in greatest linear dimension and in cnv size , measured by fa , and the mean change in foveal thickness as determined by oct , after 3 , 6 , and 12 months versus baseline were analyzed . no statistically significant differences between treatment groups or visits were observed within the same group ( anova ; p>0.05 ) ( table 3 ) . regarding the number of retreatments in the two groups , no statistically significant intergroup differences were detected the mean number per patient being 1.8 ( sd = 1.5 ) in group a and 0.9 ( sd = 1.4 ) in group b ( mann whitney u - test ; p>0.05 ) . the rap relapse rate was 80.0% in group a and 40.0% in group b , and the mean time to retreatment was 3.1 months ( sd = 1.4 ) and 3.8 months ( sd = 0.0 ) , respectively ( mann whitney u - test ; p>0.05 ) ( table 4 ) . forty - five percent of the patients experienced some ae ( six subjects in group a and three in group b ) , with no statistically significant differences found between the two groups ( chi - square test ; p>0.05 ) . none of the aes reported during the trial were considered to be related to the study drug . most of the aes had mild intensity ( 66.6% of those in group a and 100% of those in group b ) . the most frequent aes were ear and labyrinth disorders ( observed in two patients in group a and in one subject in group b ) and ocular problems ( seen in three cases in group b ) . in the course of the study , there was only one serious adverse event ( sae ) : the death of a patient in group a due to worsening of concomitant disease and not related to the study medication . a total of 20 patients ( 20 eyes under study ) were included in the trial , and all were evaluable for efficacy ( intent - to - treat sample ) and safety analysis . table 2 shows the demographic and clinical characteristics related to the study disease at baseline , according to treatment groups . seven patients ( 70% ) in group a were females and six patients ( 60% ) in group b were males . the mean age was 79.4 years ( standard deviation [ sd ] = 6.1 ) . at the time of inclusion , the most common rap stage in both groups was stage ii , which was present in six patients ( 60% ) in group a and five patients ( 50% ) in group b. a total of six patients ( three patients in group a and three patients in group b ) had received some previous treatment for cnv in the study eye . glaucoma was the most common previous ophthalmological condition in both groups in the non - study eye . prior cataract surgery on the study eye was recorded in five patients ( 50% ) and two patients ( 20% ) in groups a and b , respectively . the most frequent signs were the presence of epithelial pigment alterations ( 70% and 40% , respectively ) and macular drusen ( 70% and 60% , respectively ) . the most common angiographic pattern in the study eye was minimally classic cnv in five patients of group a ( 50% ) and occult cnv in five patients of group b ( 50% ) . oct of the study eye at the time of inclusion showed five patients ( 50% ) in group a and three patients ( 30% ) in group b to have exudative retinal detachment , while eight patients ( 80% ) and six patients ( 60% ) , respectively , presented cystoid macular edema . no statistically significant differences were observed between the two groups for any of the characteristics analyzed both being homogeneous in terms of the baseline clinical and biodemographic parameters . only two patients from group a were prematurely withdrawn from the study due to informed consent withdrawal by a patient and the onset of a severe concomitant disease which affected the participation of another patient in the clinical trial . the principal efficacy variable or endpoint of the study was the mean change in bcva after 6 and 12 months , calculated with respect to baseline va values for the patients in both treatment groups . the results obtained for groups a and b were : 1.30 ( 95% confidence interval [ ci ] : 5.90 to 8.50 ) and 7.60 ( 95% ci : 1.2014.00 ) after 6 months of follow - up , and 1.50 ( 95% ci : 8.44 to 11.44 ) and 5.60 ( 95% ci : 2.30 to 13.50 ) after 12 months of follow - up , with no statistically significant differences observed according to treatment group ( anova ; p>0.05 ) ( figure 1 ) . the percentage of patients showing improved bcva , defined as a va improvement of over five , ten , and 15 letters after 6 and 12 months of follow - up , versus baseline was determined . after 6 months of follow - up , 40% of the patients ( four patients ) in both groups presented a va increase of over five letters , 10% ( one patient ) in group a and 20% ( two patients ) in group b showed an improvement of over ten letters , and 10% ( one patient ) in group b showed a va gain of over 15 letters . after 12 months of follow - up , 50% of the patients ( five patients ) in group a and 40% ( four patients ) in group b showed a va increase of over five letters , 30% ( three patients ) in group a and 20% ( two patients ) in group b showed a gain of over ten letters , and 20% ( two patients ) in both groups showed an improvement of over 15 letters . no statistically significant differences were found between the two treatment groups or between the visits analyzed for each group ( chi - square test ; p>0.05 ) ( figure 2 ) . regarding the evolution of va , both groups showed statistically significant differences in the mean bcva value versus baseline at visits 4 and 5 ( months 4 and 5 of the trial ) in group a , and at visits 4 , 6 , 7 , and 11 ( months 4 , 6 , 7 , and 11 of the trial ) in group b ( student s t - test ; p<0.05 ) . the percentage of patients presenting a slight decrease in va after 6 and 12 months of follow - up ( defined as a loss of < 15 letters vs baseline ) was 10% ( one patient ) in both groups at 6 months , while a slight decrease was only present in 10% of the patients ( one patient ) in group b after 12 months . no significant differences were observed according to treatment group or between the visits analyzed for each group ( chi - square test ; p>0.05 ) . there were no statistically significant differences between the treatment groups regarding the presence of exudative retinal detachment determined by oct during the study with respect to baseline ( chi - square test ; p>0.05 ) . mean change in greatest linear dimension and in cnv size , measured by fa , and the mean change in foveal thickness as determined by oct , after 3 , 6 , and 12 months versus baseline were analyzed . no statistically significant differences between treatment groups or visits were observed within the same group ( anova ; p>0.05 ) ( table 3 ) . regarding the number of retreatments in the two groups , no statistically significant intergroup differences were detected the mean number per patient being 1.8 ( sd = 1.5 ) in group a and 0.9 ( sd = 1.4 ) in group b ( mann whitney u - test ; p>0.05 ) . the rap relapse rate was 80.0% in group a and 40.0% in group b , and the mean time to retreatment was 3.1 months ( sd = 1.4 ) and 3.8 months ( sd = 0.0 ) , respectively ( mann whitney u - test ; p>0.05 ) ( table 4 ) . forty - five percent of the patients experienced some ae ( six subjects in group a and three in group b ) , with no statistically significant differences found between the two groups ( chi - square test ; p>0.05 ) . none of the aes reported during the trial were considered to be related to the study drug . most of the aes had mild intensity ( 66.6% of those in group a and 100% of those in group b ) . the most frequent aes were ear and labyrinth disorders ( observed in two patients in group a and in one subject in group b ) and ocular problems ( seen in three cases in group b ) . in the course of the study , there was only one serious adverse event ( sae ) : the death of a patient in group a due to worsening of concomitant disease and not related to the study medication . rap is considered to be a subtype of neovascular armd,1 differing from the latter in terms of the course of condition and response to treatment,7 with a poorer prognosis due to the absence of standardized treatment up to date.1,9,23 therefore , any strategy offering stabilization or improvement constitutes an advancement in the management of the disease . in this sense , the contribution of this study with respect to previous studies on rap is based upon its design . the present study is a prospective one with stratified randomization for both treatment regimens ( ranibizumab in monotherapy or ranibizumab plus pdt with verteporfin ) according to the stage of the disease , resulting in comparable , homogeneous groups . in the present study , although no significant differences were observed between the groups regarding the analyzed efficacy variables , both treatment groups showed a tendency toward an increase in mean bcva scores versus baseline values , after both 6 and 12 months of follow - up . this improvement in va was greater in group b than in patients administered monotherapy ( at 12 months : 5.60 letters vs 1.50 letters ) ( figure 1 ) . after 12 months , 20% of the patients in both treatment groups showed a va improvement of over three lines , and 30% and 20% , respectively , showed more than two lines of gain in va versus the baseline visit ( figure 2 ) . starting in the fourth month of the study , a statistically significant increase in mean bcva was noted versus baseline in both groups . at this point , ranibizumab loading phase had already ended , and thus , at least three intravitreous injections of the drug had been administered in both groups . similar results were reported in a retrospective case review of 26 patients treated with ranibizumab 0.5 mg,18 where 31% of patients showed a bcva increment of over three lines after receiving the first three intravitreous injections of the loading phase . in the present study and throughout the duration of follow - up , no statistically significant differences were observed between the treatment groups regarding the morphological changes in the retina as determined by fa or icg ( surface of the lesion or of the cnv ) or in central foveal thickness as evidenced by oct . however , on analyzing the mean change in foveal thickness , a decrease was observed in months 3 , 6 , and 12 versus baseline , thus suggesting a tendency toward improvement in both treatment groups , with comparatively greater improvement found in the ranibizumab monotherapy group . rouvas et al24 have published a randomized prospective trial with a 12-month follow - up , comparing the efficacy of three treatments : ranibizumab 0.5 mg in monotherapy ( n=13 ) or in combination with pdt with verteporfin ( n=13 ) , and a combination of intravitreous triamcinolone with pdt with verteporfin ( n=11 ) ; the criterion used for retreatment was the presence of sub- or intraretinal exudate . in a similar way to our study , the above - mentioned study reported va stabilization in all treatment groups at the end of follow - up , with no statistically significant differences between groups . in this context , combination of triamcinolone and pdt showed the best results , with no differences between the other two treatment groups . likewise , the authors detected no differences in terms of the number of retreatments required by the patients administered ranibizumab in monotherapy ( 3.07 ) or in combination with pdt ( 0.46).24 in the present study , the mean number of retreatments in monotherapy group was almost one half after 12 months follow - up ( 1.8 ) . this difference between monotherapy and combined therapy may be due to the time of retreatment administration according to protocol ; intravitreal injection of ranibizumab could be administered every 30 days , while ranibizumab plus pdt should be administered every 90 days . regarding foveal thickness , rouvas et al24 reported a decrease in all treatment groups , though significance was only reached in groups receiving combined therapy ( ranibizumab with pdt and triamcinolone with pdt ) , which differed from our results wherein patients administered ranibizumab in monotherapy showed the most important anatomical changes . despite the differences between the two trials in terms of design , patient profile , method used for va evaluation , and the treatment scheme employed , the results of the present study were similar to those published by rouvas et al.24 the following should be noted : the latter study did not perform stratified randomization to therapy according to rap stage ; the va measurements were based on a nonstandardized snellen chart ; all of the analyzed patients were nave to treatment for cnv in the study eye ; and the groups were not homogeneous in terms of rap stage . in the present study , 30% of the patients in both groups had received some previous treatment for cnv in the study eye a fact that may lead to a lower improvement observed during follow - up in terms of va and foveal thickness . in this trend , reche - frutos et al27 had reported results from a prospective trial performed in non - nave patients with rap treated with ranibizumab in monotherapy at different stages . their results suggested that a higher percentage of patients with previous treatment and a lower baseline va could indicate a longer time of disease progression and greater structural involvement of the retina ; so , a poorer response could be expected . in reche - frutos et al s study , all patients with no relapse of rap after 12 months were in stage iia of rap , which indicates that monotherapy with ranibizumab could provide better response in patients with more superficial lesions . confirming these findings , in the present study , only 20% of patients in the group treated with monotherapy showed no relapse in rap lesion ; this group showed majorly rap stage ii at baseline . more recently , saito et al28 reported a retrospective case series review on patients with rap who were nave to treatment and majorly in rap stage ii and were treated with a combination of ranibizumab intravitreal injection and pdt with verteporfin ; an increase of three lines or more in bcva was reached in 50% of the treated eyes after 12 months of follow - up , in comparison with the results of the present study where only 20% of eyes treated with the combined treatment showed a similar increase in bcva at 12 months . present data indicate that both studied treatments stabilized va and foveal thickness after 6 and 12 months of follow - up , as shown by positive values obtained in the mean change in bcva and negative values in the mean change in foveal thickness for both treatment groups . the results are in agreement with the results reported in a number of studies on ranibizumab in monotherapy1820,27 or in combination with pdt.24,28,29 in this sense , the observed functional improvement ( va ) proved greater for the combination of ranibizumab plus pdt with verteporfin , while from the anatomical perspective ( foveal thickness ) , the greatest reduction corresponded to the group administered ranibizumab in monotherapy . this finding differs from the observations of other studies in which functional and anatomical improvement showed the same trend.1820,24,28,30 among other factors , this discrepancy may be due to the different profiles of the patients included in these studies , where subjects had not received previous treatment in the study eye and the initial rap stage differed . for a long time , pdt with verteporfin has been considered the gold standard treatment for rap,7,9,12 despite the scanty encouraging results obtained . however , pdt in combination with other drugs ( antiangiogenic agents or corticosteroids)14,15,17,2224 might be a better approach for the treatment of advanced rap lesions . results obtained from recent studies seem to indicate that combined therapy consisting of intravitreal ranibizumab and pdt might require fewer treatments than an anti - vegf agent in monotherapy.28,31 on the other hand , studies with bevacizumab22,23 in monotherapy or combined with verteporfin report results similar to those obtained in the present study , pointing to the combination treatment as a viable alternative in the management of rap . regarding safety , no serious aes related to the study drug were reported , and both groups showed a similar safety profile . these observations coincide with the available literature on ranibizumab 0.5 mg used to treat this disorder.1820,27,30 the main limitation of this study is that only 20 of the 30 initially planned subjects could be recruited due to the difficulty in identifying eligible patients . nevertheless , the total number of subjects per group was similar to that of the only randomized trial published to date on ranibizumab and related to this condition.24 in addition , the stratified randomization according to rap stage , the inclusion of patients previously treated for cnv , and the use of standardized methods to assess efficacy and safety give validity to the present study results obtained with ranibizumab in monotherapy or combined with pdt with verteporfin in the treatment of rap . results obtained in the present study warrant the need for randomized trials including a larger number of patients per treatment group , with a more representative profile of this disease , as seen by retinal specialists in clinical practice , and which may provide enough evidence for establishing an optimum treatment for rap . present study findings suggest that ranibizumab may be a valid therapeutic alternative in the management of rap .

purposeto compare the effects of intravitreal ranibizumab in monotherapy ( group a ) and combined with photodynamic therapy ( pdt ) with verteporfin ( group b ) in retinal angiomatous proliferation ( rap ) treatment.methodsthis was a multicentric , prospective , randomized clinical study conducted with parallel groups . the study eye in both groups received ranibizumab on days 1 , 30 , and 60 ( loading dose ) ; group b received pdt additionally on day 1 . early treatment diabetic retinopathy study ( etdrs ) visual acuity ( va ) testing and optical coherence tomography were performed monthly , and fluorescein angiography and indocyanine green angiography were performed quarterly . retreatment criteria were leakage in fluorescein angiography or indocyanine green angiography , mean foveal thickness increase 100 m , or va decrease 5 letters.resultstwenty patients were recruited ( ten patients in each group ) . six eyes had previous treatment ( three eyes in group a and three eyes in group b ) , so only 14 eyes were nave . at 12-month follow - up , mean va improved + 1.5 letters in group a and + 5.6 letters in group b ( analysis of variance test ; p>0.05 ) . two patients ( 20% ) in both groups gained 15 letters ( chi - square test ; p>0.05 ) . mean changes in greatest linear dimension and in foveal thickness were not statistically significant between groups of treatment ( analysis of variance test ; p>0.05 ) . mean retreatments per patient were 1.8 ( group a ) and 0.9 ( group b ) ( mann whitney u - test ; p>0.05 ) . one patient died due to underlying disease not related to study medication.conclusionintravitreal ranibizumab administered in monotherapy or combined with pdt was efficacious in terms of va stabilization in patients with rap .

Introduction Methods Study design Treatment scheme Efficacy and safety evaluation Statistical analysis Results Description of the patient baseline characteristics Efficacy results Safety results Discussion Conclusion

retinal angiomatous proliferation ( rap ) has recently been described as a variant of exudative - type age - related macular degeneration ( armd ) , characterized by the initial presence of new intraretinal capillaries that grow toward the subretinal space and choroid.13 three stages of the disease are distinguished : stage i , characterized by the presence of intraretinal neovascularization ; stage ii , in which new capillaries are formed in the subretinal space ( subretinal neovascularization ) ; and stage iii , characterized by choroid neovascularization ( cnv ) with retinochoroidal anastomosis.1 approximately 10%15% of all eyes with exudative armd present rap.1,4,5 the diagnosis of rap is complex , since in most cases , fluorescein angiography ( fa ) is used , revealing a blurred area of exudate within the intra- or subretinal space that is usually classified as occult cnv . given the difficulty of its detection , it is estimated that rap could represent almost a quarter of all cases of occult or minimally classical cnv.1,6 the natural course of rap differs from that in typical exudative armd , and its prognosis in relation to treatment response is poorer.7,8 different therapeutic approaches have been evaluated in rap treatment,9,10 such as surgery,11,12 laser photocoagulation,8 transpupillary thermotherapy,13 photodynamic therapy ( pdt ) with verteporfin,1416 and the intravitreous injection of triamcinolone,12,17 among others . pdt with verteporfin has been shown to be effective in the treatment of cnv associated with exudative armd.16 the existing data suggest that combined administration of pdt with verteporfin and an antiangiogenic or anti - inflammatory drug could offer advantages over monotherapy , slowing or completely arresting the neovascularization process found in rap.22,24 the purpose of the present study was to obtain efficacy and safety data on ranibizumab in monotherapy and in combination with pdt with verteporfin for the treatment of rap during 1 year of follow - up . the following groups were established : group a ( ranibizumab [ lucentis ; novartis , basel , switzerland ] 0.5 mg in monotherapy , administered as an intravitreal injection ) and group b ( combination of ranibizumab 0.5 mg and pdt with verteporfin [ visudyne ; novartis , basel , switzerland ] ) . in group b , the study included patients aged 50 years , diagnosed with rap in stages i iii , with best - corrected visual acuity ( bcva ) in the study eye between 73 and 24 letters , measured by early treatment diabetic retinopathy study ( etdrs ) charts at a distance of 4 m or snellen equivalent . group a received ranibizumab 0.5 mg on days 1 , 30 , and 60 of the trial ( loading phase ) . when needed , patients in both groups received retreatment in case of leakage detected on fa or icga , loss of over five letters in visual acuity ( va ) , or a mean increase in macular thickness of 100 m as measured by optical coherence tomography ( oct ) . comparison of the quantitative variables was based on the use of parametric ( student s t - test or analysis of variance [ anova ] ) or nonparametric tests ( mann whitney u - test ) , depending on the characteristics of the study variables . the following groups were established : group a ( ranibizumab [ lucentis ; novartis , basel , switzerland ] 0.5 mg in monotherapy , administered as an intravitreal injection ) and group b ( combination of ranibizumab 0.5 mg and pdt with verteporfin [ visudyne ; novartis , basel , switzerland ] ) . in group b , the study included patients aged 50 years , diagnosed with rap in stages i iii , with best - corrected visual acuity ( bcva ) in the study eye between 73 and 24 letters , measured by early treatment diabetic retinopathy study ( etdrs ) charts at a distance of 4 m or snellen equivalent . group a received ranibizumab 0.5 mg on days 1 , 30 , and 60 of the trial ( loading phase ) . when needed , patients in both groups received retreatment in case of leakage detected on fa or icga , loss of over five letters in visual acuity ( va ) , or a mean increase in macular thickness of 100 m as measured by optical coherence tomography ( oct ) . comparison of the quantitative variables was based on the use of parametric ( student s t - test or analysis of variance [ anova ] ) or nonparametric tests ( mann whitney u - test ) , depending on the characteristics of the study variables . at the time of inclusion , the most common rap stage in both groups was stage ii , which was present in six patients ( 60% ) in group a and five patients ( 50% ) in group b. a total of six patients ( three patients in group a and three patients in group b ) had received some previous treatment for cnv in the study eye . prior cataract surgery on the study eye was recorded in five patients ( 50% ) and two patients ( 20% ) in groups a and b , respectively . the most common angiographic pattern in the study eye was minimally classic cnv in five patients of group a ( 50% ) and occult cnv in five patients of group b ( 50% ) . oct of the study eye at the time of inclusion showed five patients ( 50% ) in group a and three patients ( 30% ) in group b to have exudative retinal detachment , while eight patients ( 80% ) and six patients ( 60% ) , respectively , presented cystoid macular edema . the results obtained for groups a and b were : 1.30 ( 95% confidence interval [ ci ] : 5.90 to 8.50 ) and 7.60 ( 95% ci : 1.2014.00 ) after 6 months of follow - up , and 1.50 ( 95% ci : 8.44 to 11.44 ) and 5.60 ( 95% ci : 2.30 to 13.50 ) after 12 months of follow - up , with no statistically significant differences observed according to treatment group ( anova ; p>0.05 ) ( figure 1 ) . after 6 months of follow - up , 40% of the patients ( four patients ) in both groups presented a va increase of over five letters , 10% ( one patient ) in group a and 20% ( two patients ) in group b showed an improvement of over ten letters , and 10% ( one patient ) in group b showed a va gain of over 15 letters . after 12 months of follow - up , 50% of the patients ( five patients ) in group a and 40% ( four patients ) in group b showed a va increase of over five letters , 30% ( three patients ) in group a and 20% ( two patients ) in group b showed a gain of over ten letters , and 20% ( two patients ) in both groups showed an improvement of over 15 letters . no statistically significant differences were found between the two treatment groups or between the visits analyzed for each group ( chi - square test ; p>0.05 ) ( figure 2 ) . regarding the evolution of va , both groups showed statistically significant differences in the mean bcva value versus baseline at visits 4 and 5 ( months 4 and 5 of the trial ) in group a , and at visits 4 , 6 , 7 , and 11 ( months 4 , 6 , 7 , and 11 of the trial ) in group b ( student s t - test ; p<0.05 ) . the percentage of patients presenting a slight decrease in va after 6 and 12 months of follow - up ( defined as a loss of < 15 letters vs baseline ) was 10% ( one patient ) in both groups at 6 months , while a slight decrease was only present in 10% of the patients ( one patient ) in group b after 12 months . no significant differences were observed according to treatment group or between the visits analyzed for each group ( chi - square test ; p>0.05 ) . there were no statistically significant differences between the treatment groups regarding the presence of exudative retinal detachment determined by oct during the study with respect to baseline ( chi - square test ; p>0.05 ) . mean change in greatest linear dimension and in cnv size , measured by fa , and the mean change in foveal thickness as determined by oct , after 3 , 6 , and 12 months versus baseline were analyzed . regarding the number of retreatments in the two groups , no statistically significant intergroup differences were detected the mean number per patient being 1.8 ( sd = 1.5 ) in group a and 0.9 ( sd = 1.4 ) in group b ( mann whitney u - test ; p>0.05 ) . the rap relapse rate was 80.0% in group a and 40.0% in group b , and the mean time to retreatment was 3.1 months ( sd = 1.4 ) and 3.8 months ( sd = 0.0 ) , respectively ( mann whitney u - test ; p>0.05 ) ( table 4 ) . forty - five percent of the patients experienced some ae ( six subjects in group a and three in group b ) , with no statistically significant differences found between the two groups ( chi - square test ; p>0.05 ) . the most frequent aes were ear and labyrinth disorders ( observed in two patients in group a and in one subject in group b ) and ocular problems ( seen in three cases in group b ) . in the course of the study , there was only one serious adverse event ( sae ) : the death of a patient in group a due to worsening of concomitant disease and not related to the study medication . at the time of inclusion , the most common rap stage in both groups was stage ii , which was present in six patients ( 60% ) in group a and five patients ( 50% ) in group b. a total of six patients ( three patients in group a and three patients in group b ) had received some previous treatment for cnv in the study eye . prior cataract surgery on the study eye was recorded in five patients ( 50% ) and two patients ( 20% ) in groups a and b , respectively . the most common angiographic pattern in the study eye was minimally classic cnv in five patients of group a ( 50% ) and occult cnv in five patients of group b ( 50% ) . oct of the study eye at the time of inclusion showed five patients ( 50% ) in group a and three patients ( 30% ) in group b to have exudative retinal detachment , while eight patients ( 80% ) and six patients ( 60% ) , respectively , presented cystoid macular edema . the results obtained for groups a and b were : 1.30 ( 95% confidence interval [ ci ] : 5.90 to 8.50 ) and 7.60 ( 95% ci : 1.2014.00 ) after 6 months of follow - up , and 1.50 ( 95% ci : 8.44 to 11.44 ) and 5.60 ( 95% ci : 2.30 to 13.50 ) after 12 months of follow - up , with no statistically significant differences observed according to treatment group ( anova ; p>0.05 ) ( figure 1 ) . after 6 months of follow - up , 40% of the patients ( four patients ) in both groups presented a va increase of over five letters , 10% ( one patient ) in group a and 20% ( two patients ) in group b showed an improvement of over ten letters , and 10% ( one patient ) in group b showed a va gain of over 15 letters . after 12 months of follow - up , 50% of the patients ( five patients ) in group a and 40% ( four patients ) in group b showed a va increase of over five letters , 30% ( three patients ) in group a and 20% ( two patients ) in group b showed a gain of over ten letters , and 20% ( two patients ) in both groups showed an improvement of over 15 letters . no statistically significant differences were found between the two treatment groups or between the visits analyzed for each group ( chi - square test ; p>0.05 ) ( figure 2 ) . regarding the evolution of va , both groups showed statistically significant differences in the mean bcva value versus baseline at visits 4 and 5 ( months 4 and 5 of the trial ) in group a , and at visits 4 , 6 , 7 , and 11 ( months 4 , 6 , 7 , and 11 of the trial ) in group b ( student s t - test ; p<0.05 ) . the percentage of patients presenting a slight decrease in va after 6 and 12 months of follow - up ( defined as a loss of < 15 letters vs baseline ) was 10% ( one patient ) in both groups at 6 months , while a slight decrease was only present in 10% of the patients ( one patient ) in group b after 12 months . no significant differences were observed according to treatment group or between the visits analyzed for each group ( chi - square test ; p>0.05 ) . there were no statistically significant differences between the treatment groups regarding the presence of exudative retinal detachment determined by oct during the study with respect to baseline ( chi - square test ; p>0.05 ) . mean change in greatest linear dimension and in cnv size , measured by fa , and the mean change in foveal thickness as determined by oct , after 3 , 6 , and 12 months versus baseline were analyzed . regarding the number of retreatments in the two groups , no statistically significant intergroup differences were detected the mean number per patient being 1.8 ( sd = 1.5 ) in group a and 0.9 ( sd = 1.4 ) in group b ( mann whitney u - test ; p>0.05 ) . the rap relapse rate was 80.0% in group a and 40.0% in group b , and the mean time to retreatment was 3.1 months ( sd = 1.4 ) and 3.8 months ( sd = 0.0 ) , respectively ( mann whitney u - test ; p>0.05 ) ( table 4 ) . forty - five percent of the patients experienced some ae ( six subjects in group a and three in group b ) , with no statistically significant differences found between the two groups ( chi - square test ; p>0.05 ) . the most frequent aes were ear and labyrinth disorders ( observed in two patients in group a and in one subject in group b ) and ocular problems ( seen in three cases in group b ) . in the course of the study , there was only one serious adverse event ( sae ) : the death of a patient in group a due to worsening of concomitant disease and not related to the study medication . rouvas et al24 have published a randomized prospective trial with a 12-month follow - up , comparing the efficacy of three treatments : ranibizumab 0.5 mg in monotherapy ( n=13 ) or in combination with pdt with verteporfin ( n=13 ) , and a combination of intravitreous triamcinolone with pdt with verteporfin ( n=11 ) ; the criterion used for retreatment was the presence of sub- or intraretinal exudate . in a similar way to our study , the above - mentioned study reported va stabilization in all treatment groups at the end of follow - up , with no statistically significant differences between groups . likewise , the authors detected no differences in terms of the number of retreatments required by the patients administered ranibizumab in monotherapy ( 3.07 ) or in combination with pdt ( 0.46).24 in the present study , the mean number of retreatments in monotherapy group was almost one half after 12 months follow - up ( 1.8 ) . regarding foveal thickness , rouvas et al24 reported a decrease in all treatment groups , though significance was only reached in groups receiving combined therapy ( ranibizumab with pdt and triamcinolone with pdt ) , which differed from our results wherein patients administered ranibizumab in monotherapy showed the most important anatomical changes . despite the differences between the two trials in terms of design , patient profile , method used for va evaluation , and the treatment scheme employed , the results of the present study were similar to those published by rouvas et al.24 the following should be noted : the latter study did not perform stratified randomization to therapy according to rap stage ; the va measurements were based on a nonstandardized snellen chart ; all of the analyzed patients were nave to treatment for cnv in the study eye ; and the groups were not homogeneous in terms of rap stage . in the present study , 30% of the patients in both groups had received some previous treatment for cnv in the study eye a fact that may lead to a lower improvement observed during follow - up in terms of va and foveal thickness . more recently , saito et al28 reported a retrospective case series review on patients with rap who were nave to treatment and majorly in rap stage ii and were treated with a combination of ranibizumab intravitreal injection and pdt with verteporfin ; an increase of three lines or more in bcva was reached in 50% of the treated eyes after 12 months of follow - up , in comparison with the results of the present study where only 20% of eyes treated with the combined treatment showed a similar increase in bcva at 12 months . the results are in agreement with the results reported in a number of studies on ranibizumab in monotherapy1820,27 or in combination with pdt.24,28,29 in this sense , the observed functional improvement ( va ) proved greater for the combination of ranibizumab plus pdt with verteporfin , while from the anatomical perspective ( foveal thickness ) , the greatest reduction corresponded to the group administered ranibizumab in monotherapy . results obtained from recent studies seem to indicate that combined therapy consisting of intravitreal ranibizumab and pdt might require fewer treatments than an anti - vegf agent in monotherapy.28,31 on the other hand , studies with bevacizumab22,23 in monotherapy or combined with verteporfin report results similar to those obtained in the present study , pointing to the combination treatment as a viable alternative in the management of rap . nevertheless , the total number of subjects per group was similar to that of the only randomized trial published to date on ranibizumab and related to this condition.24 in addition , the stratified randomization according to rap stage , the inclusion of patients previously treated for cnv , and the use of standardized methods to assess efficacy and safety give validity to the present study results obtained with ranibizumab in monotherapy or combined with pdt with verteporfin in the treatment of rap .

pelvic inflammatory disease ( pid ) is a spectrum of upper genital tract infections that includes endometritis , salpingitis , tuboovarian abscess , and/or pelvic peritonitis . typically , acute pid is caused by ascending spread of microorganisms from the vagina and/or endocervix to the endometrium , fallopian tubes , and/or adjacent structures [ 13 ] . acute salpingitis is the most important component of the pid spectrum because of its impact on future fertility . pid is one of the most frequent and important infections that occur among nonpregnant women of reproductive age and remains a major public health problem [ 48 ] . among women , unfortunately , women who acquire acute pid are at risk for long - term sequelae including tubal factor infertility , ectopic pregnancy , chronic pelvic pain , and recurrent pid [ 913 ] . in addition , the estimated annual health care cost for pid and its complications in the united states is over $ 2 billion . currently , an estimated 770,000 cases of acute pid are diagnosed annually in the united states . a recent analysis by the centers for disease control and prevention ( cdc ) of trends in the incidence of pid demonstrated that from 1985 to 2001 rates of both hospitalized and ambulatory cases of acute pid declined ( 68% and 47% , resp . ) . recently , subclinical pid has been recognized as an important entity which is common among women with lower genital tract infections , especially chlamydia trachomatis , neisseria gonorrhoeae , and bacterial vaginosis ( bv ) [ 14 , 15 ] . subclinical pid is as likely as clinically recognized acute pid and is responsible for a greater proportion of pid - related sequelae than clinically recognized disease . secondly , is concern that the continued increases in c. trachomatis infections reported by the cdc in the united states will be associated with an increase in both clinical and subclinical pid . over the past 25 years , important advances have occurred in understanding the etiology , pathogenesis , and treatment of acute pid . as a result , major paradigm shifts have occurred in our approach to the treatment of acute pid . in the past pid today , the polymicrobic etiology of pid is well established and has led to utilization of broad spectrum antimicrobial regimens for treatment of acute pid [ 1 , 2 , 17 , 18 ] . prevention of the significant long - term complications associated with pid requires development of effective treatment strategies . such treatment regimens are dependent upon an understanding of the microbiologic etiology of acute pid . firstly , most studies have utilized specimens obtained from the lower genital tract ( primarily cervix ) and not the upper genital tract ( endometrial cavity , fallopian tubes ) which is the actual site of infection . secondly , most investigations primarily focused on the sexually transmitted pathogens n. gonorrhoeae and/or c. trachomati , s and few studies have assessed the role of non - std pathogens , especially anaerobic bacteria . thirdly , even fewer investigations have addressed the putative role of mycoplasma genitalium in the etiology of pid . pid results from the intracannicular ascending spread of microorganisms from the cervix and/or vagina into the upper genital tract . prior to the mid-1970s , pid was believed to be a monoetiologic infection due primarily to n. gonorrhoeae . based initially upon culdocentesis studies of peritoneal fluid ( figure 1 ) and subsequently studies utilizing laparoscopy and/or endometrial aspirations to obtain specimens from the upper genital tract ( table 1 ) came the recognition that the etiology of acute pid is polymicrobic with a wide variety of microorganisms involved [ 1 , 2 , 1941 ] . included among these are n. gonorrhoeae , c. trachomatis , genital tract mycoplasmas ( particularly m. genitalium ) , anaerobic and aerobic bacteria which comprise the endogenous vaginal flora ( e.g. , prevotella species , black - pigmented gram - negative anaerobic rods , peptostreptococci sp . , gardnerella vaginalis , escherichia coli , haemophilus influenzae , and aerobic streptococci ) . investigations by our group conducted in the 1980s that utilized laparoscopy and/or endometrial aspirations to obtain upper genital tract specimens demonstrated that approximately two - thirds of acute pid cases were associated with n. gonorrhoeae and/or c. trachomatis ( figure 2 ) . in nearly one - third only anaerobic and aerobic bacteria are recovered . in addition , half of the women with n. gonorrhoeae and/or c. trachomatis had concomitant anaerobic and/or aerobic bacteria recovered . more recently , in the pelvic inflammatory disease evaluation and clinical health ( peach ) study , the largest treatment trial of mild to moderate acute pid in the us , n. gonorrhoeae and c. trachomatis were recovered in less than one - third of patients . many of the nongonococcal , nonchlamydial microorganisms recovered from the upper genital tract in acute pid are similar to those associated with bacterial vaginosis ( bv ) , a complex perturbation of the vaginal flora leading to loss of hydrogen peroxide producing lactobacillus and overgrowth of g. vaginalis , prevotella sp . bivius , p. disiens , and p. capillosus ) , mobiluncus sp . , black - pigmented anaerobic gram - negative rods , alpha - hemolytic streptococci , and mycoplasmas . multiple investigations have demonstrated an association between bv and acute pid [ 31 , 35 , 4351 ] . in addition , use of a broad - range 16srdna polymerase chain reaction to identify uncultivable bacteria has identified bacterial 16s sequences of anaerobic bacteria associated with bv in the fallopian tube of women with laparoscopically confirmed acute pid . although m. genitalium was identified in the early 1980s as a cause of nongonococcal urethritis in men , its role in genital tract infections in women remained unclear , due in large part to difficulty in culturing this organism . with the advent of polymerase chain reaction ( pcr ) technology , m. genitalium has been associated with cervicitis [ 53 , 54 ] and has been demonstrated as an etiologic agent in nongonococcal nonchlamydial pid [ 3639 ] . haggerty et al . detected m. genitalium in 15% of women in the peach study , a rate similar to that seen in uk women ( 13% ) and west african women ( 16% ) . these rates of m. genitalium are similar to those seen for c. trachomatis and n. gonorrhoeae in the peach study of urban women in the united states . a recent analysis from the peach study noted that rates of short - term failure ( persistent endometritis and pelvic pain ) , infertility , recurrent pid , and chronic pelvic pain were high among women with endometrial m. genitalium at baseline . subsequently , it has been demonstrated that women with m. genitalium infection ( similar to those with chlamydial infection ) present with fewer clinical signs and symptoms of acute pid than those with gonococcal infection . a pathogenic role of m. genitalium in pid is further supported by studies demonstrating that m. genitalium induces salpingitis in experimental monkey studies and adheres to human fallopian tube epithelial cells , in organ culture , causing damage to ciliated cells . this term was initially applied to women with documented tubal factor infertility associated with evidence of chronic inflammatory residua characteristic of pid who denied a history of being diagnosed or treated for acute pid . preliminary work by our group has suggested that the microorganisms ( e.g. , n. gonorrhoeae , c. trachomatis , and bacterial vaginosis ) associated with subclinical pid are the same putative agents recovered from women with clinically apparent acute pid . the therapeutic goals for treatment of acute pid include both short - term outcomes such as clinical cure and microbiologic cure and preventions of long - term sequelae such as infertility , ectopic pregnancy , recurrent infection , and chronic pelvic pain . although the incidence rates of pid have declined , no reduction in the adverse reproductive outcomes associated with pid ( infertility , ectopic pregnancy , and chronic pelvic pain ) has been demonstrated . while some antibiotic regimens have been successful in producing initial clinical and microbiologic cure with short - term followup , only a few studies have determined the efficacy of these treatment regimens for eliminating endometrial or fallopian tube infection . in addition , few studies have attempted to assess the incidence of long - term sequelae ( e.g. , tubal factor infertility , ectopic pregnancy and chronic pelvic pain ) following treatment with these antibiotic regimens [ 1 , 10 , 11 , 42 ] . in the preantibiotic era most cases of acute pid managed by conservative supportive care resolved spontaneously with studies demonstrating that approximately 85% of patients with acute pid improved clinically without the need for surgical intervention . the introduction of antibiotics into clinical practice led to improvement in the prognosis for acute pid , and mortality was nearly eliminated . studies assessing fertility rates following acute pid showed a general improvement in fertility with the mean pregnancy rate increasing from 27.9% ( range 24%43% ) in the preantibiotic era to 73.1% ( range 24%81% ) in the post - antibiotic era . while this finding is satisfying , these results are still far from adequate . substantial evidence supports the role of n. gonorrhoeae , c. trachomatis , anaerobic bacteria , and facultative bacteria in the pathogenesis of acute pid [ 15 , 9 ] . not only are n. gonorrhoeae and c. trachomatis frequently recovered from the upper genital tract in women with pid , excellent data demonstrates the role these pathogens play in producing tubal damage and in the development of the adverse sequelae of pid ( e.g. , infertility , ectopic pregnancy ) [ 5760 ] . thus , antimicrobial regimens for the treatment of acute pid must be effective against these std organisms . while some antimicrobial regimens that do not provide adequate coverage against n. gonorrhoeae and/or c. trachomatis have been shown to have excellent clinical cure rates , microbiologic cure rates are less impressive ( or lacking ) , and long - term outcome data are not available [ 17 , 18 , 6164 ] . the cdc in its 2010 treatment recommendations notes that all regimens used to treat acute pid should provide adequate coverage against n. gonorrhoeae and c. trachomatis , as they are both commonly present and have the propensity to produce tubal damage directly ( n. gonorrhoeae ) or indirectly via the host immune response ( c. trachomatis ) . the putative role of nongonococcal nonchlamydial bacteria , especially anaerobes and more recently m. genitalium , in the pathogenesis of acute pid and whether antimicrobial regimens for treatment of pid should provide coverage against these microorganisms is more controversial . some propose that anaerobic coverage is only required in patients with severe pid , especially those with tuboovarian abscesses . others suggest that anaerobic coverage should be provided to all women with acute pid . clearly anaerobic bacteria have been demonstrated in the upper genital tract of women with acute pid with anaerobic bacteria recovered from the upper genital tract in 13% to 78% of women with pid [ 2835 ] . in addition , anaerobes ( e.g. , bacteroides fragilis ) have caused tubal damage in vitro studies . bacterial vaginosis ( bv ) has been noted to be frequently present in women presenting with acute pid [ 1 , 43 , 51 ] . in the peach study , moreover , in the peach study women with acute endometritis on endometrial biopsy were commonly infected with bv - associated microorganisms in their upper genital tract ( g. vaginalis 30.5% , anaerobic gram - negative rods 31.7% , and anaerobic gram - positive cocci 22% ) . multiple previous studies [ 31 , 4349 ] support the findings of the peach study conclusions that bv is associated with acute pid . in addition , the gyn infectious follow - through ( gift ) study , a longitudinal study of women with bv , demonstrated that the presence of bv - related microorganisms significantly increased the risk for acquiring pid . the peach study authors concluded that bv - associated organisms are very commonly present in women with mild - to - moderately severe pid and suggested that treatment regimens for all women with pid include antimicrobial agents effective against anaerobes associated with bv . in a similar vein , the cdc notes that until treatment regimens that do not adequately cover these bv - associated anaerobes have been demonstrated in clinical trial to prevent the long - term sequelae of pid as efficaciously as regimens which provide effective coverage for these microbes , use of regimens with antianaerobic activity should be considered . limited data suggest that failure to cover anaerobes in women with acute pid may predispose them to development of long - term sequelae . in the 1970s when single agent monotherapy was the standard for treatment of pid , chow et al . subsequently , our group reported that anaerobic bacteria persisted in the endometrial cavities of women with pid treated with ciprofloxacin despite apparent clinical cure . this finding is analogous to the finding by our group that failure to include an antimicrobial agent effective against c. trachomatis resulted in persistent chlamydial infection in the endometrial cavity . in a proof of concept study , eckert and coworkers demonstrated that women at high risk for pid but without a clinical diagnosis of pid improved with antimicrobial regimens that provided anaerobic coverage as measured by clinical improvement and resolution of histologic endometritis . neither the 2010 cdc sexually transmitted disease treatment guidelines nor the 2007 european guideline for management of pelvic inflammatory disease strongly advocate for anaerobic coverage in the treatment of acute pid . however , because of the substantial evidence that anaerobes are commonly recovered from women with mild - to - moderate and severe pid , and that failure to eradicate anaerobes from the upper genital tract may lead to tubal damage , it seems prudent to do so . firstly , as noted above , until those regimens that do not provide adequate anaerobic coverage have been shown to prevent adverse sequelae as well as those that do , it seems advisable to provide anaerobic coverage . a second strong reason for providing anaerobic coverage is the frequent ( up to 70% ) occurrence of bv in women with pid . severe pid , as determined by laparoscopy , not clinically , is an important determinant of future infertility [ 10 , 68 ] . thus , unless severe tubal disease has been excluded at laparoscopy , coverage for anaerobes may have important implications for the future reproductive health of these women . on the other hand , reservation regarding the need for anaerobic coverage for acute pid has been raised . the peach trial compared inpatient with outpatient treatment regimens in which patients were randomized to intravenous cefoxitin and doxycycline for a minimum of 48 hours ( followed by oral doxycycline for a total of 14 days ) or to a single dose of cefoxitin plus 2 weeks of oral doxycycline . in the ambulatory arm , the single dose of cefoxitin probably had little impact on anaerobic bacteria , whereas in the hospitalized arm patients received 48 hours of anaerobic therapy . no superiority was noted for either antimicrobial regimen , calling into question the need for anaerobic therapy in women with mild - to - moderate pid . in a recent editorial , eschenbach also questioned a putative role for anaerobes in the pathogenesis of mild - to - moderate acute pid and suggested that although anaerobes may be present in the fallopian tubes , their role in the infectious process is not entirely clear . however , concern remains about the importance of anaerobes in the pathogenesis and treatment of acute pid . failing to provide anaerobic coverage in pid treatment regimen is problematic because there is limited data in support of the efficacy of such an approach . hopefully , additional studies will address this issue and provide further insight into the role of anaerobes in pid . although recent reviews of pid treatment trials have noted that most antibiotic regimens , with the exception of the doxycycline and metronidazole regimen , result in fairly similar excellent clinical and microbiologic ( primarily cervical n. gonorrhoeae and c. trachomatis ) cure rates [ 17 , 18 , 63 , 64 ] , the search continues for treatment regimen(s ) that optimize prevention of infertility , ectopic pregnancy , chronic pelvic pain , and recurrent infection . three major determinants for preservation of post - pid fertility have been identified [ 3 , 69 ] . these are ( 1 ) short duration of symptoms ( < 72 hours ) prior to institution of therapy ; ( 2 ) repetitive episodes of pid ; ( 3 ) nongonococcal pid [ 16 , 70 , 71 ] . early diagnosis and treatment are crucial for preserving fertility and the effectiveness of antibiotic therapy is dependent upon the interval from the onset of symptoms to the initiation of treatment . in an updated analysis of the lund , sweden cohort of women with laparoscopically confirmed pid , hillis and colleagues demonstrated that women treated with 3 days of symptoms had a significantly greater infertility rate compared to those <3 days from symptom onset ( 19.7% versus 8.3% ) . in their cohort of laparoscopically confirmed cases of pid , westrom and colleagues reported that reinfection was an important predictor of subsequent tubal factor infertility . in the most recent update of this cohort with 1,309 pid cases and 451 control patients who attempted to conceive , noted that the rate of infertility is directly proportional to both the number of episodes and severity of tubal inflammation seen at laparoscopy . each episode roughly doubles the rate of infertility ; with one , two , or three or more episodes of pid infertility rates were 8.0% , 19.5% , and 40% , respectively . among women with a single episode of pid , future fertility was associated with the severity of pid ( at laparoscopy ) ranging from 0.6% with mild disease to 6.2% and 21.4% for moderate and severe pid , respectively . studies based on the swedish cohort [ 16 , 70 ] have also demonstrated that women with chlamydial pid and nongonococcal nonchlamydial pid fared more poorly after treatment than those with gonococcal pid . most likely for chlamydial pid , it is the delayed commencement of treatment associated with mild slow onset of symptoms . nongonococcal nonchlamydial pid is more often associated with severe pid which is associated with a worse prognosis for future fertility . despite the controversy regarding the role of anaerobic bacteria and m. genitalium in the pathogenesis of acute pid , the polymicrobic nature of pid is widely acknowledged [ 1 , 2 ] . as a consequence , pid is treated with antibiotics which provide coverage against a broad spectrum of potential pathogens . in 2010 the center for disease control and prevention updated their guidelines for treatment of acute pid ( tables 2 and 3 ) . according to the cdc 2010 guidelines these guidelines recommend that all treatment regimens should be effective against n. gonorrhoeae and c. trachomatis even in the presence of negative endocervical screening for these organisms . although the cdc notes that the need to eradicate anaerobes from women with pid has not been definitively determined , as reviewed above , they suggest that until regimens without adequate coverage for anaerobes have been shown to prevent long - term sequelae as successfully as those that include anaerobic coverage , coverage of anaerobes should be considered in the treatment of acute pid . as noted by the cdc multiple randomized clinical treatment trials have demonstrated efficacy of both parenteral and oral regimens . in table 4 , the short - term clinical and microbiologic efficacy of oral and parenteral treatments regimens for pid are summarized . after excluding the metronidazole - doxycycline regimen ( clinical and microbiologic cure rates 75% and 71% , resp . ) , the pooled clinical cure rates ranged from 88% to 99% , and the pooled microbiologic cure rates ranged from 89% to 100% . it is important that empiric treatment be initiated as soon as a presumptive diagnosis of acute pid is made because prevention of long - term sequelae is determined to a large extent by early administration ( < 72 hours ) of appropriate antimicrobial therapy . in addition , selection of a treatment regimen should consider availability , cost , patient acceptance , and antimicrobial acceptability [ 1 , 72 ] . because parenteral antibiotics do not necessarily require hospitalization , antibiotic regimens for the treatment of acute pid are categorized as follows : regimens requiring more than a single parenteral dose as initial therapy are parenteral and regimens that are primarily oral with or without an initial single parenteral dose are considered oral . as noted in table 4 , several parenteral antimicrobial regimens have excellent short - term clinical and microbiological efficacy . most of the literature supports the combination of ( 1 ) cefoxitin or cefotetan plus doxycycline and ( 2 ) clindamycin plus gentamicin . these two regimens remain the parenteral regimens recommended by the cdc for the treatment of pid . however , cefotetan is not currently marketed in the united states . according to the cdc , there is limited data available supporting a role of other second or third generation parenteral cephalosporins ( e.g. , ceftizoxime , cefotaxime , or ceftriaxone ) as effective therapy for acute pid and/or replacements for cefotetan or cefoxitin . moreover , these antimicrobial agents are less active against anaerobic bacteria than cefoxitin or cefotetan . intravenous infusion of doxycycline frequently causes pain and , thus , doxycycline should be administered orally whenever possible . however , oral doxycycline ( 100 mg twice a day ) should be continued to complete a 14-day course of therapy . in cases involving tuboovarian abscess , either clindamycin ( 450 mg orally four times a day ) or metronidazole ( 500 mg orally every 6 hours ) should be used for continued therapy in order to provide more effective coverage against anaerobic bacteria . there is concern over the increasing resistance of anaerobes , especially the bacteroides fragilis group , to clindamycin [ 73 , 74 ] . however , based on multiple clinical studies and extensive successful results with clindamycin containing regimens , clindamycin remains as a component in one of the recommended parenteral treatment regimens in both the cdc and european guidelines for treatment of pid . however , it is efficacious in the treatment of other pelvic and abdominal infections and is an option in parenteral regimen b. with this regimen , parenteral therapy may be discontinued 24 hours after clinical therapy . while the cdc suggests that either doxycycline 100 mg orally twice a day or clindamycin 450 mg orally four times a day to complete a total of 14 days of therapy may be used , in the author 's opinion clindamycin oral therapy is preferred because of its better anaerobic coverage . in the presence of severe pid , especially tuboovarian abscess , clindamycin continued therapy is recommended by the cdc . as noted by walker and wiesenfeld , there has been renewed interest in alternative agents , particularly ampicillin - sulbactam for anaerobic coverage . unlike clindamycin in addition , ampicillin - sulbactam is effective for n. gonorrhoeae . to provide adequate coverage for c. trachomatis , concomitant administration of doxycycline is recommended . following clinical improvement , oral therapy with doxycycline 100 mg twice a day to complete 14 days of therapy should be continued . with severe disease , especially toa , metronidazole 500 mg orally four times daily should be commenced as well . while not included in the cdc 2010 recommended or alternative regimens for the treatment of pid , several factors have led clinicians to use azithromycin for the treatment of acute pid . these include widespread use in treating chlamydial infection , enhanced compliance due to its long half - life , and studies demonstrating the anti - inflammatory effects of macrolide antibiotics including azithromycin which appear to enhance host defense mechanisms and restrict local inflammation [ 17 , 18 , 75 , 76 ] . a randomized clinical trial in the united kingdom among 300 women with laparoscopically confirmed pid demonstrated excellent short - term clinical care rates with azithromycin monotherapy for one week ( 500 mg iv daily for one or two days followed by 250 mg for 5 - 6 days ) or in combination with a 12 day course of metronidazole . the microbiologic cure rates were also excellent ( > 95% ) for n. gonorrhoeae , c. trachomatis , m. hominis , and anaerobes with these regimens . however , there was a large dropout rate with only one - third of the patients completing the study per protocol which , as noted by haggerty and ness reduced the validity and generalizability of the microbiological cure evaluation . in addition , the anaerobic bacteria were only recovered from 27 ( 9% ) of the patients , a rate substantially lower than noted in other studies . the 2007 european guideline for the management of pelvic inflammatory disease contains similar recommendations . as alternative regimens ofloxacin 400 mg twice daily plus i.v . metronidazole 500 mg three times daily for 14 days or i.v . because anaerobes are probably of relatively greater importance in patients with severe pid and some studies have demonstrated good clinical response without the use of metronidazole , the european guideline suggests that metronidazole may be discontinued in those patients with mild or moderate pid who are unable to tolerate it . they further note that ofloxacin and moxifloxacin should be avoided in patients who are at high risk of gonococcal pid due to increasing quinolone resistance by n. gonorrhoeae . over the past 20 years , a new paradigm has emerged with a dramatic shift from hospital - based parenteral antibiotic regimens to oral ambulatory - based regimens [ 6 , 7 ] . initially , this shift was largely driven by the emergence of managed care and other economic factors without the benefit of clinical studies demonstrating that oral therapy was as effective as parenteral regimens , especially for prevention of long - term sequelae . the peach study has provided evidence supporting the use of oral regimens on an ambulatory basis for the treatment of mild and moderately severe acute pid [ 42 , 78 ] . peach , the largest randomized clinical treatment trial of acute pid in the united states , compared inpatient parenteral therapy ( intravenous cefoxitin and oral or intravenous doxycycline during 48-hour hospitalization followed by oral doxycycline to complete a 14 day course ) with outpatient oral therapy ( a single intramuscular dose of cefoxitin with doxycycline administration orally for 14-days ) . of most significance , peach not only assessed short - term but also long - term outcomes for over 800 patients ( 398 inpatient and 410 outpatient ) with mild - to - moderately severe pid . the short - term clinical cure rates at 30 days were excellent in both groups , with roughly 3% of women in each group requiring additional treatment . at a mean followup of 35 months , the pregnancy rates were 42.0% and 41.7% with the outpatient and inpatient regimens , respectively . long - term outcomes including infertility , ectopic pregnancy , recurrent pid , and chronic pelvic pain were also similar in both groups . however , as emphasized by haggerty and ness , despite high rates of clinical cure and eradication of n. gonorrhoeae and c. trachomatis , the rates of infertility ( 17% ) , recurrent pid ( 14% ) , and chronic pelvic pain ( 37% ) were disappointingly high . while data from the peach study suggests that neither the route nor site of treatment administration affects short - term or long - term outcomes among women with mild - to - moderately severe acute pid [ 42 , 78 ] , higher rates of post treatment histologic endometritis were present among the women in the outpatient oral group . we have shown that ongoing subclinical pid ( as defined by histologic acute endometritis ) is frequently present in women with untreated lower genital tract infections , and that persistent endometrial infection with c. trachomatis and anaerobes may lead to subsequent tubal damage and increased infertility among women with inadequately treated acute pid . similarly , among women enrolled in the peach study , 23 out of 56 ( 41% ) with m. genitalium identified in either the cervix and/or endometrium at baseline had m. genitalium persistently identified 30 days following treatment ( inadequate to treat this organism ) . moreover , women with persistent endometrial m. genitalium were 4.5 times more likely to experience short - term treatment failure ( i.e. , histologic endometritis and persistent pelvic pain at the 30-day follow - up visit ) . as noted by the cdc , outpatient oral therapy can be considered for treatment of women with mild - to - moderately severe acute pid . the oral regimens listed in table 3 provide coverage against the major etiologic agents of acute pid . which of the cephalosporins is the optimum selection is unclear . on the one hand cefoxitin has better anaerobic coverage , while ceftriaxone has better coverage against n. gonorrhoeae . the dose of ceftriaxone was increased to 250 mg i m in the 2010 cdc guidelines . the extent of efficacy against anaerobic bacteria with a single dose of cefoxitin is questionable . however , in the peach study single dose cefoxitin was effective in obtaining clinical response [ 42 , 78 ] . the cdc and walker and wiesenfeld have noted that theoretical limitations in coverage of anaerobes by recommended cephalosporins may require addition of metronidazole to the oral treatment recommendations . in addition , metronidazole will effectively treat bacterial vaginosis , which as noted above is frequently associated with pid . there is no published data on the use of oral cephalosporins for treatment of acute pid . several alternative regimens have been the subject of at least one clinical trial and contain broad spectrum coverage . these include ( 1 ) amoxicillin / clavulanic acid and doxycycline and ( 2 ) azithromycin monotherapy or a combination of ceftriaxone 250 mg i m single dose with azithromycin 1 gram orally once a week for two weeks . if one of these alternative regimens is selected , the cdc suggests the addition of metronidazole should be considered to cover anaerobic bacteria which are suspected as etiologic agents in pid and to effectively treat concomitant bv . with the emergence of quinolone - resistant n. gonorrhoeae , regimens that include a quinolone agent are no longer recommended by the cdc for treatment of acute pid . they note that in situations where single dose parenteral cephalosporin is not feasible , use of fluoroquinolones ( levofloxacin 500 mg orally once a day or ofloxacin 400 mg orally twice a day for 14 days ) with or without metronidazole ( 500 mg twice daily for 14 days ) can be considered if community prevalence and individual risk for gonorrhea are low . if this approach is selected , the cdc stresses that diagnostic tests for n. gonorrhoeae must be performed prior to initiating treatment . if n. gonorrhoeae is detected , treatment should be based on the results of antimicrobial susceptibility . with quinolone - resistant n. gonorrhoeae or if susceptibility can not be assessed ( e.g. , nucleic acid amplification test ) use of a parenteral cephalosporin is recommended . if use of a cephalosporin is not feasible , azithromycin 2 grams as a single dose can be added to a quinolone - based pid treatment regimen . patients treated with an oral regimen should demonstrate substantial clinical improvement within three days following initiation of treatment . clinical improvement is determined by defervescence , reduction in direct or rebound abdominal tenderness , and/or reduction in uterine , adnexal , and cervical motion tenderness . when patients fail to improve within this window , hospitalization is usually required for additional diagnostic tests ( e.g. , rule out toa ) , parenteral antibiotic therapy , and/or surgical intervention . while in the past , and to a lesser extent today , some clinicians have recommended that all patients with pid be hospitalized for parenteral antibiotics and bed rest , the peach study clearly demonstrated that in women with mild - to - moderately severe pid , outpatient oral therapy results in similar short- and long - term clinical outcomes as inpatient therapy . as a result , the cdc notes that a decision regarding the need for hospitalization should be based on the judgment of the health - care provider and whether the patient meets any of the cdc suggested criteria for hospitalizations ( table 5 ) . limited studies have demonstrated that pregnant women with pid have high rates of fetal wastage and preterm delivery , supporting the appropriateness of hospitalization [ 81 , 82 ] . similarly , ample data supports hospitalization of women with toas in order to maximize antimicrobial dosing and close monitoring for early recognition of severe sepsis or of leaking / rupture of the abscess . the absence of data to support benefit from hospitalization for adolescent girls with pid led the cdc to not list adolescence among the criteria for hospitalization and to suggest that a decision to hospitalize adolescents with pid should be based on the same criteria used for older women . in fact , subanalysis of the outcome data of the peach study stratified by age demonstrated that fertility outcomes of the adolescents were similar in the inpatient and outpatient treatment arms . however , some clinicians continue to advocate that all adolescents and never pregnant young women should be hospitalized for treatment . they argue that adolescence is a proxy for poor compliance , high - risk sexual activity , delayed care , and high antimicrobial failure rates . whereas the presence of hiv infection or immunosuppression has previously been an indicator for hospitalization and parenteral therapy , currently it is recommended that hiv - positive women with acute pid can be treated similarly to hiv - negative women . although hiv - infected women who develop pid may have more severe clinical presentations and are more likely to have toas [ 8486 ] , there is no evidence to suggest that immunocompromised women benefit from hospitalization or parenteral therapy for uncomplicated pid [ 17 , 87 , 88 ] . with the renewed interest in the iud as a contraceptive choice for young women , pid will be seen in women using iuds . as noted by walker and wiesenfeld , there does not exist any data to indicate that selection of treatment regimens should be influenced by the presence of an iud . in the past , clinicians generally removed iuds to optimize the treatment of pid . this was primarily based on concerns that as a foreign body , removal of the iud enhanced clinical response . only a few studies have addressed this issue and the results are conflicting . in a small randomized study of 46 women in sweden , soderberg , and lindgren reported no differences in response to treatment whether the iud was removed or left in place . on the other hand , altunyurt and colleagues , in a randomized trial from turkey , noted that clinical improvement ( e.g. , absence of pelvic pain , vaginal discharge , and pelvic tenderness ) was more common in the group whose iuds were removed . if the provider elects to leave the iud in place while pid is being treated , close clinical followup is important . according to the cdc , male sex partners of women diagnosed with acute pid should be examined and treated if they had sexual contact with the patient during the preceding 60 days . if the last episode of sexual intercourse was > 60 days prior to onset of symptoms , the last sexual partner should be treated . women diagnosed with acute pid should refrain from sexual intercourse until treatment is completed and they and their partner(s ) are asymptomatic . sex partners of women with pid should be treated empirically with regimens effective against n. gonorrhoeae and c. trachomatis . in those settings where only women are treated , arrangements should be undertaken to either provide care or appropriate referral for male sex partners . expedited partner treatment or enhanced patient referral are acceptable alternative approaches for the treatment of male partners of women who have pid with chlamydial or gonococcal infection . treatment strategies for women with acute pid should be based on the polymicrobial nature of this infection . the microorganisms recovered from the upper genital tract of women with acute pid include n. gonorrhoeae , c. trachomatis , and anaerobic and aerobic bacteria common to the endogenous vaginal flora and genital mycoplasmas , especially m. genitalium . several parenteral antimicrobial regimens have been shown to provide very good short - term clinical and microbiological efficacy ; these include clindamycin plus gentamicin , cefoxitin plus doxycycline , and cefotetan plus doxycycline . oral therapy for acute pid is currently the most commonly used approach , in response to both economic issues and the evidence from the peach study demonstrating that both short - term and long - term outcomes were similar for the oral and parenteral regimens . due to the increased quinolone resistance of n. gonorrhoeae , choices of oral regimens are more limited . the addition of oral metronidazole to this regimen is suggested by some experts including this author to provide improved anaerobic coverage and at least to treat bv which is present in up to 70% of women with acute pid . currently regimens recommended by the cdc for the treatment of acute pid provide suboptimal antimicrobial activity against m. genitalium . mycoplasma lack a cell wall and thus are resistant to beta - lactam antibiotics ( e.g. , cefoxitin , cefotetan , ceftriaxone ) . in addition , m. genitalium is associated with persistent nongonococcal urethritis treated with tetracyclines . variable resistance to fluoroquinolones recently , a newer fluoroquinolone , moxifloxacin , has demonstrated excellent activity against m. genitalium [ 91 , 93 ] . while m. genitalium has demonstrated susceptibility to macrolides , azithromycin resistance has recently been reported .

pelvic inflammatory disease ( pid ) , one of the most common infections in nonpregnant women of reproductive age , remains an important public health problem . it is associated with major long - term sequelae , including tubal factor infertility , ectopic pregnancy , and chronic pelvic pain . in addition , treatment of acute pid and its complications incurs substantial health care costs . prevention of these long - term sequelae is dependent upon development of treatment strategies based on knowledge of the microbiologic etiology of acute pid . it is well accepted that acute pid is a polymicrobic infection . the sexually transmitted organisms , neisseria gonorrhoeae and chlamydia trachomatis , are present in many cases , and microorganisms comprising the endogenous vaginal and cervical flora are frequently associated with pid . this includes anaerobic and facultative bacteria , similar to those associated with bacterial vaginosis . genital tract mycoplasmas , most importantly mycoplasma genitalium , have recently also been implicated as a cause of acute pid . as a consequence , treatment regimens for acute pid should provide broad spectrum coverage that is effective against these microorganisms .

1. Introduction 2. Etiology of PID 3. Treatment Concepts 4. Antimicrobial Treatment Regimens 5. Conclusion

pelvic inflammatory disease ( pid ) is a spectrum of upper genital tract infections that includes endometritis , salpingitis , tuboovarian abscess , and/or pelvic peritonitis . pid is one of the most frequent and important infections that occur among nonpregnant women of reproductive age and remains a major public health problem [ 48 ] . among women , unfortunately , women who acquire acute pid are at risk for long - term sequelae including tubal factor infertility , ectopic pregnancy , chronic pelvic pain , and recurrent pid [ 913 ] . in addition , the estimated annual health care cost for pid and its complications in the united states is over $ 2 billion . a recent analysis by the centers for disease control and prevention ( cdc ) of trends in the incidence of pid demonstrated that from 1985 to 2001 rates of both hospitalized and ambulatory cases of acute pid declined ( 68% and 47% , resp . ) recently , subclinical pid has been recognized as an important entity which is common among women with lower genital tract infections , especially chlamydia trachomatis , neisseria gonorrhoeae , and bacterial vaginosis ( bv ) [ 14 , 15 ] . subclinical pid is as likely as clinically recognized acute pid and is responsible for a greater proportion of pid - related sequelae than clinically recognized disease . over the past 25 years , important advances have occurred in understanding the etiology , pathogenesis , and treatment of acute pid . as a result , major paradigm shifts have occurred in our approach to the treatment of acute pid . in the past pid today , the polymicrobic etiology of pid is well established and has led to utilization of broad spectrum antimicrobial regimens for treatment of acute pid [ 1 , 2 , 17 , 18 ] . prevention of the significant long - term complications associated with pid requires development of effective treatment strategies . such treatment regimens are dependent upon an understanding of the microbiologic etiology of acute pid . secondly , most investigations primarily focused on the sexually transmitted pathogens n. gonorrhoeae and/or c. trachomati , s and few studies have assessed the role of non - std pathogens , especially anaerobic bacteria . based initially upon culdocentesis studies of peritoneal fluid ( figure 1 ) and subsequently studies utilizing laparoscopy and/or endometrial aspirations to obtain specimens from the upper genital tract ( table 1 ) came the recognition that the etiology of acute pid is polymicrobic with a wide variety of microorganisms involved [ 1 , 2 , 1941 ] . included among these are n. gonorrhoeae , c. trachomatis , genital tract mycoplasmas ( particularly m. genitalium ) , anaerobic and aerobic bacteria which comprise the endogenous vaginal flora ( e.g. investigations by our group conducted in the 1980s that utilized laparoscopy and/or endometrial aspirations to obtain upper genital tract specimens demonstrated that approximately two - thirds of acute pid cases were associated with n. gonorrhoeae and/or c. trachomatis ( figure 2 ) . in addition , half of the women with n. gonorrhoeae and/or c. trachomatis had concomitant anaerobic and/or aerobic bacteria recovered . more recently , in the pelvic inflammatory disease evaluation and clinical health ( peach ) study , the largest treatment trial of mild to moderate acute pid in the us , n. gonorrhoeae and c. trachomatis were recovered in less than one - third of patients . many of the nongonococcal , nonchlamydial microorganisms recovered from the upper genital tract in acute pid are similar to those associated with bacterial vaginosis ( bv ) , a complex perturbation of the vaginal flora leading to loss of hydrogen peroxide producing lactobacillus and overgrowth of g. vaginalis , prevotella sp . in addition , use of a broad - range 16srdna polymerase chain reaction to identify uncultivable bacteria has identified bacterial 16s sequences of anaerobic bacteria associated with bv in the fallopian tube of women with laparoscopically confirmed acute pid . although m. genitalium was identified in the early 1980s as a cause of nongonococcal urethritis in men , its role in genital tract infections in women remained unclear , due in large part to difficulty in culturing this organism . a recent analysis from the peach study noted that rates of short - term failure ( persistent endometritis and pelvic pain ) , infertility , recurrent pid , and chronic pelvic pain were high among women with endometrial m. genitalium at baseline . subsequently , it has been demonstrated that women with m. genitalium infection ( similar to those with chlamydial infection ) present with fewer clinical signs and symptoms of acute pid than those with gonococcal infection . this term was initially applied to women with documented tubal factor infertility associated with evidence of chronic inflammatory residua characteristic of pid who denied a history of being diagnosed or treated for acute pid . , n. gonorrhoeae , c. trachomatis , and bacterial vaginosis ) associated with subclinical pid are the same putative agents recovered from women with clinically apparent acute pid . the therapeutic goals for treatment of acute pid include both short - term outcomes such as clinical cure and microbiologic cure and preventions of long - term sequelae such as infertility , ectopic pregnancy , recurrent infection , and chronic pelvic pain . although the incidence rates of pid have declined , no reduction in the adverse reproductive outcomes associated with pid ( infertility , ectopic pregnancy , and chronic pelvic pain ) has been demonstrated . while some antibiotic regimens have been successful in producing initial clinical and microbiologic cure with short - term followup , only a few studies have determined the efficacy of these treatment regimens for eliminating endometrial or fallopian tube infection . in addition , few studies have attempted to assess the incidence of long - term sequelae ( e.g. , tubal factor infertility , ectopic pregnancy and chronic pelvic pain ) following treatment with these antibiotic regimens [ 1 , 10 , 11 , 42 ] . the introduction of antibiotics into clinical practice led to improvement in the prognosis for acute pid , and mortality was nearly eliminated . substantial evidence supports the role of n. gonorrhoeae , c. trachomatis , anaerobic bacteria , and facultative bacteria in the pathogenesis of acute pid [ 15 , 9 ] . not only are n. gonorrhoeae and c. trachomatis frequently recovered from the upper genital tract in women with pid , excellent data demonstrates the role these pathogens play in producing tubal damage and in the development of the adverse sequelae of pid ( e.g. , infertility , ectopic pregnancy ) [ 5760 ] . thus , antimicrobial regimens for the treatment of acute pid must be effective against these std organisms . while some antimicrobial regimens that do not provide adequate coverage against n. gonorrhoeae and/or c. trachomatis have been shown to have excellent clinical cure rates , microbiologic cure rates are less impressive ( or lacking ) , and long - term outcome data are not available [ 17 , 18 , 6164 ] . the cdc in its 2010 treatment recommendations notes that all regimens used to treat acute pid should provide adequate coverage against n. gonorrhoeae and c. trachomatis , as they are both commonly present and have the propensity to produce tubal damage directly ( n. gonorrhoeae ) or indirectly via the host immune response ( c. trachomatis ) . the putative role of nongonococcal nonchlamydial bacteria , especially anaerobes and more recently m. genitalium , in the pathogenesis of acute pid and whether antimicrobial regimens for treatment of pid should provide coverage against these microorganisms is more controversial . clearly anaerobic bacteria have been demonstrated in the upper genital tract of women with acute pid with anaerobic bacteria recovered from the upper genital tract in 13% to 78% of women with pid [ 2835 ] . in addition , anaerobes ( e.g. bacterial vaginosis ( bv ) has been noted to be frequently present in women presenting with acute pid [ 1 , 43 , 51 ] . multiple previous studies [ 31 , 4349 ] support the findings of the peach study conclusions that bv is associated with acute pid . the peach study authors concluded that bv - associated organisms are very commonly present in women with mild - to - moderately severe pid and suggested that treatment regimens for all women with pid include antimicrobial agents effective against anaerobes associated with bv . in a similar vein , the cdc notes that until treatment regimens that do not adequately cover these bv - associated anaerobes have been demonstrated in clinical trial to prevent the long - term sequelae of pid as efficaciously as regimens which provide effective coverage for these microbes , use of regimens with antianaerobic activity should be considered . limited data suggest that failure to cover anaerobes in women with acute pid may predispose them to development of long - term sequelae . neither the 2010 cdc sexually transmitted disease treatment guidelines nor the 2007 european guideline for management of pelvic inflammatory disease strongly advocate for anaerobic coverage in the treatment of acute pid . however , because of the substantial evidence that anaerobes are commonly recovered from women with mild - to - moderate and severe pid , and that failure to eradicate anaerobes from the upper genital tract may lead to tubal damage , it seems prudent to do so . on the other hand , reservation regarding the need for anaerobic coverage for acute pid has been raised . in a recent editorial , eschenbach also questioned a putative role for anaerobes in the pathogenesis of mild - to - moderate acute pid and suggested that although anaerobes may be present in the fallopian tubes , their role in the infectious process is not entirely clear . however , concern remains about the importance of anaerobes in the pathogenesis and treatment of acute pid . although recent reviews of pid treatment trials have noted that most antibiotic regimens , with the exception of the doxycycline and metronidazole regimen , result in fairly similar excellent clinical and microbiologic ( primarily cervical n. gonorrhoeae and c. trachomatis ) cure rates [ 17 , 18 , 63 , 64 ] , the search continues for treatment regimen(s ) that optimize prevention of infertility , ectopic pregnancy , chronic pelvic pain , and recurrent infection . early diagnosis and treatment are crucial for preserving fertility and the effectiveness of antibiotic therapy is dependent upon the interval from the onset of symptoms to the initiation of treatment . in their cohort of laparoscopically confirmed cases of pid , westrom and colleagues reported that reinfection was an important predictor of subsequent tubal factor infertility . studies based on the swedish cohort [ 16 , 70 ] have also demonstrated that women with chlamydial pid and nongonococcal nonchlamydial pid fared more poorly after treatment than those with gonococcal pid . most likely for chlamydial pid , it is the delayed commencement of treatment associated with mild slow onset of symptoms . nongonococcal nonchlamydial pid is more often associated with severe pid which is associated with a worse prognosis for future fertility . despite the controversy regarding the role of anaerobic bacteria and m. genitalium in the pathogenesis of acute pid , the polymicrobic nature of pid is widely acknowledged [ 1 , 2 ] . as a consequence , pid is treated with antibiotics which provide coverage against a broad spectrum of potential pathogens . in 2010 the center for disease control and prevention updated their guidelines for treatment of acute pid ( tables 2 and 3 ) . according to the cdc 2010 guidelines these guidelines recommend that all treatment regimens should be effective against n. gonorrhoeae and c. trachomatis even in the presence of negative endocervical screening for these organisms . although the cdc notes that the need to eradicate anaerobes from women with pid has not been definitively determined , as reviewed above , they suggest that until regimens without adequate coverage for anaerobes have been shown to prevent long - term sequelae as successfully as those that include anaerobic coverage , coverage of anaerobes should be considered in the treatment of acute pid . in table 4 , the short - term clinical and microbiologic efficacy of oral and parenteral treatments regimens for pid are summarized . it is important that empiric treatment be initiated as soon as a presumptive diagnosis of acute pid is made because prevention of long - term sequelae is determined to a large extent by early administration ( < 72 hours ) of appropriate antimicrobial therapy . in addition , selection of a treatment regimen should consider availability , cost , patient acceptance , and antimicrobial acceptability [ 1 , 72 ] . because parenteral antibiotics do not necessarily require hospitalization , antibiotic regimens for the treatment of acute pid are categorized as follows : regimens requiring more than a single parenteral dose as initial therapy are parenteral and regimens that are primarily oral with or without an initial single parenteral dose are considered oral . , ceftizoxime , cefotaxime , or ceftriaxone ) as effective therapy for acute pid and/or replacements for cefotetan or cefoxitin . however , based on multiple clinical studies and extensive successful results with clindamycin containing regimens , clindamycin remains as a component in one of the recommended parenteral treatment regimens in both the cdc and european guidelines for treatment of pid . however , it is efficacious in the treatment of other pelvic and abdominal infections and is an option in parenteral regimen b. with this regimen , parenteral therapy may be discontinued 24 hours after clinical therapy . unlike clindamycin in addition , ampicillin - sulbactam is effective for n. gonorrhoeae . while not included in the cdc 2010 recommended or alternative regimens for the treatment of pid , several factors have led clinicians to use azithromycin for the treatment of acute pid . the microbiologic cure rates were also excellent ( > 95% ) for n. gonorrhoeae , c. trachomatis , m. hominis , and anaerobes with these regimens . in addition , the anaerobic bacteria were only recovered from 27 ( 9% ) of the patients , a rate substantially lower than noted in other studies . initially , this shift was largely driven by the emergence of managed care and other economic factors without the benefit of clinical studies demonstrating that oral therapy was as effective as parenteral regimens , especially for prevention of long - term sequelae . long - term outcomes including infertility , ectopic pregnancy , recurrent pid , and chronic pelvic pain were also similar in both groups . however , as emphasized by haggerty and ness , despite high rates of clinical cure and eradication of n. gonorrhoeae and c. trachomatis , the rates of infertility ( 17% ) , recurrent pid ( 14% ) , and chronic pelvic pain ( 37% ) were disappointingly high . while data from the peach study suggests that neither the route nor site of treatment administration affects short - term or long - term outcomes among women with mild - to - moderately severe acute pid [ 42 , 78 ] , higher rates of post treatment histologic endometritis were present among the women in the outpatient oral group . we have shown that ongoing subclinical pid ( as defined by histologic acute endometritis ) is frequently present in women with untreated lower genital tract infections , and that persistent endometrial infection with c. trachomatis and anaerobes may lead to subsequent tubal damage and increased infertility among women with inadequately treated acute pid . in addition , metronidazole will effectively treat bacterial vaginosis , which as noted above is frequently associated with pid . there is no published data on the use of oral cephalosporins for treatment of acute pid . if one of these alternative regimens is selected , the cdc suggests the addition of metronidazole should be considered to cover anaerobic bacteria which are suspected as etiologic agents in pid and to effectively treat concomitant bv . with the emergence of quinolone - resistant n. gonorrhoeae , regimens that include a quinolone agent are no longer recommended by the cdc for treatment of acute pid . while in the past , and to a lesser extent today , some clinicians have recommended that all patients with pid be hospitalized for parenteral antibiotics and bed rest , the peach study clearly demonstrated that in women with mild - to - moderately severe pid , outpatient oral therapy results in similar short- and long - term clinical outcomes as inpatient therapy . as a result , the cdc notes that a decision regarding the need for hospitalization should be based on the judgment of the health - care provider and whether the patient meets any of the cdc suggested criteria for hospitalizations ( table 5 ) . the absence of data to support benefit from hospitalization for adolescent girls with pid led the cdc to not list adolescence among the criteria for hospitalization and to suggest that a decision to hospitalize adolescents with pid should be based on the same criteria used for older women . they argue that adolescence is a proxy for poor compliance , high - risk sexual activity , delayed care , and high antimicrobial failure rates . whereas the presence of hiv infection or immunosuppression has previously been an indicator for hospitalization and parenteral therapy , currently it is recommended that hiv - positive women with acute pid can be treated similarly to hiv - negative women . this was primarily based on concerns that as a foreign body , removal of the iud enhanced clinical response . according to the cdc , male sex partners of women diagnosed with acute pid should be examined and treated if they had sexual contact with the patient during the preceding 60 days . women diagnosed with acute pid should refrain from sexual intercourse until treatment is completed and they and their partner(s ) are asymptomatic . sex partners of women with pid should be treated empirically with regimens effective against n. gonorrhoeae and c. trachomatis . treatment strategies for women with acute pid should be based on the polymicrobial nature of this infection . the microorganisms recovered from the upper genital tract of women with acute pid include n. gonorrhoeae , c. trachomatis , and anaerobic and aerobic bacteria common to the endogenous vaginal flora and genital mycoplasmas , especially m. genitalium . several parenteral antimicrobial regimens have been shown to provide very good short - term clinical and microbiological efficacy ; these include clindamycin plus gentamicin , cefoxitin plus doxycycline , and cefotetan plus doxycycline . oral therapy for acute pid is currently the most commonly used approach , in response to both economic issues and the evidence from the peach study demonstrating that both short - term and long - term outcomes were similar for the oral and parenteral regimens . currently regimens recommended by the cdc for the treatment of acute pid provide suboptimal antimicrobial activity against m. genitalium . in addition , m. genitalium is associated with persistent nongonococcal urethritis treated with tetracyclines .

legal scholars , scientists , and commentators lament the onslaught of behavioral genetics and neuroscience in the criminal courtroom . fueled largely by anecdotal evidence about the use of bioscience in criminal cases , or media reports of high - profile cases , there is a growing belief that neuroscience has become a mainstay of the us criminal justice system . and while scholars increasingly self - identify as part of the growing fields of law and neuroscience or law and the biosciences , to date only small - scale studies have been conducted on the use of neuroscience and behavioral genetics in the us criminal justice system . one recent study involved an empirical analysis of just those cases in which neuroimaging had been reported in a judicial opinion , with 23 analysed cases . other studies have qualitatively , but not quantitatively , assessed the use and impact of neurobiological evidence in criminal law , again relying almost exclusively on discussion of that evidence in published judicial opinions . any examination of the impact of behavioral genetics and neuroscience on the us criminal justice system must begin with a more accurate understanding of how that evidence is currently being used . to better ground the interest and commentary on the use of neuroscience and behavioral genetics ( hereinafter neurobiological evidence ) in criminal law , this article summarizes some findings from the widest scale empirical study on the use of neurobiological evidence in us criminal law to date . over the past decade , over 1585 judicial opinions issued between 2005 and 2012 discuss the use of neurobiological evidence by criminal defendants to bolster their criminal defense . in 2012 alone , over 250 judicial opinions more than double the number in 2007cited defendants arguing in some form or another that their brains made them do it. approximately 5 per cent of all murder trials and 25 per cent of death penalty trials feature criminal defendants making a bid for lower responsibility or lighter punishment using neurobiological data . while these claims often overstate the science , used responsibly neurobiological evidence has the potential to improve the accuracy and decrease errors in the criminal justice system . much of the scholarship on neurobiological evidence in criminal law has focused exclusively on either behavioral genetics or neuroscience . first , scientific developments increasingly link findings from behavioral genetics to neural correlates . as a result , the emerging scientific inquiry into human behavior is trending toward a neurobiological approach over a purely genetic or neuroscientific one . moreover , the research in these fields foretells a more integrated future in human behavioral research , whereby genetics and neuroscience are linked rather than compartmentalized . as one telling example , the march 2010 issue of the seminal journal behavior genetics dedicated a special issue to pathways between gene , brain , and behavior . the 15 articles included in the volume represented the diversity of methodologies applied to the complexity of pathways linking genes , brain , and behavior . the introductory chapter concluded that the breadth of studies proves that tracing the pathways between biology and behavior requires expertise in genetics , neuroscience , psychology , and psychiatry . the integration is reflected in the use of behavioral genetics and neuroscience in the criminal justice system . legal practitioners take a multifaceted approach to characterizing defendants behaviors by introducing genetic , neurological , and environmental contributions . monamine oxidase a ( maoa ) , the first gene environment interaction associated with temperament and antisocial behavior , is a well - studied example . although maoa was first characterized as a genetic polymorphism , which together with environmental triggers is associated with behavioral variation in antisocial personality , more recent studies link the genetic and neurological correlates of maoa . joshua buckholtz et al . published a study utilizing a combined genetic and imaging approach to the study of maoa , which implicates a neural circuit for variation in human personality under genetic control . already , a multifaceted criminal defense using maoa genotyping , and neuroimaging has been introduced into criminal cases . even assuming differences in scientific methodology and results between behavioral genetics and neuroscience , the substantive legal claims raised are nearly identical when either science is used in a criminal case . as a result in this study sample , anytime behavioral genetics was raised ; a neuroscientific claim was also advanced . while neuroscience by far dominates the scientific evidence introduced , the results here include cases where both neuroscience and behavioral genetics are sometimes introduced . with a few notable exceptions , scientists are on the sidelines of these developments in criminal law . publicly engaged scientists often decry the use of neurobiological evidence criminal law , and call for an outright ban on its use . this is driven in part by concerns about significant methodological obstacles in the study of human behavior . what does aggression mean ? does criminal behavior include petty crimes as well as violent ones ? even when scientists do establish a robust and consistent definition for a specific trait they face difficulties in finding reliable measures of those traits in a manner that satisfies acceptable standards of scientific validity , including reproducibility . measuring complex behavioral traits has been difficult , and as a result difficult to replicate in other studies as well . research into the biological contributions to human behavior face an additional complication : behaviors , which are complex traits and involve multiple genes , regulated by widely varying cellular and neurological mechanisms , are subject to substantial environmental influence . these scientific concerns underscore the study of complex behavioral traits of interest to the criminal law . criminal. any scientific claim about the biological contributions to criminal conduct begins with a narrower focus on the behaviors often implicated by criminal conduct including violence , aggression , and drug and alcohol abuse . studies of those traits and related traits have likewise yielded only preliminary data that have yet to be well replicated or understood . but decrying the use of neurobiological evidence in criminal law seems both futile and counterproductive ; neuroscience is already entrenched in the us legal system . and used appropriately , it holds promise of improving decision - making in law . but just as neuroscientists go too far in calling for an outright ban , defense attorneys are guilty of overstating the science . criminal defendants regularly use neuroscience at every stage of the criminal process , from pretrial , to trial , and sentencing determinations . prosecutors , too , have seized upon cognitive neuroscience to argue that defendants are incorrigible and should be given longer sentences . rather than standing in the way , neuroscientists should educate the public about the responsible use of neuroscience in the courtroom . to empirically ground the dialog about the use of neuroscience in the us criminal justice system , this article is the first comprehensive empirical study of the use of neurobiological evidence in us criminal justice cases . drawing from data analysed from 1585 coded criminal cases , in which judicial opinions were written and published in westlaw during 200512 , this study presents some surprising results and trends in emerging use of neurobiological evidence in criminal law . the cases collected , coded , and analysed in this study demonstrate a rising use of neurobiological research in criminal law . that use continues to be haphazard , ad hoc , and often ill conceived . while scientists caution that the neurobiological evidence at issue is weak , particularly for making claims about individuals rather than studying between - group differences , their cautionary advice has largely gone unheeded . even the gravest decisions , including assessments bearing on deservingness for capital punishment , are beginning to turn on this research . defense attorneys have introduced behavioral genetics and neuroscience in attempts to exculpate criminal defendants , to bolster preexisting legal defenses , and to mitigate defendants ' culpability and punishment . prosecutors have seized upon the double - edged potential of a claimed neurobiological evidence to denigrate defendants ' characters and to demonstrate defendants ' likely future dangerousness . as the science continues to develop , its potential use in criminal investigations , interrogations , and predictions of dangerousness will undoubtedly rise . the discovery of more specific biological and neurological contributions to violence , aggressiveness , impulsivity , substance abuse , even though highly contestable and indeterminate as a scientific matter , has foreshadowed an inevitable reexamination of the us criminal justice system . indeed , the united states supreme court has already become involved in evaluating the relevance of neurobiological evidence to criminal culpability : in september of 2006 , it granted certiorari to address , in part , whether a defendant 's genetic predisposition to violence should inform whether he should be sentenced to death for first - degree murder . liberty , justice , privacy , and the structure and purpose of the us criminal justice system are all at issue . a careful and systematic study of the use , perception , current and likely impact of neurobiological evidence on criminal law is essential before its application further expands . seventeen law students and three undergraduate students were trained over time on a coding book developed by the author that included 84 variables . the students included second and third - year law students , with varying scientific background . the 84 variables are detailed in the coding book with specificity to ensure uniform coding . these variables included , for example , purely identifying information about the case , the purpose for which the behavioral genetics or neuroscience evidence was introduced , the outcome of the case ( successful or unsuccessful for the criminal defendant ) , the state where the case was heard , and the types of attorneys or representation present . coders were first given the coding book , and asked to code a set of four test cases . after coding the four cases , they were trained in detail on the meaning of each variable through an intensive in - person training session with farahany . they were then given a second set of test cases to code , and discrepancies were reviewed and discussed with farahany . coders were then were given cases in sets of 10 to code , which were spot - checked by farahany . there was less than 5 per cent disagreement between coders , and typically pertaining only to the nature of the claim raised . farahany reviewed all coding with spot - checking , and resolved any conflicts that arose between the coders . cases in the study were selected from searches of the westlaw legal database using the keywords and variations on the words : neuroscience , frontal lobe , hereditary , head injury , pet scan , eeg , fmri , ct scan , brain disorder , cognitive impairment , meg , nirs , brain scan , brain , diffusion tensor , heritable , hereditary , genetic , biological , memory , frontemporal , and qeeg . the westlaw databases used included all us supreme court , all federal court and all state court . the broad search terms used search yielded over 10,000 opinions per year , which were then scanned for relevance . after excluding cases that did not bear on the use of neurobiological evidence by a criminal defendant , 1585 cases were included in the study , and all opinions in those cases majority , plurality , concurring , and dissenting opinions for a total of 1800 judicial opinions , were coded . cases in which the scientific evidence focused on the victim or forensic identification were excluded . westlaw 's inclusion criteria for judicial opinions are proprietary , unpublished , and may have changed during the study time period . these variations may account for some of the year - to - year differences in the number of opinions discovered . moreover , the cases contained therein are primarily appellate opinions , since trial opinions at the state level are often jury verdicts without written judicial opinions . consequently , the opinions coded may reflect defendants failed attempts at using neuroscientific evidence at trial , failure to by defense counsel to investigate or introduce neurobiological evidence at trial , or newly discovered evidence on appeal . the sample may be skewed toward defendants who have already fared poorly in the criminal justice system with their claims . moreover , more than 90 per cent of criminal cases in the united states never go to trial . most individuals who are charged with a crime forego their constitutional right to a trial and plead guilty in exchange for a plea agreement . of those cases that do go to trial , while many are appealed , many more are not . of cases that are appealed , there are narrow legal grounds available for overturning a conviction or setting aside a sentence and procedurally the cases must be raised in that manner . moreover , investigation into neurobiological contributions to criminal behavior can be costly . in cases where the defendant has adequate resources , or able to secure resources from the state , or as pro bono services these skews may mean the sample of cases here underreports the actual use of neuroscience in the criminal courtroom . and that the cases are skewed toward unsuccessful use of neurobiological evidence . hence , while the present empirical study significantly advances the ethical , legal and social discussions with respect to the use of neurobiological evidence in us criminal law , it is still a narrow view of the overall issue most studies on the use of neurobiological evidence in criminal cases claim it 's used almost exclusively to mitigate capital punishment and with limited success . the findings discussed herein suggest both a broader and potentially more successful use of neurobiological evidence in us criminal law . significant differences by state also appear , including the extent to which the evidence has been introduced , and the ultimate success of that evidence . these differences can be explained in part by population differences , and varying legal regimes across states ( eg the availability of capital punishment , the type of insanity defense available in the state , or the relevance of a diminished capacity defense ) . importantly , neurobiological evidence is being used for purposes not yet discussed or analysed by scholars . for example , while many scholars have discussed the implications of using neurobiological evidence for mitigation of criminal punishment , virtually no author has discussed the implications of using it to assess the competency of a criminal defendant . and yet the empirical analysis herein illustrates that the second most common use of biological neurobiological evidence in criminal cases is to challenge competency . the implications of this use , and the relevance of behavioral genetics and neuroscience to competency determinations , are critical areas for further exploration . finally , while attempts to introduce neurobiological testimony have been relatively unsuccessful to date , the attempts may have been more successful than most scholars believe . depending on the type of claim that a criminal defendant raises , testimony by an expert on the matter may serve as powerful evidence that impacts the outcome of the case for the defendant . in short , the fundamental assumptions guiding the current ethical , legal and social inquiry into the use of neurobiological evidence in criminal law are limited and potentially flawed . the goal of this study is to broaden the dialog and bring empirical evidence to bear on the discussion . the number of judicial opinions discussing the use of neurobiological evidence by criminal defendants is increasing year over year ( see graph 1 ) . contrary to popular belief , many of these cases are not capital homicide cases ( the prosecutor did not seek the death penalty , or it was unavailable as a sentence in that jurisdiction ) . defense attorneys are introducing neurobiological evidence across the board in serious felony cases , and not just in bifurcated capital sentencing hearings following a conviction of first - degree murder . ( homicide ( capital ) are murder cases in which the prosecutor sought the death penalty . homicide ( not capital ) are some degree of homicide ( murder , manslaughter ) cases in which the death penalty was not at issue . other felony cases are those in which the defendant was not charged with homicide . ) this image / content is not covered by the terms of the creative commons licence of this publication . for permission to reuse , please contact the rights holder . what started as about 100 judicial opinions per year discussing neurobiological evidence in criminal law in 2005 climbed to around 250300 opinions in 2012 . opinions earlier in the study often discuss neurobiological evidence as part of a laundry list of other types of scientific evidence introduced . in later opinions , judges spilled substantial ink discussing the neurobiological evidence often in significant detail and with citations to scientific literature and the experts who testified in the case ( see graph 2 ) . this suggests a shift in both the frequency and the nature of how such evidence is being evaluated by judges and juries in criminal cases . substantive is one paragraph or more of the opinion discussing the neurobiological evidence introduced by a criminal defendant . ) this image / content is not covered by the terms of the creative commons licence of this publication . for permission to reuse one surprising result is how broadly neurobiological evidence is being by criminal defendants at trial . the popular mantra in academic circles is that the use of neurobiological evidence is primarily a phenomenon limited to capital cases , as mitigating evidence for sentencing . in the sample of opinions studied here , only about 40 per cent of the cases were capital , and a staggering 60 per cent of cases were other serious felony cases . drilling down further by looking at the most serious crime a defendant was charged with in the sample , it becomes clear that across felony cases neurobiological evidence is being used as part of criminal defenses ( see graph 3 ) . most serious offense charged with when neurobiological evidence raised in non - capital cases , 200512 . an evaluation of the most serious criminal charge ( per case ) that the defendant was charged with to illustrate the range of felony cases impacted by neurobiological evidence . this image / content is not covered by the terms of the creative commons licence of this publication . for permission to reuse in the 60 per cent of non - capital cases where neurobiological evidence is introduced , neurobiological evidence is also introduced in drug possession and trafficking cases , violent assaults , robbery , fraud , and more . although this sample likely underrepresents the prevalence of neurobiological evidence used in criminal cases due to the methodological barriers discussed supra , a conservative estimate based on this sample alone is that neurobiological evidence is introduced in at least five to 6 per cent of murder trials in the usa , and 14 per cent of other felony offenses . one explanation for differences in the findings of this study versus other empirical studies on the use of neurobiological evidence is a difference in what the neurobiological evidence includes medical history ( such the use of past medical records or medical history of head injuries or brain damage ) , neuropsychological testing ( through interviews , battery of testing , and/or evaluation of the defendant ) , brain scanning of the defendant , or assertions that the defendant suffers from brain or head injury . notably , only about 15 per cent of the cases where neurobiological evidence was raised in the sample had any form of brain scanning discussed in the opinion ( see graph 4 ) . a large proportion of the cases ( nearly 40 per cent ) have no discussion of neurological testing in the opinion , even though the defendant staked their defense in part on a claim that his brain made him do it. of course , it 's entirely possible that the judicial opinion did not discuss the specifics of testing that actually was introduced in the criminal case , so this is a conservative estimate of testing introduced . ( the number of opinions per year in which each time of neurobiological testing is discussed . interview+ = neuropsychological testing / evaluation , as well as medical or other history of head / brain trauma ; scan+ = some form of brain scanning evidence , neuropsychological testing / evaluation , as well as medical or other history of head / brain trauma ; history only = medical or other history of head / brain trauma but no other form of testing discussed in opinion ; no neurotesting = no discussion of any form of neurological testing introduced in opinion . ) this image / content is not covered by the terms of the creative commons licence of this publication . for permission to reuse , please contact the rights holder . in the 15 per cent of cases that included a discussion of brain scanning in the opinion , the type of scanning was most often mri or cat scans , rather than more sophisticated functional neuroimaging such as eeg , spect , or fmri scanning ( see graph 5 ) . functional magnetic resonance imaging ( fmri ) was discussed in about 2 per cent of the 15 per cent of scanning cases , but in each of the cases the fmri evidence was not admitted into the case for further consideration because of concerns about scientific reliability , credibility , or relevance . ( in the opinions where neuroimaging was introduced , the type of neuroimaging . spect = single - photon emission computed tomography ; qeeg = quantitative electroencephalography ; fmri = functional magnetic resonance imaging ; unknown = brain scanning discussed but type not mentioned ; beam = sometimes known as qeeg , brain electrical activity mapping ; cat = computed tomography ; eeg = electroencephalography ; mri = magnetic resonance imaging ; pet = positron emission tomography . ) author , 2016 . this image / content is not covered by the terms of the creative commons licence of this publication . for permission to reuse one surprising finding is the extent to which neurobiological evidence is used in pretrial proceedings ( see graph 6 ) . in pretrial proceedings , the subjective mental state and competency of the defendant can be contested . at this phase of trial , neurobiological evidence may offer a way to improve subjective competency evaluations . judges typically engage in a colloquy with a defendant and rely upon their own perception of the defendant , together with mental health evaluations , to rule on the defendants competency . neuropsychological testing , neurological history , and neuroimaging may meaningfully and appropriately improve such judgments and perceptions . distribution of neurobiological evidence - based claims in capital and non - capital cases , 20052012 . e.g. for mental retardation , 1% of the claims raised in the study sample were for a claim of mental retardation in a non - capital case . ) this image / content is not covered by the terms of the creative commons licence of this publication . for permission to reuse the test for competency to stand trial is whether a criminal defendant has sufficient present ability to consult with his lawyer with a reasonable degree of rational understanding . at any point during a trial , the competency of a defendant can be challenged , but it often arises as a pretrial issue , to address whether the defendant has the present ability to proceed . finding a criminal defendant incompetent to stand trial rather , he may be indefinitely detained in a psychiatric facility until he is rendered competent , or if he never is , remain there until he no longer serves as a present danger to himself or to others . the data here show a frequent use of neurobiological evidence to challenge defendants competency during criminal proceedings . in 15 per cent of the neurobiological evidence - based claims raised in the study sample , the defendant argued that something unique about his brain rendered him incompetent to proceed during the criminal case . of the 15 per cent of all of the claims in the study pertaining to competency , 77 per cent of those challenged the competency of the defendant to stand trial . a smaller but increasing proportion of claims focused upon the defendant 's lack of competency to waive his rights , to plead guilty , or to have confessed to the crime or crimes at issue ( see graph 7 ) . this image / content is not covered by the terms of the creative commons licence of this publication . for permission to reuse consider for example the case of miguel angel ruiz , who was found incompetent to stand trial based on neuropsychological testimony about his brain disorders . in february 2007 , 17-year - old miguel angel ruiz ( ruiz ) was charged with murdering his mother . in january of 2008 , a trial court suspended criminal proceedings against ruiz to assess his competency to stand trial . after a hearing and a subsequent jury verdict finding ruiz competent to stand trial , the trial judge set aside the verdict and concluded that there was no reasonable , credible evidence to support a finding that ruiz was competent . ruiz presented testimony from two neuropsychologists who had performed a battery of tests on him . the prosecution introduced evidence from correctional officers who had interacted with ruiz while he was awaiting trial . one of the defense experts diagnosed ruiz with a severe language disorder that arose from an organic brain - processing deficit that interfered with his language skills . as a result , he had severe inability to converse , explain , or impart information , all critical elements of being able to assist in his own defense . another expert concluded similarly , that ruiz had a developmental language disorder , specifically that the left part of his brain , which deals with language skills , did not develop as well as the right part of the brain , which deals with nonverbal skills. the expert believed this was a congenital disorder because a brain injury after birth would have affected his motor skills , which were intact. after administering standard tests to determine if ruiz was faking his disorder , the expert testified that there was no evidence of malingering . i 'm not inclined to set aside a jury 's decision lightly or unadvisedly but quite frankly when i received the verdict after hearing the evidence , i was surprised . i just could n't believe the jury could return a finding of competency based on the evidence i heard the testimony of both experts in the court 's mind is very persuasive and as a result , i 'm setting aside the jury verdict in this matter. the california court of appeals for the fifth district affirmed the trial court 's decision to set aside the jury 's verdict on finding the defendant competent to stand trial . while highly unusual to set aside a jury 's finding of fact , neurobiological evidence appears to have powerful factual and persuasive appeal to judges . the result is that in a substantial portion of cases where competency is raised , credible evidence of neurobiological impairment may enable more legitimately impaired defendants to obtain a favorable finding of incompetent to proceed . similarly and relatedly , the right to due process under the united states constitution requires that a defendant 's guilty plea be knowing , voluntary , and intelligent . traditionally , the bar to withdrawing a guilty plea has been quite difficult to surmount . indeed , defendants are using neurobiological evidence to argue that based on their neurological functioning at the time of entering their guilty plea , they did not enter that plea in a knowing , voluntary and intelligent manner . in 2007 , for example , richard hodges pleaded guilty to possession of cocaine and residential burglary . as the judge engaged in what is known as a plea colloquy , hodges occasionally appeared lost , asked questions about matters not relevant to the plea process , and exhibited confusion . they found no neurological reasons for richard 's problems and determined his learning abilities were within normal limits . after hearing all of the expert testimony , the judge concluded that hodges was not suffering from an organic brain disorder . in some cases , the court has set aside a guilty plea and remanded the case for an evidentiary hearing on claims of competency to enter a guilty plea . in other cases , the court has rejected the neurobiological evidence as contrary to his perceptions of competency based on the plea colloquy . to be sure , attempts to withdraw guilty pleas still largely fail . but it 's interesting to note the extent to which claims about the involuntariness or lack of competency to have entered a guilty plea are grounded in neurobiology . this is an area to watch over time to see if framing these issues as brain disorders will have an effect on case outcomes . in a much smaller portion of the defendants a particular difficulty for defendants raising these claims is the inability to go backward in time using present neuropsychiatric testing to understand the defendant 's competency when questioned months or years prior to being evaluated . daniel thomas , for example , was stopped for a traffic violation when the police officer noticed the smell of marijuana and a large blanket draped over what seemed to be a big square object in the back seat . later , he sought to suppress the evidence of his written confession , arguing that when he waived his right to remain silent he did not do so voluntarily , knowingly , or intelligently because he was suffering from severe migraine headaches and brain injury . a neuropsychologist testified at thomas 's suppression hearing that a brain injury could cause a degree of cognitive impairment that under stress would manifest more symptoms . these symptoms could include problems with attention , concentration , and memory . in combination with stress and sleep - depravation , thomas 's brain injury might impair his attention and concentration , and that poor attention in turn could impair his ability to process information . the judge acknowledged that the expert accurately described a person with traumatic brain injury but believed other evidence established that thomas had adequate memory and intelligence to voluntarily , knowingly , and intelligently waive of his miranda rights . the judge pointed to the time lapse before he wrote out his confession , and the counterexpert who said that given the defendant 's educational and professional background such a deficit could not be considered a significant impairment . based on the objective observations of the defendant 's capacities , the court was unconvinced that defendant 's brain injury impeded his ability to knowingly and intelligently waive his rights . this appears to be a rather typical outcome in these cases . because the present neurobiological evidence may have little bearing on the defendant 's actual competency to have confessed at the time of the crime , courts may give neurobiology little weight particularly in comparison to other circumstantial evidence that bears on the defendant 's likely capacities at the time . although it captures popular imagination , the insanity defense is raised infrequently and notoriously difficult to prove . although the precise requirement of the insanity defense varies by jurisdiction , an approach common to many states is the requirement that the defendant have a complete lack of understanding of the difference between right and wrong . this legal standard can be exceedingly difficult to establish , since mental illness typically results in some degree rather than complete impairment of understanding . jurors also tend to overcredit claims that a defendant is faking ( malingering ) their impairment . although the first issue , the legal standard of insanity , is nearly impossible to overcome , to whether the defendant is malingering is an area that neurobiological evidence may inform . it is for this second purpose bolstering claims of mental illness that defendants used neurobiological evidence in about four per cent of the claims analysed in the study . consider the case of mr chavez , who seemed to have a psychotic break while carrying a metal pole . perched at a busy intersection on the sidewalk and armed with a metal pipe , he menacingly approached cars as they stopped at a red light harassing them by asking is this your car ? the victims later testified that chavez looked kind of crazy and evil looking. when the police arrived , chavez told the officer to shoot him as he was going to smash him. he was ultimately arrested . at trial , his defense counsel argued that chavez was legally insane during the incident and introduced expert testimony that he was suffering from schizopaffective disorder of a bipolar type and a manic break at the time of the offense . a neuropsychologist testified that chavez had a dysfunction in the frontal lobe region of his brain with an iq of 79 . a pet scan of defendant 's brain revealed certain abnormalities in the left lateral frontal area . the state 's expert countered that chavez 's mental illness had no impact on his ability to understand the nature and quality of his acts , and to know that his acts were wrong when he committed them , focusing on the legal concept of insanity . with regard to the incident involving the deputy , the state 's expert opined that the defendant intentionally attacked the deputy because he did n't like what he was being told , defendant 's awareness of what he was doing and that it was wrong . this example underscores that while neurobiological evidence may bolster a finding of mental illness or impairment , the legal standard for insanity may still remain an insurmountable hurdle for most defendants . and yet , neurobiological evidence can and has assisted some criminal defendants with their claims of legal insanity . a particularly telling case is highlighted in the 2011 opinion in the case of thomas curtis vs. the state of indiana . in december 2009 , curtis was charged with murdering his wife . in 2010 , curtis filed a notice of intent to introduce the insanity defense and the trial court appointed a psychologist and a psychiatrist to evaluate him . both experts testified that they believed curtis was insane at the time he killed his wife . both the psychologist and psychiatrist who evaluated curtis thought he was unable to appreciate the wrongfulness of his conduct . organic brain injury. upon review of the trial judge 's decision to enter a gbmi verdict the appellate court found that there was neither expert nor lay opinion that curtis was sane at the time he killed his wife . instead , the trial court had been swayed by the inadequacy of available treatment noting that in a state hospital curtis would be kicked out as soon they can , and then there 's no guarantees. the trial court rejected the insanity defense after concluding that the defendant could continue to be a danger to society because of an inadequate state mental health system. although sympathetic to the trial court 's concern , the appellate court credited the uncontroverted evidence of legal insanity , reversed the judgment of gbmi , and remanded the case for an entry of not guilty by reason of insanity and the appropriate commitment proceedings . the trial court 's concern is echoed throughout the study opinions . while neurobiological evidence may be compelling evidence to bolster a finding of incompetency or insanity , without adequate mental health treatment options available courts and juries are left to struggle with how to appropriately weigh the evidence while also safeguarding the community writ large from an admitted and potentially untreated or untreatable dangerous neurobiological predisposition . neurobiological evidence has been considerably less helpful in determining whether a defendant has committed a crime . neurobiological evidence is often used to challenge the folk psychological beliefs underlying criminal law : that actions are voluntary and the product of conscious choice . the alternative that actions arise from unconscious predispositions over which an individual has little control has made little inroads in criminal law . most basically , the concepts of voluntariness and intentionality in law do n't map well onto how those concepts are understood by scientists . nor does a theory that individuals are automatons and unable to control their actions align well with our subjective experiences of self - directed decision - making . finally , the science usually population - level science ( such as a correlation between neurological or genetic variations and behavioral variations across a population)doesn't tell us much about why any particular individual behaved as they did . as a result , attempts to use neurobiological evidence for determinations of guilt or innocence seems to make far less of an impact than attempts in pretrial and sentencing determinations . in about 4 per cent of the study - sample claims ( 7 per cent of the judicial opinions ) defendants argued that their neurobiology made them act involuntary . typically , this involved a claim of involuntary conduct following the voluntary ingestion of drugs or alcohol . prosecutors start out ahead as a matter of law in proving that a defendant acted voluntarily . this presumption means that the defense that a person acted involuntarily will only succeed in a narrow set of circumstances . involuntariness is recognized if a defendant 's actions were a reflex or convulsion , a bodily movement arising from unconsciousness , sleep , hypnosis , or by some factor other than actor 's will . these automatism cases are legal freaks , but a growing chorus of scientists argues that they should serve as a paradigm ; decision - making is primarily perhaps , unbeknownst to the chorus , those claims have been featured in criminal courtrooms . judges are certainly not allowing all of these claims into the courtroom as one opinion about a high - speed car chase makes plain . in this case , a police officer saw a known former criminal offender with an outstanding warrant for his arrest at a gas station , and approached him when the defendant returned to his car . the officer asked the defendant to step out of the car but the defendant instead instructed the driver to take off . what ensued was a high - speed car chase culminating in the driver stopping in the middle of the street and backing his car into the police officer 's car to injure him . the defendant was arrested and convicted with resisting arrest and assaulting the officer . on appeal he argued that the trial court judge made a legal error in excluding relevant expert testimony regarding his head injuries and subsequent blackouts . eight weeks prior to this incident a tree limb fell on his head . at the time he had a cat scan and mri performed on his head and neck . because of his head injury , the defendant claimed that he was acting unconsciously in a blacked out state of shock during the police chase . the trial judge excluded the neurobiological evidence because it was misleading and irrelevant . a mere failure to remember an event does not excuse its occurrence unless the failure to remember signifies involuntary or unconscious conduct . instead , the experts agreed that while the defendant had been hit on the head the mri and other evidence did not support neurological trauma , blackouts , or states of unconsciousness . nearly 40 per cent of the opinions addressing automatism claims do not discuss any neurological testing of the criminal defendant . instead , the claims appear to be rooted in past head injuries or other trauma the defendant alleges to have suffered . it 's all the more important that neuroscientists start talking about the responsible use of neurobiological evidence in law , because it has already influenced jury decision - making about defendants mental states . in approximately 10 per cent of all the claims raised in the study , the defendant argued that neurobiological evidence showed he lacked the mental state necessary to commit the crime . the typical defendant uses neurobiological information to argue that he acted impulsively rather than with the premeditation or purpose contemplated by the crime definition . so claimed john gunther who was charged with first - degree murder of his mother in 2008 . gunther used a metal pipe to bludgeon his mother to death and she died of blunt force trauma to her head . he did so apparently to steal her money , television , vcr , and jewelry to purchase drugs . he admitted to killing his mother and threatening to do so many time but denied planning or intending to kill her. in support of his claim , two experts testified on his behalf . the first , a clinical psychologist and neuropsychologist , reviewed gunther 's medical records and evidence of head trauma in 2007 and again in 2008 . this medical history together with a series of neuropsychological tests the expert administered supported his conclusion that gunther had damage to his frontal lobe possibly reducing his ability to premeditate or deliberate , and instead causing him to act impulsively on the night he killed his mother. a second expert concurred echoing that gunther 's brain injuries could explain killing his mother as impulsive and affect[ing ] his ability to form mental states such as specific intent to kill his mother , premeditation or deliberation. the jury nevertheless found gunther guilty of first - degree murder , which requires deliberation and premeditation . in reviewing the jury 's determination on appeal the court found the jury reached a reasonable conclusion : here , the jury reasonably could conclude that gunther planned to murder his mother . he repeatedly told several people , over an extended period of time , that he hated her . gunther 's conversations were sufficiently frequent and detailed as to signal his intent to murder his mother , which he eventually did. this opinion is consistent with how other courts approach neurobiological evidence when used to challenge mental state . presented with circumstantial evidence consistent with planning and premeditation and with conflicting neurobiological evidence , judges and juries tend to credit the circumstantial evidence over the neurobiological . this may be in part because of the near impossibility of understanding the defendant 's mental state at the time the crime was committed . neurobiological evaluations can not take us backward in time to understand what the defendant was thinking , feeling , or why the defendant acted as he did . but the missing link between predisposition evidence and the causes and intentions of any specific actions can not presently be overcome . moreover , concepts like mental states are narrower in criminal law than the lay perspective may assume . mental state in criminal law is about the intentionality with respect to the specific act in question . did the defendant mean to swing the pipe ( purpose of the act ) , understanding the person he was swinging the pipe at was another human being ( the circumstances ) , and that the impact of swinging a pipe at another person would be to cause grave bodily suffering or injury ( consequences ) ? with this narrow understanding of the mental state necessary to convict a criminal defendant , it becomes apparent that at least as the law is presently understood neurobiological evidence may provide little support . in only the rare case will neurobiological evidence address the purpose of the act , an understanding of the circumstances or anticipation of the consequences . more direct dialog between neuroscientists and attorneys could better inform both groups about how concepts like voluntariness and mental state differ in both law and science , which could lead to more responsible testimony by neuroscientists in criminal cases about the ( ir ) relevance of neurobiological evidence to determining the voluntariness or mental state of the defendant . greater engagement by leading neuroscientists in the legal process would substantially improve both judges and jurors understanding of the limitations of science in answering the questions that law poses in addressing voluntariness and mental state . fundamentally , neurobiological evidence is fueling a societal debate about why we punish people who commit crimes . if so , would this goal be better served by rehabilitating and reintegrating into society those who commit crimes ? although neurobiological evidence can not answer these philosophical questions for us , it can provide empirical evidence about human behavior that bear on these discussions . and yet , neurobiological data may tell us little about any particular defendant and whether they are deserving of punishment . it 's this concern that studies do not tell us why a particular person behaved as they did that seems to motivate many scientists to oppose the use of neurobiological evidence in sentencing . but regardless of whether scientists agree or not about the appropriate role of neurobiological evidence in criminal law , neurobiological evidence seems clearly entrenched in sentencing decisions . developmental neuroscience has served as the empirical basis for recent constitutional prohibitions against the execution of or life imprisonment of juveniles . and there may be a coming tsunami of neurobiological evidence - backed sentencing claims at trial . trial attorneys have already been found ineffective at trial because they failed to investigate a defendant 's probable neurological abnormality ( even though defendants rarely prevail otherwise on such claims ) . nearly half of those claims were the defendant arguing he received ineffective assistance of counsel by failing to introduce neurobiological evidence at sentencing . more than half of the sentencing claims were for capital cases , while the remaining 42 per cent were non - capital cases . to establish a defendant received ineffective assistance of counsel he must show that his attorney acted below an objective standard of reasonableness , and there was a reasonable probability that but for counsel 's unprofessional errors the outcome of the case likely would have been different . in fact , even when defense counsel has slept through substantial portions of a trial , judges have ruled that defendants did not receive ineffective assistance of counsel . it 's particularly notable then that judges have already found that failing to investigate a reasonable probability of a brain abnormality constitutes ineffective assistance of counsel . even in cases with horrific facts , judges have found neurobiological evidence an essential component of counsel investigation . in one case , a defendant and his coworker went to a bar in arizona where they consumed almost two - dozen beers . when they left the bar , they picked up a female victim walking along the side of the road . eventually , things turned badly between these three and one of the defendants turned a knife on the woman . he sexually assaulted her , slit her throat , stabbed her over 30 times and then left her mutilated body in the desert . he was convicted by a jury of first - degree murder and sentenced to death . he presented volumes of new evidence on appeal about his brain damage and neuropsychological deficits . the court found a reasonable probability that the sentencing judge would have imposed a sentence less than death had the defendant 's counsel obtained and presented an expert evaluation of his neuropsychological functioning . powerful evidence of mitigation and therefore found that the defendant 's trial counsel had rendered ineffective assistance of counsel by failing to investigate it . defense counsel are ineffective if they fail to mount a defense at all , sleep through an entire ( but not just parts of ) a trial , or if they fail to investigate a probable neurological abnormality in a defendant . one of these things is not like the others , and its oddity makes clear that neurobiological evidence is an embedded part of the criminal process . to further underscore this point , consider that it may be entirely reasonable to choose not to introduce neurological evidence because of it 's double - edged potential . when the defendant connie was convicted of burglary , battery , kidnapping , sexual battery with great force , and first - degree murder of 77-year - old woman in her own home , his neurobiological evidence - based defense did n't just fail , it did so miserably . in the closing statement of the trial , the prosecutor summed up the neurobiological evidence he introduced saying : so , what are we left with ? a doctor [ ] comes in and tells you he could n't help it , he was born that way . this man was born evil , born bad , he 's going to be that way for now on and there 's nothing i can do except identify it for you . he 's got diffuse brain damage and he goes around raping women and beating them up ... how much weight do you give to he just does it because he does it ? the jury voted 11 - 1 in favor of the death penalty , and the judge followed their recommendation . it 's entirely unsurprising that even upon discovering neurobiological evidence defense attorneys may often choose to forego using it as part of mitigation . once a criminal defendant has been released from prison he can be involuntarily civilly committed if he continues to serve as a danger to himself or the community . this is most prevalent in cases of sexually violent predators , which requires proving that a person has a past conviction of a sexual offense , is likely to reoffend , and has a diagnosed mental disorder that makes the person a danger to the health and safety of others . some of the brain abnormality evidence introduced by a criminal defendant at trial can cut against him at a civil commitment hearing . for example , when a defendant committed a series of sexually violent attacks and was convicted and sentenced to 15 years in state prison , he was sent to a state hospital as a mentally disordered sex offender instead . while there , he failed the treatment programs because of his repeated sexual advances to female staff members . in a later civil commitment proceeding , the court weighed heavily that the defendant had suffered a serious head injury when struck in the head by the butt of a shotgun . a year and a half after the injury , his behavior became more aggressive , and the experts concluded that the brain injury was likely a factor in his crimes . based on this evidence , the court concluded that the defendant 's capacity to control his violent sexual tendencies would be seriously impaired if released into the community and committed him to confined institutionalization . ninety - one of the cases in the study pertained to juveniles ( about 6 per cent ) . the developing brain theory is about the juvenile brain generally , rather than specifically about the particular offender . these defendants argue that the juvenile brain is still developing that the frontal lobe region is still underdeveloped and that the brain is not fully myelinated and that as a result juveniles should be treated less harshly than adults . the fact that the brain is still developing means a juvenile has less capacity for self - restraint , but also means that their criminal conduct is not representative of how they will behave as an adult with a fully developed brain . in a triology of cases , the united states supreme court has cited to evidence about the developing juvenile brain to find it unconstitutional under the eighth amendment of the united states constitution to executive juveniles , to impose life without the possibility of parole for non - homicidal offenders , or to have a mandatory scheme of life imprisonment without the possibility of parole . since the latest of these cases , miller v. alabama , there is considerable confusion and debate by lower courts about the meaning of that ruling and the extent to which a judge must consider neuroscience when sentencing a juvenile offender . generally , between 20 and 30 per cent of defendants enjoy some success on appeal , in part because of neurobiological evidence , in capital case and non - capital case alike . although a one - to - one comparison of matched cases where neurobiological evidence was not introduced can not be done to accurately understand how neuroscience impacts case outcomes , the success rate on appeal in these cases appears to be higher than in criminal appeals in general . comparing the reversal rates in these cases versus all criminal appellate cases , the reversal rate in cases with neurobiological evidence is higher . in a 2010 study of the estimate 69,348 criminal appeals in the us , in only about 12 per cent of the cases did the appellate court reverse , remand , or modify a component of the trial courts decision . whereas the success rate in death penalty study cases was 23 per cent ( merits and non - merits cases together ) , compared to the 18.6 per cent success rate in death - penalty merits appeals overall . the success rate in non - capital cases also appears to be higher in the study cases the general reversal rate in non - capital cases was 7.7 per cent ( merits cases ) and 2.3 per cent ( without a review of the merits ) , while the overall reversal rate was 20 per cent in the non - capital cases in the study . + means the defendant achieved a positive outcome on appeal , whether as a reversal , remand , or modification of a component of the trial courts decision . we have not been able to locate any comparable data against which the relative success rate in juvenile cases with and without neurobiological cases can be compared . the study cases show that , in general , the juvenile defendants fared better than adult defendants , in some categories achieving as high as a 38 per cent favorable outcome on appeal ( see graph 9 ) . favorable means the defendant achieved a positive outcome on appeal , whether as a reversal , remand , or modification of a component of the trial courts decision . the use of neurobiological evidence in criminal cases may draw serious criticism and justifiable concern by scientists . but neurobiological evidence also has improved the criminal justice system through better competency determinations and reconsiderations about the role of punishment in society . and neurobiological evidence at times replaces what was even shoddier evidence that we relied about to make inferences about the individual capacities and behavior of a criminal defendant . given the recent rulings about the neurobiological evidence and ineffective assistance of counsel , it 's safe to assume that neurobiological evidence is now a mainstay of our criminal justice system . as a result , it 's time for a more nuanced dialog between neuroscientists , legal decision - makers , and the public about the role of neurobiological evidence in the criminal courtroom . it 's no longer productive to call for outright bans ; neuroscientists should help to improve public understanding about what neurobiological evidence can and can not tell us about human behavior . at the same time , the dialog about how neurobiological evidence is being used in criminal cases by legal scholars , commentators , and the media should account for the differences between popular perception and the results of this study . some successful efforts are already underway to improve public understanding of law and cognitive neuroscience . to name a few , the dana foundation and the american association for the advancement of science have launched a neuroscience in society series that has hosted a number of pertinent events to inform judges and the public about advances in cognitive neuroscience . the john d. and catherine t. macarthur foundation funded a multiyear project on law and neuroscience , which includes an educational component . the royal society in london has issued a four - part series of accessible reports on the developments in neuroscience and their implications for society , public policy , and law . the presidential commission for the study of bioethical issues has issued a two - part report entitled grey matters , that includes a detailed chapter and recommendations on law and neuroscience . dozens of worldwide academic conferences have been held on the topic . and a recent pbs special entitled brains on trial engaged neuroscientists , philosophers , and lawyers to educate the public about these developing trends . but more can and should be done to engage the public on these issues neuroscientists should be at the forefront of this conversation as experts in criminal courtrooms , in public presentations , through accessible writing for public audiences , or by filing amicus briefs in legal cases where neurobiological evidence is at issue . neurobiological evidence has profound implications for some of the most significant decisions we make in law and policy . it 's time we better understand how it 's being used and start to address how it may be better used in our criminal justice system .

the goal of this study was to examine the growing use of neurological and behavioral genetic evidence by criminal defendants in us criminal law . judicial opinions issued between 200512 that discussed the use of neuroscience or behavioral genetics by criminal defendants were identified , coded and analysed . criminal defendants are increasingly introducing such evidence to challenge defendants competency , the effectiveness of defense counsel at trial , and to mitigate punishment .

INTRODCUTION BACKGROUND METHODOLOGICAL APPROACH RESULTS CONCLUSION

legal scholars , scientists , and commentators lament the onslaught of behavioral genetics and neuroscience in the criminal courtroom . fueled largely by anecdotal evidence about the use of bioscience in criminal cases , or media reports of high - profile cases , there is a growing belief that neuroscience has become a mainstay of the us criminal justice system . and while scholars increasingly self - identify as part of the growing fields of law and neuroscience or law and the biosciences , to date only small - scale studies have been conducted on the use of neuroscience and behavioral genetics in the us criminal justice system . other studies have qualitatively , but not quantitatively , assessed the use and impact of neurobiological evidence in criminal law , again relying almost exclusively on discussion of that evidence in published judicial opinions . any examination of the impact of behavioral genetics and neuroscience on the us criminal justice system must begin with a more accurate understanding of how that evidence is currently being used . to better ground the interest and commentary on the use of neuroscience and behavioral genetics ( hereinafter neurobiological evidence ) in criminal law , this article summarizes some findings from the widest scale empirical study on the use of neurobiological evidence in us criminal law to date . over the past decade , over 1585 judicial opinions issued between 2005 and 2012 discuss the use of neurobiological evidence by criminal defendants to bolster their criminal defense . much of the scholarship on neurobiological evidence in criminal law has focused exclusively on either behavioral genetics or neuroscience . as a result , the emerging scientific inquiry into human behavior is trending toward a neurobiological approach over a purely genetic or neuroscientific one . moreover , the research in these fields foretells a more integrated future in human behavioral research , whereby genetics and neuroscience are linked rather than compartmentalized . as one telling example , the march 2010 issue of the seminal journal behavior genetics dedicated a special issue to pathways between gene , brain , and behavior . the introductory chapter concluded that the breadth of studies proves that tracing the pathways between biology and behavior requires expertise in genetics , neuroscience , psychology , and psychiatry . the integration is reflected in the use of behavioral genetics and neuroscience in the criminal justice system . legal practitioners take a multifaceted approach to characterizing defendants behaviors by introducing genetic , neurological , and environmental contributions . even assuming differences in scientific methodology and results between behavioral genetics and neuroscience , the substantive legal claims raised are nearly identical when either science is used in a criminal case . as a result in this study sample , anytime behavioral genetics was raised ; a neuroscientific claim was also advanced . while neuroscience by far dominates the scientific evidence introduced , the results here include cases where both neuroscience and behavioral genetics are sometimes introduced . with a few notable exceptions , scientists are on the sidelines of these developments in criminal law . publicly engaged scientists often decry the use of neurobiological evidence criminal law , and call for an outright ban on its use . measuring complex behavioral traits has been difficult , and as a result difficult to replicate in other studies as well . any scientific claim about the biological contributions to criminal conduct begins with a narrower focus on the behaviors often implicated by criminal conduct including violence , aggression , and drug and alcohol abuse . but decrying the use of neurobiological evidence in criminal law seems both futile and counterproductive ; neuroscience is already entrenched in the us legal system . criminal defendants regularly use neuroscience at every stage of the criminal process , from pretrial , to trial , and sentencing determinations . rather than standing in the way , neuroscientists should educate the public about the responsible use of neuroscience in the courtroom . to empirically ground the dialog about the use of neuroscience in the us criminal justice system , this article is the first comprehensive empirical study of the use of neurobiological evidence in us criminal justice cases . drawing from data analysed from 1585 coded criminal cases , in which judicial opinions were written and published in westlaw during 200512 , this study presents some surprising results and trends in emerging use of neurobiological evidence in criminal law . the cases collected , coded , and analysed in this study demonstrate a rising use of neurobiological research in criminal law . that use continues to be haphazard , ad hoc , and often ill conceived . defense attorneys have introduced behavioral genetics and neuroscience in attempts to exculpate criminal defendants , to bolster preexisting legal defenses , and to mitigate defendants ' culpability and punishment . prosecutors have seized upon the double - edged potential of a claimed neurobiological evidence to denigrate defendants ' characters and to demonstrate defendants ' likely future dangerousness . as the science continues to develop , its potential use in criminal investigations , interrogations , and predictions of dangerousness will undoubtedly rise . the discovery of more specific biological and neurological contributions to violence , aggressiveness , impulsivity , substance abuse , even though highly contestable and indeterminate as a scientific matter , has foreshadowed an inevitable reexamination of the us criminal justice system . indeed , the united states supreme court has already become involved in evaluating the relevance of neurobiological evidence to criminal culpability : in september of 2006 , it granted certiorari to address , in part , whether a defendant 's genetic predisposition to violence should inform whether he should be sentenced to death for first - degree murder . liberty , justice , privacy , and the structure and purpose of the us criminal justice system are all at issue . a careful and systematic study of the use , perception , current and likely impact of neurobiological evidence on criminal law is essential before its application further expands . these variables included , for example , purely identifying information about the case , the purpose for which the behavioral genetics or neuroscience evidence was introduced , the outcome of the case ( successful or unsuccessful for the criminal defendant ) , the state where the case was heard , and the types of attorneys or representation present . coders were first given the coding book , and asked to code a set of four test cases . there was less than 5 per cent disagreement between coders , and typically pertaining only to the nature of the claim raised . cases in the study were selected from searches of the westlaw legal database using the keywords and variations on the words : neuroscience , frontal lobe , hereditary , head injury , pet scan , eeg , fmri , ct scan , brain disorder , cognitive impairment , meg , nirs , brain scan , brain , diffusion tensor , heritable , hereditary , genetic , biological , memory , frontemporal , and qeeg . after excluding cases that did not bear on the use of neurobiological evidence by a criminal defendant , 1585 cases were included in the study , and all opinions in those cases majority , plurality , concurring , and dissenting opinions for a total of 1800 judicial opinions , were coded . westlaw 's inclusion criteria for judicial opinions are proprietary , unpublished , and may have changed during the study time period . moreover , the cases contained therein are primarily appellate opinions , since trial opinions at the state level are often jury verdicts without written judicial opinions . consequently , the opinions coded may reflect defendants failed attempts at using neuroscientific evidence at trial , failure to by defense counsel to investigate or introduce neurobiological evidence at trial , or newly discovered evidence on appeal . in cases where the defendant has adequate resources , or able to secure resources from the state , or as pro bono services these skews may mean the sample of cases here underreports the actual use of neuroscience in the criminal courtroom . and that the cases are skewed toward unsuccessful use of neurobiological evidence . hence , while the present empirical study significantly advances the ethical , legal and social discussions with respect to the use of neurobiological evidence in us criminal law , it is still a narrow view of the overall issue most studies on the use of neurobiological evidence in criminal cases claim it 's used almost exclusively to mitigate capital punishment and with limited success . the findings discussed herein suggest both a broader and potentially more successful use of neurobiological evidence in us criminal law . significant differences by state also appear , including the extent to which the evidence has been introduced , and the ultimate success of that evidence . these differences can be explained in part by population differences , and varying legal regimes across states ( eg the availability of capital punishment , the type of insanity defense available in the state , or the relevance of a diminished capacity defense ) . for example , while many scholars have discussed the implications of using neurobiological evidence for mitigation of criminal punishment , virtually no author has discussed the implications of using it to assess the competency of a criminal defendant . and yet the empirical analysis herein illustrates that the second most common use of biological neurobiological evidence in criminal cases is to challenge competency . the implications of this use , and the relevance of behavioral genetics and neuroscience to competency determinations , are critical areas for further exploration . in short , the fundamental assumptions guiding the current ethical , legal and social inquiry into the use of neurobiological evidence in criminal law are limited and potentially flawed . the goal of this study is to broaden the dialog and bring empirical evidence to bear on the discussion . the number of judicial opinions discussing the use of neurobiological evidence by criminal defendants is increasing year over year ( see graph 1 ) . defense attorneys are introducing neurobiological evidence across the board in serious felony cases , and not just in bifurcated capital sentencing hearings following a conviction of first - degree murder . this image / content is not covered by the terms of the creative commons licence of this publication . what started as about 100 judicial opinions per year discussing neurobiological evidence in criminal law in 2005 climbed to around 250300 opinions in 2012 . for permission to reuse one surprising result is how broadly neurobiological evidence is being by criminal defendants at trial . the popular mantra in academic circles is that the use of neurobiological evidence is primarily a phenomenon limited to capital cases , as mitigating evidence for sentencing . one explanation for differences in the findings of this study versus other empirical studies on the use of neurobiological evidence is a difference in what the neurobiological evidence includes medical history ( such the use of past medical records or medical history of head injuries or brain damage ) , neuropsychological testing ( through interviews , battery of testing , and/or evaluation of the defendant ) , brain scanning of the defendant , or assertions that the defendant suffers from brain or head injury . a large proportion of the cases ( nearly 40 per cent ) have no discussion of neurological testing in the opinion , even though the defendant staked their defense in part on a claim that his brain made him do it. interview+ = neuropsychological testing / evaluation , as well as medical or other history of head / brain trauma ; scan+ = some form of brain scanning evidence , neuropsychological testing / evaluation , as well as medical or other history of head / brain trauma ; history only = medical or other history of head / brain trauma but no other form of testing discussed in opinion ; no neurotesting = no discussion of any form of neurological testing introduced in opinion . ) in the 15 per cent of cases that included a discussion of brain scanning in the opinion , the type of scanning was most often mri or cat scans , rather than more sophisticated functional neuroimaging such as eeg , spect , or fmri scanning ( see graph 5 ) . ( in the opinions where neuroimaging was introduced , the type of neuroimaging . this image / content is not covered by the terms of the creative commons licence of this publication . in pretrial proceedings , the subjective mental state and competency of the defendant can be contested . judges typically engage in a colloquy with a defendant and rely upon their own perception of the defendant , together with mental health evaluations , to rule on the defendants competency . at any point during a trial , the competency of a defendant can be challenged , but it often arises as a pretrial issue , to address whether the defendant has the present ability to proceed . the data here show a frequent use of neurobiological evidence to challenge defendants competency during criminal proceedings . this image / content is not covered by the terms of the creative commons licence of this publication . after a hearing and a subsequent jury verdict finding ruiz competent to stand trial , the trial judge set aside the verdict and concluded that there was no reasonable , credible evidence to support a finding that ruiz was competent . after administering standard tests to determine if ruiz was faking his disorder , the expert testified that there was no evidence of malingering . similarly and relatedly , the right to due process under the united states constitution requires that a defendant 's guilty plea be knowing , voluntary , and intelligent . traditionally , the bar to withdrawing a guilty plea has been quite difficult to surmount . indeed , defendants are using neurobiological evidence to argue that based on their neurological functioning at the time of entering their guilty plea , they did not enter that plea in a knowing , voluntary and intelligent manner . after hearing all of the expert testimony , the judge concluded that hodges was not suffering from an organic brain disorder . in some cases , the court has set aside a guilty plea and remanded the case for an evidentiary hearing on claims of competency to enter a guilty plea . in other cases , the court has rejected the neurobiological evidence as contrary to his perceptions of competency based on the plea colloquy . in combination with stress and sleep - depravation , thomas 's brain injury might impair his attention and concentration , and that poor attention in turn could impair his ability to process information . the judge acknowledged that the expert accurately described a person with traumatic brain injury but believed other evidence established that thomas had adequate memory and intelligence to voluntarily , knowingly , and intelligently waive of his miranda rights . the judge pointed to the time lapse before he wrote out his confession , and the counterexpert who said that given the defendant 's educational and professional background such a deficit could not be considered a significant impairment . based on the objective observations of the defendant 's capacities , the court was unconvinced that defendant 's brain injury impeded his ability to knowingly and intelligently waive his rights . although it captures popular imagination , the insanity defense is raised infrequently and notoriously difficult to prove . at trial , his defense counsel argued that chavez was legally insane during the incident and introduced expert testimony that he was suffering from schizopaffective disorder of a bipolar type and a manic break at the time of the offense . the state 's expert countered that chavez 's mental illness had no impact on his ability to understand the nature and quality of his acts , and to know that his acts were wrong when he committed them , focusing on the legal concept of insanity . and yet , neurobiological evidence can and has assisted some criminal defendants with their claims of legal insanity . instead , the trial court had been swayed by the inadequacy of available treatment noting that in a state hospital curtis would be kicked out as soon they can , and then there 's no guarantees. although sympathetic to the trial court 's concern , the appellate court credited the uncontroverted evidence of legal insanity , reversed the judgment of gbmi , and remanded the case for an entry of not guilty by reason of insanity and the appropriate commitment proceedings . neurobiological evidence is often used to challenge the folk psychological beliefs underlying criminal law : that actions are voluntary and the product of conscious choice . most basically , the concepts of voluntariness and intentionality in law do n't map well onto how those concepts are understood by scientists . finally , the science usually population - level science ( such as a correlation between neurological or genetic variations and behavioral variations across a population)doesn't tell us much about why any particular individual behaved as they did . in about 4 per cent of the study - sample claims ( 7 per cent of the judicial opinions ) defendants argued that their neurobiology made them act involuntary . because of his head injury , the defendant claimed that he was acting unconsciously in a blacked out state of shock during the police chase . it 's all the more important that neuroscientists start talking about the responsible use of neurobiological evidence in law , because it has already influenced jury decision - making about defendants mental states . in approximately 10 per cent of all the claims raised in the study , the defendant argued that neurobiological evidence showed he lacked the mental state necessary to commit the crime . this opinion is consistent with how other courts approach neurobiological evidence when used to challenge mental state . moreover , concepts like mental states are narrower in criminal law than the lay perspective may assume . did the defendant mean to swing the pipe ( purpose of the act ) , understanding the person he was swinging the pipe at was another human being ( the circumstances ) , and that the impact of swinging a pipe at another person would be to cause grave bodily suffering or injury ( consequences ) ? more direct dialog between neuroscientists and attorneys could better inform both groups about how concepts like voluntariness and mental state differ in both law and science , which could lead to more responsible testimony by neuroscientists in criminal cases about the ( ir ) relevance of neurobiological evidence to determining the voluntariness or mental state of the defendant . it 's this concern that studies do not tell us why a particular person behaved as they did that seems to motivate many scientists to oppose the use of neurobiological evidence in sentencing . trial attorneys have already been found ineffective at trial because they failed to investigate a defendant 's probable neurological abnormality ( even though defendants rarely prevail otherwise on such claims ) . in fact , even when defense counsel has slept through substantial portions of a trial , judges have ruled that defendants did not receive ineffective assistance of counsel . defense counsel are ineffective if they fail to mount a defense at all , sleep through an entire ( but not just parts of ) a trial , or if they fail to investigate a probable neurological abnormality in a defendant . one of these things is not like the others , and its oddity makes clear that neurobiological evidence is an embedded part of the criminal process . when the defendant connie was convicted of burglary , battery , kidnapping , sexual battery with great force , and first - degree murder of 77-year - old woman in her own home , his neurobiological evidence - based defense did n't just fail , it did so miserably . in the closing statement of the trial , the prosecutor summed up the neurobiological evidence he introduced saying : so , what are we left with ? in a later civil commitment proceeding , the court weighed heavily that the defendant had suffered a serious head injury when struck in the head by the butt of a shotgun . in a triology of cases , the united states supreme court has cited to evidence about the developing juvenile brain to find it unconstitutional under the eighth amendment of the united states constitution to executive juveniles , to impose life without the possibility of parole for non - homicidal offenders , or to have a mandatory scheme of life imprisonment without the possibility of parole . although a one - to - one comparison of matched cases where neurobiological evidence was not introduced can not be done to accurately understand how neuroscience impacts case outcomes , the success rate on appeal in these cases appears to be higher than in criminal appeals in general . comparing the reversal rates in these cases versus all criminal appellate cases , the reversal rate in cases with neurobiological evidence is higher . the use of neurobiological evidence in criminal cases may draw serious criticism and justifiable concern by scientists . at the same time , the dialog about how neurobiological evidence is being used in criminal cases by legal scholars , commentators , and the media should account for the differences between popular perception and the results of this study . to name a few , the dana foundation and the american association for the advancement of science have launched a neuroscience in society series that has hosted a number of pertinent events to inform judges and the public about advances in cognitive neuroscience . the royal society in london has issued a four - part series of accessible reports on the developments in neuroscience and their implications for society , public policy , and law .

since 1982 , when hepatitis c ( hcv ) infection became a reportable virus , it has become the most common blood - borne infection in the united states and worldwide . in the united states , estimates of prevalence range from 1.3% to 2.0% , with more than 5.2 million people living with chronic hcv.1 whereas new cases of hcv infection have drastically declined over the past three decades due to awareness and public health policies , underdiagnosis and undertreatment have contributed to a continuing challenge in combating chronic hcv.2 in addition , hcv infection largely affects an aging population , driving liver disease to become the twelfth leading cause of death in the united states and the fourth leading cause of death in persons aged 4564 years . even these numbers , although significant , likely underestimate the mortality from liver disease in the us.3 by many estimates , the burden of liver disease due to chronic hcv will dramatically increase over the next two decades until a significant majority of individuals are cured of their infection . the goal of treatment is to reduce the high morbidity and mortality associated with the natural history of chronic infection particularly , the development of hepatic fibrosis , cirrhosis , and it complications , such as portal hypertension and hepatocellular carcinoma . the landscape of hcv therapy has dramatically changed since initial trials with interferon monotherapy in the 1980s . clinical trials with interferon followed by addition of ribavirin ( rbv ) and use of pegylated interferon- ( peg ) showed steadily improved sustained virologic response ( svr ) rates in the 1990s and early 2000s . these therapeutic trials were aided by advances in molecular diagnostics that allowed for accurate assessment of virologic response . even though progress was tangible , with svr rates steadily improving from less than 20% to nearly 70% in select patients , wide application of these therapies was greatly limited due to treatment contraindications , side effects , and low svr rates in individuals with advanced fibrosis and prior treatment failures.4,5 greater understanding of the hcv genome and the life cycle of the hcv virion has revealed new targets for therapy that act directly on the viral machinery to inhibit replication . consequently , treatment of hcv has now evolved beyond stimulation of the host immune system and nonspecific targeting of viral replication . coined direct - acting antivirals ( daas ) , these new drugs include a diverse class of compounds that take advantage of this understanding of the hcv genome and lifecycle . among the proteins that are encoded in the hcv rna , the serine - like protease encoded in the ns3 region and the rna - dependent rna polymerase encoded in the ns5b region have been targets for the daa compounds.6,7 an ns3 serine protease and a cofactor , ns4a , allow for posttranslational cleavage of the 3,000 amino acid polyprotein produced from hcv rna and host ribosomes . once cleaved , this polyprotein forms four structural and six ns proteins key in viral rna replication and new viral particle assembly . protease inhibitors.8 boceprevir ( boc ) and telaprevir ( tvr ) , the first - generation protease inhibitors , combined with peg / rbv brought improvement in overall svr rates from approximately 40% to 60% in treatment nave individuals , while treatment duration ranged from 2448 weeks , depending on hcv rna decline throughout therapy . in addition to the common side effects seen with peg / rbv , boc and tvr compounded treatment difficulty with unique side effects of their own.912 with boc , anemia , neutropenia , and dysgeusia were most common side effects , whereas anemia , skin rash , and anorectal symptoms were more frequent with tvr . anemia was recognized as a common adverse event , occurring in about 50% of individuals treated with boc and 40% of those treated with tvr . anemia frequently led to the use of erythropoietin - stimulating agents , blood transfusions , or treatment discontinuation with subsequent treatment failure in a significant number of individuals . in addition , tvr - based therapy resulted in a more than 20% higher incidence of rash and pruritus compared to placebo . although 90% of rash events related to tvr were mild or moderate and did not progress , 6% of all patients required treatment discontinuation . gastrointestinal symptoms are another common group of adverse events for patients treated with boc or tvr . dysgeusia was present in 35%44% of patients treated with boc , and anorectal symptoms such as anal pruritus and rectal burning were seen in 26% of patients treated with tvr.13 these new drugs have several factors that make them advantageous , including high potency , multigenotypic coverage , and an intermediate - to - high barrier to resistance . some second - wave protease inhibitors are being tested in combination with ritonavir , a cyp3a inhibitor , to reduce overall protease inhibitor exposure while maintaining potent antiviral activity and attempting to minimize toxicities . furthermore , these agents are being tested in combination as all - oral regimens with and without rbv , paving the way for interferon - free as well as rbv - free regimens . there are two distinct groups , the nucleoside polymerase inhibitors ( nis ) and nonnucleoside polymerase inhibitors ( nnis ) . the nis are analogs of natural substrates that interact with the catalytic site for ns5b , incorporate into the elongating chain of hcv rna , and subsequently cause chain termination of viral rna synthesis . since the active site of the polymerase is highly conserved through replication , viruses with mutations of the active site tend to lose the ability to replicate , giving the nis a high barrier to resistance.14,15 nnis , on the other hand , bind to several allosteric sites on the ns5b protein and change the confirmation of the active site , which in turn prevents effective viral rna synthesis . the efficacy of antiviral activity varies by hcv genotype and subtype , and these novel drugs vary among themselves in the extent to which resistant variants develop.16 the ns5a component of the replication complex is another prime target for inhibiting hcv replication . ns5a is a multifunctional protein involved with many different stages of the hcv life cycle , including rna replication and virion assembly and secretion . inhibitors of the ns5a complex have proven to be high - potency drugs , with multigenotypic efficacy and a low - to - intermediate barrier to resistance . this effect may be especially potent when used in conjunction with another class of anti - hcv therapy.17 simeprevir ( smv ) , a once - daily dosed ns3/4a protease inhibitor , in combination with peg / rbv , was approved in november 2013 by the us food and drug administration ( fda ) for treatment of hcv genotype 1 patients . the pillar study was a randomized , placebo - controlled phase ii trial for treatment - nave , noncirrhotic , hcv genotype 1 patients . the study showed svr rates of 75%86% in the smv + peg / rbv groups versus 65% in the peg / rbv group . discontinuation rates were 8%16% in smv - treated patients and 15% in the control group . the majority of patients experiencing viral breakthrough or relapse had emerging ns3 protease mutations at time of failure.18 phase iii trials of smv include quest-1 , quest-2 , and promise . quest-1 and quest-2 randomized patients to peg / rbv plus placebo versus smv + peg / rbv 150 mg daily . pooled data reveal overall svr rates 12 weeks after treatment ( svr12 ) among hcv genotype 1 patients of 80% in the smv + peg / rbv group versus 50% in the placebo group . patients with hcv genotype 1a had svr rates of 71% , versus 90% in genotype 1b patients . in fact , hcv genotype 1a patients with a q80k mutation had svr rates similar to those found in patients in the control arms receiving peg / rbv ( 58% and 52% , respectively ) . further , hcv genotype 1a patients without a q80k mutation matched the svr rates of patients with hcv genotype 1b ( 84% versus 85% ) . in quest-1 , patients were stratified by fibrosis level and hvc subgenotype ( 1a versus 1b ) . in the smv treatment group , patients with cirrhosis achieved an svr rate of 58% compared with 82% in patients without cirrhosis ( figure 1).1921 promise was a phase iii trial of treatment - experienced hcv genotype 1 patients who were randomized to receive smv or placebo , each combined with peg / rbv . this was followed by response - guided therapy for an additional 1236 weeks of peg / rbv . results showed that among previous relapsers , 79% achieved svr12 with smv therapy , compared to 39% in the placebo arm . in addition , the majority of patients ( 93% ) were eligible for shortened therapy with 83% svr12 rates . as was seen in the quest-1 trial , svr12 rates were higher in hcv genotype 1b patients ( 86% ) than in genotype 1a patients ( 70% ) . patient subpopulations with unfavorable conditions , including those with cirrhosis or advanced fibrosis ( metavir f4 ) and interleukin ( il ) 28b tt genotype , also achieved higher svr rates ( 74% and 64% versus 26% and 18% respectively ) , when compared with placebo.22 adverse event profiles were similar between the smv and placebo groups . a total of four deaths occurred in the treatment groups , but these were thought by the investigators to be unrelated to treatment . in the pooled analysis of quest 1 , quest 2 , and promise , 2% of those in the smv group had serious adverse events , versus 3% of those in the control group . a total of three patients ( 0.4% ) in the smv group had significant adverse events , which were determined to be related to smv by the study investigator ; one patient experienced major depression and two patients experienced photosensitivity reactions . other common adverse events were rash ( 28% in treatment groups versus 20% in control groups ) , influenza - like illness ( 26% in treatment groups versus 21% control groups ) , pruritus ( 22% in treatment groups versus 15% in control groups ) and nausea ( 22% in treatment groups and 18% in control groups).22,23 overall , smv + peg / rbv confers similar efficacy to its daa predecessors , boc and tvr , with the added benefit of a more convenient dosing regimen and a more favorable side - effect profile . a meta - analysis attempted to compare the relative efficacy and safety of smv - based therapy with boc / tvr - based triple therapy and concluded that smv shows a similar chance of achieving an svr with a lower probability of both the incidence of chronic hcv - related adverse events and discontinuation due to adverse events.24 it should be noted that the manufacturer of smv has recommended , as noted in the package insert , that patients with hcv genotype 1a be tested for the q80k mutation prior to treatment initiation . this has raised concerns among providers , given the expense of this test and the question as to whether or not insurance providers will reimburse for this test . this may preclude some providers from considering smv - based therapy altogether for hcv genotype 1a patients . sofosbuvir ( sof ) is a nucleotide analog with potent activity against hcv genotypes 16 . sof administered in combination with rbv is the first all - oral therapy for hcv genotypes 2 or 3 . for patients with hcv genotypes 1 , 4 , 5 , or 6 , sof in combination with peg / rbv phase ii trials showed sof was a potent and rapid suppressor of hcv genotypes 14 and 6 , with a once - daily 400 mg dose . in addition , phase ii data revealed that hcv genotypes 2 and 3 could be efficaciously treated with sof + rbv without peg , while the addition of peg to sof + rbv treatment increased the response rate for hcv genotypes 1 , 4 , and 6 and allowed the duration of therapy to be decreased to 12 weeks.2527 data from four phase iii trials form the principal basis for characterizing the safety and efficacy of sof in patients with chronic hcv infection . three trials were conducted in subjects with hcv genotype 2 or 3 , and one study evaluated genotypes 1 , 4 , 5 , and 6 . fission evaluated sof + rbv treatment for 12 weeks in treatment - nave hcv genotype 2 and 3 patients . the primary efficacy endpoint of this study was noninferiority of sof + rbv alone compared to the standard 24 weeks of peg / rbv . this endpoint was met with 67% ( 170 of 253 ) of patients achieving svr12 in the sof + rbv treatment group versus 67% ( 162 of 243 ) in the peg / rbv treatment group . subgroup analyses were generally similar for svr12 rates between the treatment groups for age , sex , race , ethnicity , baseline body mass index , il28b genotype , and baseline hcv rna . in both treatment groups , patients with hcv genotype 2 had higher svr12 rates than did patients with genotype 3 . noncirrhotic patients also had higher svr12 rates than did cirrhotic patients in both treatment groups ( figure 2).28 positron evaluated sof + rbv for 12 weeks compared with placebo in patients with hcv genotype 2 or 3 who were interferon intolerant , interferon ineligible ( ie , had medical conditions that precluded interferon therapy ) , or unwilling to take interferon . the majority of patients considered to be peg ineligible were considered to have a psychiatric comorbidity ( 58% ) , while most peg intolerance was attributed to flu - like symptoms ( 32% ) followed by prior psychiatric sequelae with peg ( 20% ) . overall svr rates were 78% in combined analysis of hcv genotype 2 and 3 patients . genotype 2 patients showed svr rates of 93% , while genotype 3 achieved svr in only 61% of patients , although svr rates reached as high as 70% in genotype 3 treatment - nave patients . this reflects the favorable response of hcv genotype 2 seen in the fission trial . of note , in hcv genotype 3 patients who underwent prior hcv treatment for more than 12 weeks , only 18% ( 2/11 ) achieved svr . cirrhotic patients with hcv genotype 2 had a 94% svr rate , while cirrhotic hcv genotype 3 patients had a 21% svr rate.29 fusion evaluated sof + rbv for 12 weeks or 16 weeks in treatment - experienced subjects for whom prior peg - based therapy had failed . a sof + rbv group with a 16-week treatment duration was included in the study to evaluate whether a longer duration would lead to improved response rates compared with a 12-week duration in this difficult - to - treat population . overall , in this treatment - experienced population , 16 weeks of therapy proved to have significant positive impact on svr rates . in hcv genotype 2 , svr rates increased from 86% to 94% , and in hcv genotype 3 , svr rates increased from 30% to 62% with the 4-weeks - longer duration of therapy . extending treatment duration by 4 weeks conferred a significant benefit to patients with cirrhosis as well . hcv genotype 2 patients with cirrhosis saw an increase in svr from 60% to 78% , while genotype 3 patients with cirrhosis saw a marked increase in response rates , from 19% to 61% ( figure 3).29 an additional trial , neutrino ( published with the positron data ) , evaluated sof + peg / rbv for 12 weeks in hcv genotype 1 , 4 , 5 , or 6 treatment - nave subjects . the primary endpoint was a null svr rate of 60% based on phase iii trials of boc and tvr , the standard recommended regimen for this population at the time of the study . hcv genotype 1 patients achieved a rate of 89% , genotype 4 patients achieved 96% svr , a single genotype 5 patient achieved svr , and 6 of 6 genotype 6 patients achieved svr . overall , 80% ( 43/54 ) patients with cirrhosis achieved svr.28 the frequency of any adverse event in patients receiving 12 weeks of sof + rbv without peg was 88% , compared to 77% in the placebo group . the most common adverse event in the sof + rbv genotype 2 and 3 pooled data was fatigue , reported in 40% of patients , followed by headache and nausea , reported in 23% and 20% of patients , respectively . the frequency of the commonly reported adverse events was similar in patients in receiving sof + rbv treatment for 12 and 16 weeks.28,29 four percent of patients receiving sof + rbv for 12 weeks experienced a serious adverse event , while 1% ( 8/566 ) patients discontinued treatment due to an adverse event . of note , there were no treatment discontinuations in the 96 patients undergoing 16 weeks of sof + rbv in the fusion study . it should also be noted that across the four phase iii studies for sof , no genotypic or phenotypic viral resistance to sof or rbv was detected . the one death among all of the sof treatment groups was determined to be caused by heroin and cocaine overdose on day 1 of treatment.28,29 with rbv monotherapy , expected hemoglobin reductions are approximately 2 g / dl . this reduction is similar to those observed in the sof + rbv groups in the fission , positron , and fusion studies . in addition , sof monotherapy was not associated with any hemoglobin reductions in the phase ii studies . in non interferon - containing treatment groups , no effect on neutrophil or platelet counts was observed.26,27 the incidence of grade 3 total bilirubin elevation ( 2.55.0 times the upper limit of normal ) in the pooled sof + rbv 12-week treatment groups was 2% . no patients with a bilirubin elevation had concomitant elevations in transaminases or clinical symptoms such as jaundice , and no patient interrupted or discontinued treatment due to elevated bilirubin.29 sofosbuvir is currently undergoing investigation in the pre liver - transplant population . infection of an uninfected , transplanted graft is universal among transplant recipients with chronic hcv . the rate of accelerated , progressive liver disease and cirrhosis in the setting of immunosuppression in the posttransplantation period is high , with rates of moderate chronic hepatitis as high as 27% after a median of 3 years and the progression to cirrhosis in 8% of patients after a median of approximately 4 years . sof + rbv has been evaluated in 61 patients awaiting liver transplant to assess hcv recurrence posttransplant . preliminary data reveal that , of 41 chronic hcv patients undergoing transplant who were given sof + rbv while awaiting transplant , 93% had undetectable hcv rna at the time of transplant and 71% of patients continued to have undetectable levels 24 weeks posttransplant.30 there is currently an ongoing trial of sof + smv with and without rbv for 12 or 24 weeks in hcv genotype 1 noncirrhotic patients who were either null - responsive to peg / rbv or treatment - nave . preliminary data showed that svr rates 8 weeks after treatment ( svr8 ) for hcv genotypes 1a and 1b were similar , and that the svr8 rate in the treatment - experienced cohort with 12 weeks of sof + smv without rbv was 93% ( 13/14 ) ( figure 4).31 daclatasvir is a first - in - class ns5a replication complex inhibitor , which has recently been tested in combination with sof , with and without rbv , in hcv genotype 1 , 2 , and 3 treatment - nave and treatment - experienced patients . ninety - two percent of patients with hcv genotype 2 and 89% of 18 patients with genotype 3 infection had a sustained virologic response at week 12 . hcv subtypes 1a and 1b did not differ with respect to svr ( 98% and 100% , respectively ) . thirty - two patients ( 15% ) in this study had clinically significant fibrosis ; however , a separate analysis in this population is not available . the most common adverse events were fatigue , headache , and nausea.32 another ns5a inhibitor , ledipasvir , has been tested in combination with sof . the lonestar trial evaluated sof + ledipasvir with and without rbv for treatment - nave and treatment - experienced hcv genotype 1 patients , including patients with cirrhosis . all but two of the 100 study participants achieved svr ( figure 5 ) . one noncirrhotic , treatment - nave patient in the group receiving sof + ledipasvir for 8 weeks relapsed . the other failure was a prior treatment failure in a patient treated with 12 weeks of sof + ledipasvir . the most common adverse events were nausea , anemia , upper respiratory tract infection , and headache.33 a combination pill containing both sof and ledipasvir has been developed by the manufacturer ( gilead sciences inc . , foster city , ca , usa ) , and was submitted as a new drug application to the fda in february 2014 . another phase ii interferon - free trial evaluated hcv genotype 1 , treatment - nave and treatment - experienced noncirrhotic patients . this trial included a protease inhibitor , abt-450 , boosted with 100 mg of ritonavir daily , combined with abt-267 or abt-333 or both , with or without rbv , for 8 , 12 , or 24 weeks . the rate of treatment failure was lower among those receiving three direct - acting agents plus rbv . it appeared that 12 weeks was an optimal duration of therapy , although the limit in power of the study precluded determination of statistical significance . the issue of justifying the cost of new therapies has been a topic of controversy among the lay press and a concern for patients . the cost of sof has been widely publicized , and the treatment has been critiqued for its sticker price of $ 28,000 usd for 28 pills , such that a 12-week course of treatment with sof alone is more than $ 84,000 . patients with compensated cirrhosis may live for more than a decade , and models suggest each individual would accrue more than $ 270,000 in expenses prior to developing end - stage liver disease.35 furthermore , according to the united network for organ sharing transplant living web site , the estimated us average of billed charges per liver transplant in 2011 was $ 577,100.36,37 stratified cost - effectiveness analyses have yet to be published on our newest therapies . even so , not only is treatment duration shortened with these therapies , but the costs associated with side effects due to treatment with boc / tvr plus peg / rbv , including blood transfusion , erythropoietin , clinic visits , and hospitalizations , are eliminated . consequently , is it likely that the general consensus , even among insurance companies , will be that the price of treating an individual per svr is actually cost - effective when factors beyond the price of the drug alone are considered . the recent approval by the us fda of the two newest daa s , smv and sof , for the treatment of chronic hcv marks another treatment milestone and will likely enable many of our warehoused simeprevir has shown a favorable protease inhibitor profile with fewer adverse effects than its two predecessors and a shorter duration when combined with peg / rbv . the currently approved regimen of smv with a peg / rbv backbone for hcv genotype 1 patients appears capable of achieving an svr that is similar to that achieved by boc / tvr + peg / rbv , but in hcv genotype 1a patients , the possibility of the q80k mutation , combined with the fact that interferon is still required , may cause providers to look elsewhere for treatment options or again shelve their patients for future therapies . some providers may seek preapproval from payers for off - label use of smv in combination with sof , which , as discussed above , has proven efficacious in a limited number of noncirrhotic , treatment - nave and treatment - experienced , hcv genotype 1a and 1b patients , for 12 weeks with or without rbv . smv may find most of its usefulness in the future as a component in off - label , all - oral regimens , but it certainly remains a therapeutic option with peg / rbv in those well - suited ( noncirrhotic , treatment - nave , il-28b cc , non sofosbuvir in combination with rbv has become the first all - oral , non - peg regimen and is the first - in - its - class ns5b polymerase inhibitor to be approved for chronic hcv . while the peg - free regimen is approved for hcv genotypes 2 and 3 , its current indication for the hcv genotype 1 population still includes a peg / rbv backbone , granted only for 12 weeks , and it brings overall svr rates from 60% to 90% . this provides a new standard of therapy and a new bar to be met for future therapies in hcv genotype 1 patients . in addition , as discussed above , numerous trials have shown that sof in combination with other daas , particularly daclatasvir and ledipasvir , with and without rbv , will be extremely effective , tolerable , and durable for chronic hcv , even among difficult - to - treat patients , with unfavorable pretreatment profiles . further , several trials , including those with daclatasvir and ledipasvir in combination with sof , may allow for the omission of rbv as a part of therapy . rbv - sparing regimens are another milestone in the future of chronic hcv therapy and will deliver yet another improvement in the safety and tolerability of treatment . safety and tolerability also becomes of the utmost importance when we contemplate therapy for our fragile pretransplant population , a cohort in whom the benefit of sof + rbv prior to transplant has shown promise in durable eradication of the virus in the posttransplant period . the clinical impact of this outcome can not yet be quantified but will likely have significant benefit on graft survival , morbidity , and mortality for patients who receive a transplant for chronic hcv . there are several factors whose importance and impact on care have yet to be established . it is not entirely clear if the development of resistance in previously treated patients will play a role in clinical decision making , ie , whether or not there will be a role in resistance profiling prior to treatment initiation in those for whom prior treatment has failed , as it appears our previous markers ( treatment nave / experienced , fibrosis stage , il-28b status ) will become less indicative of response to therapy . although tremendous strides have been made in the advancement of hcv therapy , accessibility to care and identification of those requiring treatment precludes addressing the global burden of chronic hcv . as such , while the new era of chronic hcv therapy remains in its adolescence , the global implication of the evolution in therapy is one that could conceivably mean eradication of hepatitis c.

greater understanding of the hepatitis c virus ( hcv ) genome and life cycle of the hcv virion allows for new targets for therapy that directly act on the viral machinery to inhibit replication . numerous direct - acting antivirals are in development , and four have been brought to market . simeprevir , a second - generation protease inhibitor , has been approved for hcv genotype 1 patients in combination with pegylated interferon- and ribavirin . sofosbuvir , a novel nucleotide analog , has pangenotypic coverage and has been approved for hcv genotype 1 patients with ribavirin and pegylated interferon-. for hcv genotypes 2 and 3 , an all - oral regimen of sofosbuvir with ribavirin has become the new gold standard for treatment . the efficacy and safety for these two novel therapies among various subpopulations of those infected with chronic hepatitis c are discussed in the following review . in addition , off - label and future therapeutic regimens are addressed , as well as the concerns about cost of current and future therapies .

Introduction The era of the direct-acting antivirals The evolving standard of care Simeprevir Sofosbuvir Combination all-oral therapy Concerns about cost Conclusion

since 1982 , when hepatitis c ( hcv ) infection became a reportable virus , it has become the most common blood - borne infection in the united states and worldwide . in the united states , estimates of prevalence range from 1.3% to 2.0% , with more than 5.2 million people living with chronic hcv.1 whereas new cases of hcv infection have drastically declined over the past three decades due to awareness and public health policies , underdiagnosis and undertreatment have contributed to a continuing challenge in combating chronic hcv.2 in addition , hcv infection largely affects an aging population , driving liver disease to become the twelfth leading cause of death in the united states and the fourth leading cause of death in persons aged 4564 years . clinical trials with interferon followed by addition of ribavirin ( rbv ) and use of pegylated interferon- ( peg ) showed steadily improved sustained virologic response ( svr ) rates in the 1990s and early 2000s . even though progress was tangible , with svr rates steadily improving from less than 20% to nearly 70% in select patients , wide application of these therapies was greatly limited due to treatment contraindications , side effects , and low svr rates in individuals with advanced fibrosis and prior treatment failures.4,5 greater understanding of the hcv genome and the life cycle of the hcv virion has revealed new targets for therapy that act directly on the viral machinery to inhibit replication . coined direct - acting antivirals ( daas ) , these new drugs include a diverse class of compounds that take advantage of this understanding of the hcv genome and lifecycle . among the proteins that are encoded in the hcv rna , the serine - like protease encoded in the ns3 region and the rna - dependent rna polymerase encoded in the ns5b region have been targets for the daa compounds.6,7 an ns3 serine protease and a cofactor , ns4a , allow for posttranslational cleavage of the 3,000 amino acid polyprotein produced from hcv rna and host ribosomes . protease inhibitors.8 boceprevir ( boc ) and telaprevir ( tvr ) , the first - generation protease inhibitors , combined with peg / rbv brought improvement in overall svr rates from approximately 40% to 60% in treatment nave individuals , while treatment duration ranged from 2448 weeks , depending on hcv rna decline throughout therapy . in addition to the common side effects seen with peg / rbv , boc and tvr compounded treatment difficulty with unique side effects of their own.912 with boc , anemia , neutropenia , and dysgeusia were most common side effects , whereas anemia , skin rash , and anorectal symptoms were more frequent with tvr . in addition , tvr - based therapy resulted in a more than 20% higher incidence of rash and pruritus compared to placebo . some second - wave protease inhibitors are being tested in combination with ritonavir , a cyp3a inhibitor , to reduce overall protease inhibitor exposure while maintaining potent antiviral activity and attempting to minimize toxicities . furthermore , these agents are being tested in combination as all - oral regimens with and without rbv , paving the way for interferon - free as well as rbv - free regimens . since the active site of the polymerase is highly conserved through replication , viruses with mutations of the active site tend to lose the ability to replicate , giving the nis a high barrier to resistance.14,15 nnis , on the other hand , bind to several allosteric sites on the ns5b protein and change the confirmation of the active site , which in turn prevents effective viral rna synthesis . the efficacy of antiviral activity varies by hcv genotype and subtype , and these novel drugs vary among themselves in the extent to which resistant variants develop.16 the ns5a component of the replication complex is another prime target for inhibiting hcv replication . ns5a is a multifunctional protein involved with many different stages of the hcv life cycle , including rna replication and virion assembly and secretion . inhibitors of the ns5a complex have proven to be high - potency drugs , with multigenotypic efficacy and a low - to - intermediate barrier to resistance . this effect may be especially potent when used in conjunction with another class of anti - hcv therapy.17 simeprevir ( smv ) , a once - daily dosed ns3/4a protease inhibitor , in combination with peg / rbv , was approved in november 2013 by the us food and drug administration ( fda ) for treatment of hcv genotype 1 patients . the pillar study was a randomized , placebo - controlled phase ii trial for treatment - nave , noncirrhotic , hcv genotype 1 patients . pooled data reveal overall svr rates 12 weeks after treatment ( svr12 ) among hcv genotype 1 patients of 80% in the smv + peg / rbv group versus 50% in the placebo group . patients with hcv genotype 1a had svr rates of 71% , versus 90% in genotype 1b patients . in fact , hcv genotype 1a patients with a q80k mutation had svr rates similar to those found in patients in the control arms receiving peg / rbv ( 58% and 52% , respectively ) . further , hcv genotype 1a patients without a q80k mutation matched the svr rates of patients with hcv genotype 1b ( 84% versus 85% ) . in the smv treatment group , patients with cirrhosis achieved an svr rate of 58% compared with 82% in patients without cirrhosis ( figure 1).1921 promise was a phase iii trial of treatment - experienced hcv genotype 1 patients who were randomized to receive smv or placebo , each combined with peg / rbv . in addition , the majority of patients ( 93% ) were eligible for shortened therapy with 83% svr12 rates . as was seen in the quest-1 trial , svr12 rates were higher in hcv genotype 1b patients ( 86% ) than in genotype 1a patients ( 70% ) . in the pooled analysis of quest 1 , quest 2 , and promise , 2% of those in the smv group had serious adverse events , versus 3% of those in the control group . a meta - analysis attempted to compare the relative efficacy and safety of smv - based therapy with boc / tvr - based triple therapy and concluded that smv shows a similar chance of achieving an svr with a lower probability of both the incidence of chronic hcv - related adverse events and discontinuation due to adverse events.24 it should be noted that the manufacturer of smv has recommended , as noted in the package insert , that patients with hcv genotype 1a be tested for the q80k mutation prior to treatment initiation . this may preclude some providers from considering smv - based therapy altogether for hcv genotype 1a patients . sofosbuvir ( sof ) is a nucleotide analog with potent activity against hcv genotypes 16 . sof administered in combination with rbv is the first all - oral therapy for hcv genotypes 2 or 3 . for patients with hcv genotypes 1 , 4 , 5 , or 6 , sof in combination with peg / rbv phase ii trials showed sof was a potent and rapid suppressor of hcv genotypes 14 and 6 , with a once - daily 400 mg dose . in addition , phase ii data revealed that hcv genotypes 2 and 3 could be efficaciously treated with sof + rbv without peg , while the addition of peg to sof + rbv treatment increased the response rate for hcv genotypes 1 , 4 , and 6 and allowed the duration of therapy to be decreased to 12 weeks.2527 data from four phase iii trials form the principal basis for characterizing the safety and efficacy of sof in patients with chronic hcv infection . three trials were conducted in subjects with hcv genotype 2 or 3 , and one study evaluated genotypes 1 , 4 , 5 , and 6 . fission evaluated sof + rbv treatment for 12 weeks in treatment - nave hcv genotype 2 and 3 patients . this endpoint was met with 67% ( 170 of 253 ) of patients achieving svr12 in the sof + rbv treatment group versus 67% ( 162 of 243 ) in the peg / rbv treatment group . in both treatment groups , patients with hcv genotype 2 had higher svr12 rates than did patients with genotype 3 . noncirrhotic patients also had higher svr12 rates than did cirrhotic patients in both treatment groups ( figure 2).28 positron evaluated sof + rbv for 12 weeks compared with placebo in patients with hcv genotype 2 or 3 who were interferon intolerant , interferon ineligible ( ie , had medical conditions that precluded interferon therapy ) , or unwilling to take interferon . overall svr rates were 78% in combined analysis of hcv genotype 2 and 3 patients . this reflects the favorable response of hcv genotype 2 seen in the fission trial . cirrhotic patients with hcv genotype 2 had a 94% svr rate , while cirrhotic hcv genotype 3 patients had a 21% svr rate.29 fusion evaluated sof + rbv for 12 weeks or 16 weeks in treatment - experienced subjects for whom prior peg - based therapy had failed . in hcv genotype 2 , svr rates increased from 86% to 94% , and in hcv genotype 3 , svr rates increased from 30% to 62% with the 4-weeks - longer duration of therapy . extending treatment duration by 4 weeks conferred a significant benefit to patients with cirrhosis as well . hcv genotype 2 patients with cirrhosis saw an increase in svr from 60% to 78% , while genotype 3 patients with cirrhosis saw a marked increase in response rates , from 19% to 61% ( figure 3).29 an additional trial , neutrino ( published with the positron data ) , evaluated sof + peg / rbv for 12 weeks in hcv genotype 1 , 4 , 5 , or 6 treatment - nave subjects . hcv genotype 1 patients achieved a rate of 89% , genotype 4 patients achieved 96% svr , a single genotype 5 patient achieved svr , and 6 of 6 genotype 6 patients achieved svr . overall , 80% ( 43/54 ) patients with cirrhosis achieved svr.28 the frequency of any adverse event in patients receiving 12 weeks of sof + rbv without peg was 88% , compared to 77% in the placebo group . the most common adverse event in the sof + rbv genotype 2 and 3 pooled data was fatigue , reported in 40% of patients , followed by headache and nausea , reported in 23% and 20% of patients , respectively . the frequency of the commonly reported adverse events was similar in patients in receiving sof + rbv treatment for 12 and 16 weeks.28,29 four percent of patients receiving sof + rbv for 12 weeks experienced a serious adverse event , while 1% ( 8/566 ) patients discontinued treatment due to an adverse event . of note , there were no treatment discontinuations in the 96 patients undergoing 16 weeks of sof + rbv in the fusion study . this reduction is similar to those observed in the sof + rbv groups in the fission , positron , and fusion studies . in addition , sof monotherapy was not associated with any hemoglobin reductions in the phase ii studies . no patients with a bilirubin elevation had concomitant elevations in transaminases or clinical symptoms such as jaundice , and no patient interrupted or discontinued treatment due to elevated bilirubin.29 sofosbuvir is currently undergoing investigation in the pre liver - transplant population . the rate of accelerated , progressive liver disease and cirrhosis in the setting of immunosuppression in the posttransplantation period is high , with rates of moderate chronic hepatitis as high as 27% after a median of 3 years and the progression to cirrhosis in 8% of patients after a median of approximately 4 years . preliminary data reveal that , of 41 chronic hcv patients undergoing transplant who were given sof + rbv while awaiting transplant , 93% had undetectable hcv rna at the time of transplant and 71% of patients continued to have undetectable levels 24 weeks posttransplant.30 there is currently an ongoing trial of sof + smv with and without rbv for 12 or 24 weeks in hcv genotype 1 noncirrhotic patients who were either null - responsive to peg / rbv or treatment - nave . preliminary data showed that svr rates 8 weeks after treatment ( svr8 ) for hcv genotypes 1a and 1b were similar , and that the svr8 rate in the treatment - experienced cohort with 12 weeks of sof + smv without rbv was 93% ( 13/14 ) ( figure 4).31 daclatasvir is a first - in - class ns5a replication complex inhibitor , which has recently been tested in combination with sof , with and without rbv , in hcv genotype 1 , 2 , and 3 treatment - nave and treatment - experienced patients . ninety - two percent of patients with hcv genotype 2 and 89% of 18 patients with genotype 3 infection had a sustained virologic response at week 12 . the most common adverse events were fatigue , headache , and nausea.32 another ns5a inhibitor , ledipasvir , has been tested in combination with sof . the lonestar trial evaluated sof + ledipasvir with and without rbv for treatment - nave and treatment - experienced hcv genotype 1 patients , including patients with cirrhosis . the most common adverse events were nausea , anemia , upper respiratory tract infection , and headache.33 a combination pill containing both sof and ledipasvir has been developed by the manufacturer ( gilead sciences inc . , foster city , ca , usa ) , and was submitted as a new drug application to the fda in february 2014 . another phase ii interferon - free trial evaluated hcv genotype 1 , treatment - nave and treatment - experienced noncirrhotic patients . this trial included a protease inhibitor , abt-450 , boosted with 100 mg of ritonavir daily , combined with abt-267 or abt-333 or both , with or without rbv , for 8 , 12 , or 24 weeks . the rate of treatment failure was lower among those receiving three direct - acting agents plus rbv . the issue of justifying the cost of new therapies has been a topic of controversy among the lay press and a concern for patients . the cost of sof has been widely publicized , and the treatment has been critiqued for its sticker price of $ 28,000 usd for 28 pills , such that a 12-week course of treatment with sof alone is more than $ 84,000 . patients with compensated cirrhosis may live for more than a decade , and models suggest each individual would accrue more than $ 270,000 in expenses prior to developing end - stage liver disease.35 furthermore , according to the united network for organ sharing transplant living web site , the estimated us average of billed charges per liver transplant in 2011 was $ 577,100.36,37 stratified cost - effectiveness analyses have yet to be published on our newest therapies . the recent approval by the us fda of the two newest daa s , smv and sof , for the treatment of chronic hcv marks another treatment milestone and will likely enable many of our warehoused simeprevir has shown a favorable protease inhibitor profile with fewer adverse effects than its two predecessors and a shorter duration when combined with peg / rbv . the currently approved regimen of smv with a peg / rbv backbone for hcv genotype 1 patients appears capable of achieving an svr that is similar to that achieved by boc / tvr + peg / rbv , but in hcv genotype 1a patients , the possibility of the q80k mutation , combined with the fact that interferon is still required , may cause providers to look elsewhere for treatment options or again shelve their patients for future therapies . some providers may seek preapproval from payers for off - label use of smv in combination with sof , which , as discussed above , has proven efficacious in a limited number of noncirrhotic , treatment - nave and treatment - experienced , hcv genotype 1a and 1b patients , for 12 weeks with or without rbv . smv may find most of its usefulness in the future as a component in off - label , all - oral regimens , but it certainly remains a therapeutic option with peg / rbv in those well - suited ( noncirrhotic , treatment - nave , il-28b cc , non sofosbuvir in combination with rbv has become the first all - oral , non - peg regimen and is the first - in - its - class ns5b polymerase inhibitor to be approved for chronic hcv . while the peg - free regimen is approved for hcv genotypes 2 and 3 , its current indication for the hcv genotype 1 population still includes a peg / rbv backbone , granted only for 12 weeks , and it brings overall svr rates from 60% to 90% . this provides a new standard of therapy and a new bar to be met for future therapies in hcv genotype 1 patients . in addition , as discussed above , numerous trials have shown that sof in combination with other daas , particularly daclatasvir and ledipasvir , with and without rbv , will be extremely effective , tolerable , and durable for chronic hcv , even among difficult - to - treat patients , with unfavorable pretreatment profiles . further , several trials , including those with daclatasvir and ledipasvir in combination with sof , may allow for the omission of rbv as a part of therapy . rbv - sparing regimens are another milestone in the future of chronic hcv therapy and will deliver yet another improvement in the safety and tolerability of treatment . safety and tolerability also becomes of the utmost importance when we contemplate therapy for our fragile pretransplant population , a cohort in whom the benefit of sof + rbv prior to transplant has shown promise in durable eradication of the virus in the posttransplant period . it is not entirely clear if the development of resistance in previously treated patients will play a role in clinical decision making , ie , whether or not there will be a role in resistance profiling prior to treatment initiation in those for whom prior treatment has failed , as it appears our previous markers ( treatment nave / experienced , fibrosis stage , il-28b status ) will become less indicative of response to therapy . although tremendous strides have been made in the advancement of hcv therapy , accessibility to care and identification of those requiring treatment precludes addressing the global burden of chronic hcv . as such , while the new era of chronic hcv therapy remains in its adolescence , the global implication of the evolution in therapy is one that could conceivably mean eradication of hepatitis c.

nursing workload is a major challenge of health systems and an important theme in many nursing studies . as a keyword of medical terms in mesh database , workload is defined as the volume of work an individual , a department , or other employed groups must do in a period of time . in the early 90s , nursing workload was defined as the total required nursing time , as a result of all works that should be done within a specified time period . in 1997 , needham announced that definition of nursing workload is simple for nurses and includes the volume of work produced by patients during the time . however , more precise assessment of this definition is much more complex . needham defined the direct patient care , indirect tasks , and those not concerned with patients as the required nursing time and nursing workload . this definition accepts that the conducted task is , in fact , a nurse 's responsibility , not merely the patient care . myny believes that this definition is comparable with the definition by bi and salvendy who thought workload is a function of environmental and organizational factors . moreover , several dimensions of nursing workload have been suggested including physical , cognitive , time pressure , emotional , quantitative , qualitative , and diversity dimensions . despite there being lack of a general definition about nursing workload , there is a general consensus in two cases : first , nursing workload is beyond what is done close to a patient and second , nursing workload is increasing . increased nursing workload is one of the main challenges of national and international nursing . in 1990 a study reported that nursing workload has considerably increased within 16 years in a ward . some experiences concerning increased nursing workload were reported in a study that reviewed the states of nurses in the usa , the uk , canada , scotland , and germany . a study in taiwan reported that nursing workload is twofold to sevenfold more than in the usa and other developed countries . is associated with extended consequences for nurse , patient , and organization . workload and time pressure high workload is an important dimension of nurses role ; such a stress can influence their mental and psychological health and increase the economical costs for a society . stress , job dissatisfaction and burnout , increased nursing turnover and professional employees withdrawal , impaired physical security , increased risk of desecration and nuisance to nurses , and also complaints of patients families have been reported as the major consequences of nursing workload . increased nosocomial infections , delayed analgesic administration , lack of patient education , increased hospitalization , delayed ventilator disconnection , increased iatrogenic complications , medication errors , and mortality are the important outcomes of increased nursing workload for a patient . moreover , lack of opportunity to think about required interactive manner with a certain patient and impaired nurse harmful outcomes of increased nursing workload have been studied in many researches ; in most of them , the general consensus about the role of nurse shortage and effects of imbalance in the number of patients nurses has been reviewed . carayon introduced four levels of unit , job , patient , and situation as measurement models of workload . at the unit level , the most common workload index is the patient nurse ratio . at the job level the main determinant of nursing workload at the patient level is the clinical situation of a patient . situation level explains the factors such as physical working environment , lack of appropriate and adequate provision of resources and facilities , diversity of family needs , and ineffective communications between members of a multidisciplinary team , which can all increase the situational workload . moreover , each of these levels alone has limitations and may not be able to explain workload sources in nurses . other studies in this field reviewed some individual factors such as nursing labor shortage and patient 's situation . findings of the above - mentioned studies indicated that there is no general theme of factors influencing nursing workload . myny reported that despite much interest shown on the impact of workers supplying level on the quality of care , there is less attention paid toward the factors influencing nursing workload . given the nursing shortage and complexity , better understanding of the factors influencing nursing workload is an important issue . identifying these factors might help the nursing manager to provide a suitable workload for the labor force and accordingly help them to manage many harmful consequences of increased workload . despite reports concerning heavy nursing workload in iran , no study has ever been done specifically on the factors influencing nursing workload in iran . according to the literature , naturalism paradigm and qualitative research methods see the reality based on the background facts , and accept multi - reality and numerous constructs of an event . thus , they are useful for lesser well - known study areas . therefore , this study was conducted with a qualitative approach to detect experiences iranian nurses experiences of factors influencing work load to increase . given the complexity of the concept for workload and also limitations of studies specifically addressing this issue , the study design selected was a qualitative approach through conventional content analysis method . according to elo and kyngs , qualitative content analysis is a reliable research method to create reproducible and reliable inferences which might help to get a rich and comprehensive description of the phenomenon under study . content analysis is extremely sensitive toward the content and can be applied to understand relationships and identify key processes . based on the report of polit et al . , in order to maintain the natural environment , the study was conducted where the phenomenon occurred , i.e. in general surgical , orthopedic , oncology , and intensive care unit ( icu ) wards of two university hospitals in tehran , iran . the participants were objectively selected from among clinical nurses and nursing managers . based on holloway and wheeler , the general inclusion criteria were knowledge of the phenomenon under study , willingness and ability to transfer experiences , and some more specific criteria included having at least 6 months of working experience . data saturation ended with 15 subjects including 14 women and 1 man , aged 2450 years , with bsc degree in nursing . they were 2 nursing supervisors , 1 matron , 1 staff , 10 clinical nurses , and 1 msc in biochemistry with bsc in nursing who had changed her course of study due to bad working condition and had rich experiences in this case . they also had from 8 months to 20 years of experience in various working shifts in bone marrow transplantation ( bmt ) , pediatric , neonatal , dialysis , neurology , internal , and emergency wards . data collection was carried out through conducting unstructured interviews from july 2009 to august 2010 . following completion of each interview and initial analysis , interviews were conducted again in case of ambiguity to detect more in- depth data . interviews took 1580 min , except one which took 2 h. general questions with open answers were used in the interviews , e.g. when did you feel the workload ? and then , in order to achieve more in - depth and richer data , exploratory questions were used , e.g. can you explain the situation with a real example so that i understand it ? , , the interviews were continued until unidentified data or new category was obtained , according to the study of strubert and speziale . according to elo and kyngs , for analysis in the preparation stage , the whole interview might be an appropriate background for deriving the units of meaning which were selected as the most appropriate units of analysis . each interview was read repeatedly to reach was read repeatedly for immersion in data . in the organizing stage , open encoding was conducted with a review of interviews and recording notes and some titles and subjects in their margin . grouping started after conducting a few interviews . by repeating the above - mentioned procedure for each new interview , some titles were added and complete categories emerged . comparing and merging subcategories were divided and groups with similar events were categorized as one class . each class or category was named with terms indicating its content . moreover , the reminder interpretations concerning the association between the concepts were recorded during the analysis . prolonged engagement in field and sufficient time to communicate and collect data helped to boost participants trust and interaction and to collect in - depth data . furthermore , maximum variance in the distribution of samples according to age , work experience , position , and so forth was used in this study . in order to make sure that the analyses accurately reflected experiences of the participants , member check was done within data analysis and collection , and necessary changes were applied in interpretations to enhance study credibility according to the comment of the subjects , if necessary . for providing dependability and confirmability , a part of crude data was audited by experts , including interviews and analyzed outputs ( primary codes and categories obtained ) . the study was started after obtaining permission from the ethics committee of university of tarbait modares and hospitals . in addition , sufficient explanations were given to the participants about the importance , objectives , and the study method , particularly the interview record , content process , confidentiality in all stages of the study , and mutual decision - making concerning the time and place of the interview . in addition , an introduction of the interviewer and the way that the participants could access the study findings were provided to the study subjects . based on the report of polit et al . , in order to maintain the natural environment , the study was conducted where the phenomenon occurred , i.e. in general surgical , orthopedic , oncology , and intensive care unit ( icu ) wards of two university hospitals in tehran , iran . the participants were objectively selected from among clinical nurses and nursing managers . based on holloway and wheeler , the general inclusion criteria were knowledge of the phenomenon under study , willingness and ability to transfer experiences , and some more specific criteria included having at least 6 months of working experience . data saturation ended with 15 subjects including 14 women and 1 man , aged 2450 years , with bsc degree in nursing . they were 2 nursing supervisors , 1 matron , 1 staff , 10 clinical nurses , and 1 msc in biochemistry with bsc in nursing who had changed her course of study due to bad working condition and had rich experiences in this case . they also had from 8 months to 20 years of experience in various working shifts in bone marrow transplantation ( bmt ) , pediatric , neonatal , dialysis , neurology , internal , and emergency wards . data collection was carried out through conducting unstructured interviews from july 2009 to august 2010 . following completion of each interview and initial analysis , interviews took 1580 min , except one which took 2 h. general questions with open answers were used in the interviews , e.g. when did you feel the workload ? and then , in order to achieve more in - depth and richer data , exploratory questions were used , e.g. can you explain the situation with a real example so that i understand it ? , , the interviews were continued until unidentified data or new category was obtained , according to the study of strubert and speziale . according to elo and kyngs , for analysis in the preparation stage , the whole interview might be an appropriate background for deriving the units of meaning which were selected as the most appropriate units of analysis . each interview was read repeatedly to reach was read repeatedly for immersion in data . in the organizing stage , open encoding was conducted with a review of interviews and recording notes and some titles and subjects in their margin . grouping started after conducting a few interviews . by repeating the above - mentioned procedure for each new interview , some titles were added and complete categories emerged . comparing and merging the categories related to a group reduced the primary categories . subcategories were divided and groups with similar events were categorized as one class . each class or category was named with terms indicating its content . moreover , the reminder interpretations concerning the association between the concepts were recorded during the analysis . prolonged engagement in field and sufficient time to communicate and collect data helped to boost participants trust and interaction and to collect in - depth data . furthermore , maximum variance in the distribution of samples according to age , work experience , position , and so forth was used in this study . in order to make sure that the analyses accurately reflected experiences of the participants , member check was done within data analysis and collection , and necessary changes were applied in interpretations to enhance study credibility according to the comment of the subjects , if necessary . for providing dependability and confirmability , a part of crude data was audited by experts , including interviews and analyzed outputs ( primary codes and categories obtained ) . the study was started after obtaining permission from the ethics committee of university of tarbait modares and hospitals . in addition , sufficient explanations were given to the participants about the importance , objectives , and the study method , particularly the interview record , content process , confidentiality in all stages of the study , and mutual decision - making concerning the time and place of the interview . in addition , an introduction of the interviewer and the way that the participants could access the study findings were provided to the study subjects . following data analysis , imbalanced facilities and tasks appeared as the main theme which indicated factors influencing increase in nursing workload . imbalanced facilities and tasks means disproportion between the necessary elements for responding to the patients needs with defined expectations and responsibilities for nurses . imbalanced facilities and tasks had four dimensions : insufficient resources , assignment with no preparation , assigning non - medical and non - care tasks , and needs / expectations of patients and families , which caused increased nursing workload based on the experiences and perceptions of participants . these elements were diverse and divided into two categories : worker shortage , i.e. disproportion between the number of nurses and services labor compared to the number of patients and their expectations and needs . this was a chronic issue and concern , but was experienced acutely in some circumstances such as weekends and during replacement of absent nurses . worker shortage not only increased the number of working shifts and hours illegally to a large extent , but also increased the number of patients cared by a nurse during a shift and caused increased nursing workload . 1 said : the work pressure is pretty high now and nurses are not that much ; this ward used to be run with four to five nurses , now we are only two . a clerk was added to us after a lot of insistence ; otherwise we used to do the office affairs by ourselves . shortage of facilities and equipments was one of the main experiences of nurses , including many sources such as financial aid and the necessary equipments for advanced care . lack of equipment per se could directly increase the workload , and also , waste time , slow work , and increase the complexity of the situation . for instance , as a consequence of shortage of beds , icu patients were hospitalized more in general wards , which increased the workload and working pressure . this merely was not due to increased workload caused by the nature of disease and the type of required care for a special patient , but was due to the fact that care for intensive patients in general wards took more energy and time than care for patients in the icu owing to lack of adequate facilities and equipment . 4 said : workload is extremely high where you get shortage of equipment . there is only one laryngoscope for cardiopulmonary resuscitation ( cpr ) ; at the same time , you have to go for another cpr in another ward quickly . when a resident needs gloves and we do nt have it , we must go and take a pair from the other ward . these all are tasks , means time , the simplest case is bed . when icu has no bed , intensive patient stays out with a ventilator . devices raise alarm , then nurse must go and check the device every ten minutes and pass this long corridor to check the patient and come back . it makes a difference when a nurse can directly observe patients in icu ; the path should not be that long ; then , it takes much more time to do the job . worker shortage , i.e. disproportion between the number of nurses and services labor compared to the number of patients and their expectations and needs . this was a chronic issue and concern , but was experienced acutely in some circumstances such as weekends and during replacement of absent nurses . worker shortage not only increased the number of working shifts and hours illegally to a large extent , but also increased the number of patients cared by a nurse during a shift and caused increased nursing workload . 1 said : the work pressure is pretty high now and nurses are not that much ; this ward used to be run with four to five nurses , now we are only two . a clerk was added to us after a lot of insistence ; otherwise we used to do the office affairs by ourselves . the work pressure is pretty high now and nurses are not that much ; this ward used to be run with four to five nurses , now we are only two . a clerk was added to us after a lot of insistence ; otherwise we used to do the office affairs by ourselves . shortage of facilities and equipments was one of the main experiences of nurses , including many sources such as financial aid and the necessary equipments for advanced care . lack of equipment per se could directly increase the workload , and also , waste time , slow work , and increase the complexity of the situation . for instance , as a consequence of shortage of beds , icu patients were hospitalized more in general wards , which increased the workload and working pressure . this merely was not due to increased workload caused by the nature of disease and the type of required care for a special patient , but was due to the fact that care for intensive patients in general wards took more energy and time than care for patients in the icu owing to lack of adequate facilities and equipment . 4 said : workload is extremely high where you get shortage of equipment . there is only one laryngoscope for cardiopulmonary resuscitation ( cpr ) ; at the same time , you have to go for another cpr in another ward quickly . when a resident needs gloves and we do nt have it , we must go and take a pair from the other ward . these all are tasks , means time , the simplest case is bed . when icu has no bed , intensive patient stays out with a ventilator . devices raise alarm , then nurse must go and check the device every ten minutes and pass this long corridor to check the patient and come back . it makes a difference when a nurse can directly observe patients in icu ; the path should not be that long ; then , it takes much more time to do the job . there is only one laryngoscope for cardiopulmonary resuscitation ( cpr ) ; at the same time , you have to go for another cpr in another ward quickly . when a resident needs gloves and we do nt have it , we must go and take a pair from the other ward . these all are tasks , means time , the simplest case is bed . when icu has no bed , intensive patient stays out with a ventilator . devices raise alarm , then nurse must go and check the device every ten minutes and pass this long corridor to check the patient and come back . it makes a difference when a nurse can directly observe patients in icu ; the path should not be that long ; then , it takes much more time to do the job . experiences of nurses showed that one of the main sources for workload was lack of professional preparedness and assigning responsibility without the required scientific competencies for playing the role and tasks . the experiences of nurses showed that deficiency in educating in terms of necessary scientific and professional competencies start from the university level , and not only has not been compensated during their employment , but also has continued and also intensified due to lack of adequate in - service training . eliminating novice nursing orientation courses , lack of financial support from the training department , and inappropriate quality and insufficient development and in - service training programs were the most important experiences of the participants . an educational supervisor ( no . 15 ) sated that in - service training and orientation programs used to be in such a way that nurses had a relative familiarity with icu in addition to the information related to their own particular ward ; however , nowadays nursing training programs are not enough and their programs have been eliminated . 15 said : when a new graduated nurse came here , he / she went for a nursing orientation program for two weeks in icu and also two weeks in their own ward . but now it is a human force shortage , and it is impossible to do it again . when a new graduated nurse came here , he / she went for a nursing orientation program for two weeks in icu and also two weeks in their own ward . but now it is a human force shortage , and it is impossible to do it again . the experiences of nurses showed that the outcome of deficiency in teaching professional competencies was the deficiency of knowledge and inability to take up the assigned responsibilities . lack of necessary knowledge and competency for taking responsibilities caused nurses to try various ways to do a task . 14 ) said : when you do the venipuncture for adults , you can see the veins by your eyes , but for infants it should be done by anatomical science . experience is essential , but in - service training is more important . where have we learned to do the venipuncture for infants ? when there is no training , you need to try different things over and over and spend a lot of time and energy . no one taught us about infant 's nasogastric tube ( ngt ) ; we tried it many times with the same adult method ; well , it did not work and it increased our task . when you do the venipuncture for adults , you can see the veins by your eyes , but for infants it should be done by anatomical science . experience is essential , but in - service training is more important . where have we learned to do the venipuncture for infants ? do you know how much energy and time was taken by us ? when there is no training , you need to try different things over and over and spend a lot of time and energy . no one taught us about infant 's nasogastric tube ( ngt ) ; we tried it many times with the same adult method ; well , it did not work and it increased our task . in view of some of the participants , assignment with no preparation had similar effects to insufficient resources ; assignment with no preparation was a type of qualitative shortage of workers that increased the workload of other nurses . an experienced nurse ( no . 5 ) stated the following about lack of competency in some novice nurses and tiredness caused by the workload : one new nurse has recently been added and no one knows how she works ! patient is suffering from phlebitis ; from night to morning , she does washing the line over and over with this phlebitis . i have pain; finally , the head nurse must go and see what has happened . many times i have worked here for afternoon and night shifts and i can not stand anymore with such illiterate naive new nurses ! patient is suffering from phlebitis ; from night to morning , she does washing the line over and over with this phlebitis . i have pain; finally , the head nurse must go and see what has happened . many times i have worked here for afternoon and night shifts and i can not stand anymore with such illiterate naive new nurses ! this was an important source of workload which was confirmed by all the participants . nurses believed that assigning non - medical and non - care tasks was a virtual and unprofessional job descri ption for imposing unnecessary and waste duties . this has been experienced in two forms . changing the nature of health care to competitor of care : one of the important sources for increased workload was overemphasis of managers on indirect cares such as frequent report writing , so that such measures became a competitor of care with increased nursing workload and by wasting time and energy of nurses , caused them lose the care objectives . 6 ) described his / her experience as the following : we have to generally write a lot . when you ve got 14 patients , you must write 14 reports rather than observe whether the patient received the correct medication . once something should be purchased out of hospital , then , that patient would lose that medicine . if i do nt have to write , i would find a physician and tell him to prescribe the medicine in their medical sheet ; our biggest issue is lack of time . many times , every thing is missed for patients . assigning other irrelevant tasks : nurses also experienced increased workload due to other irrelevant tasks assigned to them . this type of task was diverse and included performing unprofessional affairs by an expert and professional nurse . assigning other irrelevant tasks was also a chronic concern ; however , it had been experienced severely when administrative clerks and staff were off and their jobs were imposed on the nurses . such an irrelevant job description intensively increased nurses workload . 2 ) stated about imposing irrelevant administrative affairs of secretaries and administrative staff and increased workload : fridays ( weekends ) we might have discharge too , but there is no clerk . changing the nature of health care to competitor of care : one of the important sources for increased workload was overemphasis of managers on indirect cares such as frequent report writing , so that such measures became a competitor of care with increased nursing workload and by wasting time and energy of nurses , caused them lose the care objectives . 6 ) described his / her experience as the following : we have to generally write a lot . when you ve got 14 patients , you must write 14 reports rather than observe whether the patient received the correct medication . once something should be purchased out of hospital , then , that patient would lose that medicine . if i do nt have to write , i would find a physician and tell him to prescribe the medicine in their medical sheet ; our biggest issue is lack of time . when you ve got 14 patients , you must write 14 reports rather than observe whether the patient received the correct medication . once something should be purchased out of hospital , then , that patient would lose that medicine . if i do nt have to write , i would find a physician and tell him to prescribe the medicine in their medical sheet ; our biggest issue is lack of time . assigning other irrelevant tasks : nurses also experienced increased workload due to other irrelevant tasks assigned to them . this type of task was diverse and included performing unprofessional affairs by an expert and professional nurse . assigning other irrelevant tasks was also a chronic concern ; however , it had been experienced severely when administrative clerks and staff were off and their jobs were imposed on the nurses . such an irrelevant job description intensively increased nurses workload . 2 ) stated about imposing irrelevant administrative affairs of secretaries and administrative staff and increased workload : fridays ( weekends ) we might have discharge too , but there is no clerk . fridays ( weekends ) we might have discharge too , but there is no clerk . the experience of nurses showed the real needs of patients and families , particularly the need for the presence of a nurse and providing direct care for treatment and supporting their families , as one of the main sources of workload . findings showed that the more complicated the patient condition was , the higher was the workload . an experienced nurse in bmt ward pointed out that the patients admitted in these wards are very weak and vulnerable and are not able to have self - care ; therefore , they need an accurate care results in high workload for nurses . this participant ( no . 5 ) said : suppose you were working nonstop at morning ; you had to wash the line repeatedly , blood samples had to be taken every day . some patients did not have medicine , besides you had to wait for them to go to bathroom and take a shower , and clean their bed by yourself ( such patients were not able to do anything ) . suppose you were working nonstop at morning ; you had to wash the line repeatedly , blood samples had to be taken every day . some patients did not have medicine , besides you had to wait for them to go to bathroom and take a shower , and clean their bed by yourself ( such patients were not able to do anything ) . 14 ) stated : a patient who was not able to see was admitted due to exploded firecracker in his eyes in new year celebrations . he insisted to open his eyes ; his hands were tied not to open his bandages . there is itching , i have pain. his brothers and sisters also came by visiting me and asked repeated questions : why did you tie his hands? how much we must ask them not to cry near him and do not tell him that has been blinded ; you know these tasks are beyond normal expectations ; it takes a lot of energy . a patient who was not able to see was admitted due to exploded firecracker in his eyes in new year celebrations . he insisted to open his eyes ; his hands were tied not to open his bandages . there is itching , i have pain. his brothers and sisters also came by visiting me and asked repeated questions : why did you tie his hands? how much we must ask them not to cry near him and do not tell him that has been blinded ; you know these tasks are beyond normal expectations ; it takes a lot of energy . in their experiences , in addition to urgent and real needs , the unrealistic expectations also intensified the imbalance between workload and time and increased work pressure of nurses . 5 stated : vip patients expect to do everything in the best way , they expect to disconnect their serum when finished ; sometimes it takes some time to reach them from another room when they page you . perhaps , there is something more urgent and should do it with the first priority , but they expect to do their job as soon as they call you , whether the patient or their relatives . vip patients expect to do everything in the best way , they expect to disconnect their serum when finished ; sometimes it takes some time to reach them from another room when they page you . perhaps , there is something more urgent and should do it with the first priority , but they expect to do their job as soon as they call you , whether the patient or their relatives . these elements were diverse and divided into two categories : worker shortage , i.e. disproportion between the number of nurses and services labor compared to the number of patients and their expectations and needs . this was a chronic issue and concern , but was experienced acutely in some circumstances such as weekends and during replacement of absent nurses . worker shortage not only increased the number of working shifts and hours illegally to a large extent , but also increased the number of patients cared by a nurse during a shift and caused increased nursing workload . 1 said : the work pressure is pretty high now and nurses are not that much ; this ward used to be run with four to five nurses , now we are only two . a clerk was added to us after a lot of insistence ; otherwise we used to do the office affairs by ourselves . shortage of facilities and equipments was one of the main experiences of nurses , including many sources such as financial aid and the necessary equipments for advanced care . lack of equipment per se could directly increase the workload , and also , waste time , slow work , and increase the complexity of the situation . for instance , as a consequence of shortage of beds , icu patients were hospitalized more in general wards , which increased the workload and working pressure . this merely was not due to increased workload caused by the nature of disease and the type of required care for a special patient , but was due to the fact that care for intensive patients in general wards took more energy and time than care for patients in the icu owing to lack of adequate facilities and equipment . 4 said : workload is extremely high where you get shortage of equipment . there is only one laryngoscope for cardiopulmonary resuscitation ( cpr ) ; at the same time , you have to go for another cpr in another ward quickly . when a resident needs gloves and we do nt have it , we must go and take a pair from the other ward . devices raise alarm , then nurse must go and check the device every ten minutes and pass this long corridor to check the patient and come back . it makes a difference when a nurse can directly observe patients in icu ; the path should not be that long ; then , it takes much more time to do the job . worker shortage , i.e. disproportion between the number of nurses and services labor compared to the number of patients and their expectations and needs . this was a chronic issue and concern , but was experienced acutely in some circumstances such as weekends and during replacement of absent nurses . worker shortage not only increased the number of working shifts and hours illegally to a large extent , but also increased the number of patients cared by a nurse during a shift and caused increased nursing workload . 1 said : the work pressure is pretty high now and nurses are not that much ; this ward used to be run with four to five nurses , now we are only two . a clerk was added to us after a lot of insistence ; otherwise we used to do the office affairs by ourselves . the work pressure is pretty high now and nurses are not that much ; this ward used to be run with four to five nurses , now we are only two . a clerk was added to us after a lot of insistence ; otherwise we used to do the office affairs by ourselves . shortage of facilities and equipments was one of the main experiences of nurses , including many sources such as financial aid and the necessary equipments for advanced care . lack of equipment per se could directly increase the workload , and also , waste time , slow work , and increase the complexity of the situation . for instance , as a consequence of shortage of beds , icu patients were hospitalized more in general wards , which increased the workload and working pressure . this merely was not due to increased workload caused by the nature of disease and the type of required care for a special patient , but was due to the fact that care for intensive patients in general wards took more energy and time than care for patients in the icu owing to lack of adequate facilities and equipment . 4 said : workload is extremely high where you get shortage of equipment . there is only one laryngoscope for cardiopulmonary resuscitation ( cpr ) ; at the same time , you have to go for another cpr in another ward quickly . when a resident needs gloves and we do nt have it , we must go and take a pair from the other ward . these all are tasks , means time , the simplest case is bed . when icu has no bed , intensive patient stays out with a ventilator . devices raise alarm , then nurse must go and check the device every ten minutes and pass this long corridor to check the patient and come back . it makes a difference when a nurse can directly observe patients in icu ; the path should not be that long ; then , it takes much more time to do the job . there is only one laryngoscope for cardiopulmonary resuscitation ( cpr ) ; at the same time , you have to go for another cpr in another ward quickly . when a resident needs gloves and we do nt have it , we must go and take a pair from the other ward . these all are tasks , means time , the simplest case is bed . when icu has no bed , intensive patient stays out with a ventilator . devices raise alarm , then nurse must go and check the device every ten minutes and pass this long corridor to check the patient and come back . it makes a difference when a nurse can directly observe patients in icu ; the path should not be that long ; then , it takes much more time to do the job . experiences of nurses showed that one of the main sources for workload was lack of professional preparedness and assigning responsibility without the required scientific competencies for playing the role and tasks . the experiences of nurses showed that deficiency in educating in terms of necessary scientific and professional competencies start from the university level , and not only has not been compensated during their employment , but also has continued and also intensified due to lack of adequate in - service training . eliminating novice nursing orientation courses , lack of financial support from the training department , and inappropriate quality and insufficient development and in - service training programs were the most important experiences of the participants . an educational supervisor ( no . 15 ) sated that in - service training and orientation programs used to be in such a way that nurses had a relative familiarity with icu in addition to the information related to their own particular ward ; however , nowadays nursing training programs are not enough and their programs have been eliminated . 15 said : when a new graduated nurse came here , he / she went for a nursing orientation program for two weeks in icu and also two weeks in their own ward . but now it is a human force shortage , and it is impossible to do it again . when a new graduated nurse came here , he / she went for a nursing orientation program for two weeks in icu and also two weeks in their own ward . but now it is a human force shortage , and it is impossible to do it again . the experiences of nurses showed that the outcome of deficiency in teaching professional competencies was the deficiency of knowledge and inability to take up the assigned responsibilities . lack of necessary knowledge and competency for taking responsibilities caused nurses to try various ways to do a task . 14 ) said : when you do the venipuncture for adults , you can see the veins by your eyes , but for infants it should be done by anatomical science experience is essential , but in - service training is more important . where have we learned to do the venipuncture for infants ? do you know how much energy and time was taken by us ? when there is no training , you need to try different things over and over and spend a lot of time and energy . no one taught us about infant 's nasogastric tube ( ngt ) ; we tried it many times with the same adult method ; well , it did not work and it increased our task . when you do the venipuncture for adults , you can see the veins by your eyes , but for infants it should be done by anatomical science . experience is essential , but in - service training is more important . where have we learned to do the venipuncture for infants ? when there is no training , you need to try different things over and over and spend a lot of time and energy . no one taught us about infant 's nasogastric tube ( ngt ) ; we tried it many times with the same adult method ; well , it did not work and it increased our task . in view of some of the participants , assignment with no preparation had similar effects to insufficient resources ; assignment with no preparation was a type of qualitative shortage of workers that increased the workload of other nurses . an experienced nurse ( no . 5 ) stated the following about lack of competency in some novice nurses and tiredness caused by the workload : one new nurse has recently been added and no one knows how she works ! patient is suffering from phlebitis ; from night to morning , she does washing the line over and over with this phlebitis . i have pain; finally , the head nurse must go and see what has happened . many times i have worked here for afternoon and night shifts and i can not stand anymore with such illiterate naive new nurses ! patient is suffering from phlebitis ; from night to morning , she does washing the line over and over with this phlebitis . i have pain; finally , the head nurse must go and see what has happened . many times i have worked here for afternoon and night shifts and i can not stand anymore with such illiterate naive new nurses ! nurses believed that assigning non - medical and non - care tasks was a virtual and unprofessional job descri ption for imposing unnecessary and waste duties . this has been experienced in two forms . changing the nature of health care to competitor of care : one of the important sources for increased workload was overemphasis of managers on indirect cares such as frequent report writing , so that such measures became a competitor of care with increased nursing workload and by wasting time and energy of nurses , caused them lose the care objectives . 6 ) described his / her experience as the following : we have to generally write a lot . when you ve got 14 patients , you must write 14 reports rather than observe whether the patient received the correct medication . once something should be purchased out of hospital , then , that patient would lose that medicine . if i do nt have to write , i would find a physician and tell him to prescribe the medicine in their medical sheet ; our biggest issue is lack of time . many times , every thing is missed for patients . assigning other irrelevant tasks : nurses also experienced increased workload due to other irrelevant tasks assigned to them . this type of task was diverse and included performing unprofessional affairs by an expert and professional nurse . assigning other irrelevant tasks was also a chronic concern ; however , it had been experienced severely when administrative clerks and staff were off and their jobs were imposed on the nurses . such an irrelevant job description intensively increased nurses workload . 2 ) stated about imposing irrelevant administrative affairs of secretaries and administrative staff and increased workload : fridays ( weekends ) we might have discharge too , but there is no clerk . changing the nature of health care to competitor of care : one of the important sources for increased workload was overemphasis of managers on indirect cares such as frequent report writing , so that such measures became a competitor of care with increased nursing workload and by wasting time and energy of nurses , caused them lose the care objectives . 6 ) described his / her experience as the following : we have to generally write a lot . when you ve got 14 patients , you must write 14 reports rather than observe whether the patient received the correct medication . once something should be purchased out of hospital , then , that patient would lose that medicine . if i do nt have to write , i would find a physician and tell him to prescribe the medicine in their medical sheet ; our biggest issue is lack of time . when you ve got 14 patients , you must write 14 reports rather than observe whether the patient received the correct medication . once something should be purchased out of hospital , then , that patient would lose that medicine . if i do nt have to write , i would find a physician and tell him to prescribe the medicine in their medical sheet ; our biggest issue is lack of time . assigning other irrelevant tasks : nurses also experienced increased workload due to other irrelevant tasks assigned to them . this type of task was diverse and included performing unprofessional affairs by an expert and professional nurse . assigning other irrelevant tasks was also a chronic concern ; however , it had been experienced severely when administrative clerks and staff were off and their jobs were imposed on the nurses . 2 ) stated about imposing irrelevant administrative affairs of secretaries and administrative staff and increased workload : fridays ( weekends ) we might have discharge too , but there is no clerk . fridays ( weekends ) we might have discharge too , but there is no clerk . the experience of nurses showed the real needs of patients and families , particularly the need for the presence of a nurse and providing direct care for treatment and supporting their families , as one of the main sources of workload . findings showed that the more complicated the patient condition was , the higher was the workload . an experienced nurse in bmt ward pointed out that the patients admitted in these wards are very weak and vulnerable and are not able to have self - care ; therefore , they need an accurate care results in high workload for nurses . this participant ( no . 5 ) said : suppose you were working nonstop at morning ; you had to wash the line repeatedly , blood samples had to be taken every day . some patients did not have medicine , besides you had to wait for them to go to bathroom and take a shower , and clean their bed by yourself ( such patients were not able to do anything ) . suppose you were working nonstop at morning ; you had to wash the line repeatedly , blood samples had to be taken every day . some patients did not have medicine , besides you had to wait for them to go to bathroom and take a shower , and clean their bed by yourself ( such patients were not able to do anything ) . 14 ) stated : a patient who was not able to see was admitted due to exploded firecracker in his eyes in new year celebrations . he insisted to open his eyes ; his hands were tied not to open his bandages . there is itching , i have pain. his brothers and sisters also came by visiting me and asked repeated questions : why did you tie his hands? how much we must ask them not to cry near him and do not tell him that has been blinded ; you know these tasks are beyond normal expectations ; it takes a lot of energy . a patient who was not able to see was admitted due to exploded firecracker in his eyes in new year celebrations . he insisted to open his eyes ; his hands were tied not to open his bandages . there is itching , i have pain. his brothers and sisters also came by visiting me and asked repeated questions : why did you tie his hands? how much we must ask them not to cry near him and do not tell him that has been blinded ; you know these tasks are beyond normal expectations ; it takes a lot of energy . in their experiences , in addition to urgent and real needs , the unrealistic expectations also intensified the imbalance between workload and time and increased work pressure of nurses . 5 stated : vip patients expect to do everything in the best way , they expect to disconnect their serum when finished ; sometimes it takes some time to reach them from another room when they page you . perhaps , there is something more urgent and should do it with the first priority , but they expect to do their job as soon as they call you , whether the patient or their relatives . vip patients expect to do everything in the best way , they expect to disconnect their serum when finished ; sometimes it takes some time to reach them from another room when they page you . perhaps , there is something more urgent and should do it with the first priority , but they expect to do their job as soon as they call you , whether the patient or their relatives . the findings of the study showed that imbalanced facilities and tasks was the main theme of the nurses experiences on the factors influencing increase in nursing workload . imbalanced facilities and tasks indicates the inappropriateness between what nurses have and what others expect them to do , and also indicates lack or deficiency of elements such as workers , financial resources , facilities and equipment , knowledge , and required competency for implementing tasks . the other side of this imbalance is encountering the expectations and demands of the system , patients , and families . in addition to being in a situation with shortcomings and limitations , nurses have to do other irrelevant and non - care tasks , and fulfill the needs and expectations of patients and families . insufficiency of working resources and the high - demand tasks have already been reported as concerns of nurses . in the present study , insufficient resources indicate shortage of workers , facilities , and equipments for care and nursing duties . shortage of professional and non - professional nurses and its impact on increased workload is one of the main themes in many studies . shortage of nurse is a worldwide concern , causes limitation in worker supply and increased nursing workload , and has negative impacts on health care . in a study in iran , nurses faced with low worker supply and shortage of nonprofessional labor , which led to high workload , forced labor hours , time limitation , and excessive tiredness . changes in demographic population and increased demand due to aging and elderly population , demographic changes in nurses workforce , nursing workforce profile , valued experience , and quality of nursing staff which can transfer them to other industries have major role to play in shortage of nurses . stressful , insecure , and unacceptable working places have negative impacts on the nursing workforce . other factors influencing nursing workers shortage are organizational structure and inappropriate management practices and strategies for manpower supply , decreased manpower supply due to the pressure of costs and accordingly implementing forced overtime which increases the workload and decrease nurses control on their program and can influence the decision on whether to stay or withdraw from this profession . in the present study , shortage of financial resources , facilities , and equipment was one of the other dimensions of insufficiency of resources . current era is called the era of cost - limit policy , which means highly extended limitations . inadequate logistics , such as lack of medical equipment and computer and beds in icus , is an important factor that increases nursing workload . other studies reported that physical working environment , inappropriate supply of resources and facilities , and factors related to the technology , tools , and equipments cause increased nursing workload . for instance , parallel with the technological advancement , the nursing working hours is increased for managing the advanced technology . this type of workload is one of the reasons for nurses withdrawing from the profession . assignment with no preparation that indicates shortage in education is one of the other resource task imbalances emphasizing on lack of competency . besides , findings indicated that assignment with no preparation not only increases workload of non - competent and unqualified nurses , but also increases the working pressure of other nurses . findings suggested that this issue has a direct association with insufficiency of academic and in - service education . unlike the findings of the present study , there was a satisfied level of competency in new nurses who graduated from public and governmental universities of jordan . a literature review reported that education as a nurse 's feature is one of the factors influencing the workload . furthermore , these studies showed that when experienced nurses take the responsibility of supporting and educating inexperienced nurses , they could experience workload . assigning non - medical tasks was one of the sources of nursing workload . in a study , regardless of workload , nurses were found to mostly spend their time on indirect care . also reported that nursing managers did not believe in job description ; therefore , nurses had to do some tasks they were not supposed to do and this increased their workload . in a study , nurses of five counties reported that they had to do so many irrelevant affairs . needham quoted from barratt ( 1994 ) and reported that as a profession , nurses need to be clear regarding their role and uniqueness of their knowledge and skills that a nurse is able to do . needham also stated that there are many ambiguities concerning the role of nurses both inside the system and in the society . a study regarding the duties of nurses showed that nurses do some affairs at different times , such as cleaner , nutritionist , carrier , office worker , clerk , receptionist , and physician , and it seems that all these professions lead to increased workload for nurses . patients and families expectations and needs was the other major source of workload . literatures indicate that acuity of patient includes the amount of nurse 's standard workload that is derived from the dependency of the patient and the amount of direct care that the patient receives . if a nurse spends more time due to patient 's dependency and the population of that ward comprises many dependent patients , then higher workload would be expected . according to the workload model , patient is an important determinant for the main nursing workload for clinical set . according to the local level model , diverse needs of families can increase the workload . moreover , reports illustrate that most of the time , patients expect more direct care than what is really needed ; therefore , it leads to patient nurse dissatisfaction . emerged patterns and structures in qualitative studies are context - dependent ; this restricts the applicability of the findings . however , the maximum variation in the sampling from different parts was one of the strengths of this study , which increases the application of the findings . emerged patterns and structures in qualitative studies are context - dependent ; this restricts the applicability of the findings . however , the maximum variation in the sampling from different parts was one of the strengths of this study , which increases the application of the findings . findings of this study show that there is a deep and widespread imbalance between resources and ability and competencies of nurses on one hand and tasks and expectations on the other . findings of this study indicate that educational and clinical managers must necessarily consider allocating resources and also adopt approaches for supplying resources and educating qualified nurses . furthermore , the findings of the study can be the basis for the development of tools to measure nursing workload . given the ambiguity of the workload concept despite multiple texts , further analysis of this concept is proposed .

background : while nursing workload is a worldwide challenge , less attention has been given to the determining factors . understanding these factors is important and could help nursing managers to provide suitable working environment and to manage the adverse outcomes of nursing workload . the aim of this study was to discover nurses experiences of determinant factors of their workload.materials and methods : in this qualitative study , the participants included 15 nurses working in two hospitals in tehran , iran . the data were collected through 26 unstructured interviews and were analyzed using conventional content analysis . the rigor has been guaranteed with prolonged engagement , maximum variance sampling , member check , and audit trail.results:resourcetask imbalance was the main theme of nurses experiences . it means that there was an imbalance between necessary elements to meet patients needs in comparison with expectation and responsibility . resource task imbalance included lack of resource , assignment without preparation , assigning non - care tasks , and patients and families needs / expectations.conclusions : a deep and comprehensive imbalance between recourses and tasks and expectations has been perceived by the participants to be the main source of work overload . paying more attention to resource allocation , education of quality workforce , and job description by managers is necessary .

I M Study design and participants Data collection Analysis The strength of the study Ethical considerations R Insufficient resources Assignment with no preparation Assigning non-medical and non-care tasks Patients and families needs/expectations D Limitations and strengths C

nursing workload is a major challenge of health systems and an important theme in many nursing studies . as a keyword of medical terms in mesh database , workload is defined as the volume of work an individual , a department , or other employed groups must do in a period of time . in 1997 , needham announced that definition of nursing workload is simple for nurses and includes the volume of work produced by patients during the time . needham defined the direct patient care , indirect tasks , and those not concerned with patients as the required nursing time and nursing workload . myny believes that this definition is comparable with the definition by bi and salvendy who thought workload is a function of environmental and organizational factors . moreover , several dimensions of nursing workload have been suggested including physical , cognitive , time pressure , emotional , quantitative , qualitative , and diversity dimensions . despite there being lack of a general definition about nursing workload , there is a general consensus in two cases : first , nursing workload is beyond what is done close to a patient and second , nursing workload is increasing . increased nursing workload is one of the main challenges of national and international nursing . some experiences concerning increased nursing workload were reported in a study that reviewed the states of nurses in the usa , the uk , canada , scotland , and germany . a study in taiwan reported that nursing workload is twofold to sevenfold more than in the usa and other developed countries . workload and time pressure high workload is an important dimension of nurses role ; such a stress can influence their mental and psychological health and increase the economical costs for a society . stress , job dissatisfaction and burnout , increased nursing turnover and professional employees withdrawal , impaired physical security , increased risk of desecration and nuisance to nurses , and also complaints of patients families have been reported as the major consequences of nursing workload . increased nosocomial infections , delayed analgesic administration , lack of patient education , increased hospitalization , delayed ventilator disconnection , increased iatrogenic complications , medication errors , and mortality are the important outcomes of increased nursing workload for a patient . moreover , lack of opportunity to think about required interactive manner with a certain patient and impaired nurse harmful outcomes of increased nursing workload have been studied in many researches ; in most of them , the general consensus about the role of nurse shortage and effects of imbalance in the number of patients nurses has been reviewed . at the job level the main determinant of nursing workload at the patient level is the clinical situation of a patient . situation level explains the factors such as physical working environment , lack of appropriate and adequate provision of resources and facilities , diversity of family needs , and ineffective communications between members of a multidisciplinary team , which can all increase the situational workload . findings of the above - mentioned studies indicated that there is no general theme of factors influencing nursing workload . myny reported that despite much interest shown on the impact of workers supplying level on the quality of care , there is less attention paid toward the factors influencing nursing workload . given the nursing shortage and complexity , better understanding of the factors influencing nursing workload is an important issue . identifying these factors might help the nursing manager to provide a suitable workload for the labor force and accordingly help them to manage many harmful consequences of increased workload . according to the literature , naturalism paradigm and qualitative research methods see the reality based on the background facts , and accept multi - reality and numerous constructs of an event . therefore , this study was conducted with a qualitative approach to detect experiences iranian nurses experiences of factors influencing work load to increase . given the complexity of the concept for workload and also limitations of studies specifically addressing this issue , the study design selected was a qualitative approach through conventional content analysis method . according to elo and kyngs , qualitative content analysis is a reliable research method to create reproducible and reliable inferences which might help to get a rich and comprehensive description of the phenomenon under study . , in order to maintain the natural environment , the study was conducted where the phenomenon occurred , i.e. in general surgical , orthopedic , oncology , and intensive care unit ( icu ) wards of two university hospitals in tehran , iran . based on holloway and wheeler , the general inclusion criteria were knowledge of the phenomenon under study , willingness and ability to transfer experiences , and some more specific criteria included having at least 6 months of working experience . they were 2 nursing supervisors , 1 matron , 1 staff , 10 clinical nurses , and 1 msc in biochemistry with bsc in nursing who had changed her course of study due to bad working condition and had rich experiences in this case . , , the interviews were continued until unidentified data or new category was obtained , according to the study of strubert and speziale . furthermore , maximum variance in the distribution of samples according to age , work experience , position , and so forth was used in this study . in order to make sure that the analyses accurately reflected experiences of the participants , member check was done within data analysis and collection , and necessary changes were applied in interpretations to enhance study credibility according to the comment of the subjects , if necessary . in addition , sufficient explanations were given to the participants about the importance , objectives , and the study method , particularly the interview record , content process , confidentiality in all stages of the study , and mutual decision - making concerning the time and place of the interview . in addition , an introduction of the interviewer and the way that the participants could access the study findings were provided to the study subjects . in general surgical , orthopedic , oncology , and intensive care unit ( icu ) wards of two university hospitals in tehran , iran . the participants were objectively selected from among clinical nurses and nursing managers . based on holloway and wheeler , the general inclusion criteria were knowledge of the phenomenon under study , willingness and ability to transfer experiences , and some more specific criteria included having at least 6 months of working experience . they were 2 nursing supervisors , 1 matron , 1 staff , 10 clinical nurses , and 1 msc in biochemistry with bsc in nursing who had changed her course of study due to bad working condition and had rich experiences in this case . , , the interviews were continued until unidentified data or new category was obtained , according to the study of strubert and speziale . prolonged engagement in field and sufficient time to communicate and collect data helped to boost participants trust and interaction and to collect in - depth data . furthermore , maximum variance in the distribution of samples according to age , work experience , position , and so forth was used in this study . in order to make sure that the analyses accurately reflected experiences of the participants , member check was done within data analysis and collection , and necessary changes were applied in interpretations to enhance study credibility according to the comment of the subjects , if necessary . in addition , sufficient explanations were given to the participants about the importance , objectives , and the study method , particularly the interview record , content process , confidentiality in all stages of the study , and mutual decision - making concerning the time and place of the interview . in addition , an introduction of the interviewer and the way that the participants could access the study findings were provided to the study subjects . following data analysis , imbalanced facilities and tasks appeared as the main theme which indicated factors influencing increase in nursing workload . imbalanced facilities and tasks means disproportion between the necessary elements for responding to the patients needs with defined expectations and responsibilities for nurses . imbalanced facilities and tasks had four dimensions : insufficient resources , assignment with no preparation , assigning non - medical and non - care tasks , and needs / expectations of patients and families , which caused increased nursing workload based on the experiences and perceptions of participants . disproportion between the number of nurses and services labor compared to the number of patients and their expectations and needs . shortage of facilities and equipments was one of the main experiences of nurses , including many sources such as financial aid and the necessary equipments for advanced care . lack of equipment per se could directly increase the workload , and also , waste time , slow work , and increase the complexity of the situation . this merely was not due to increased workload caused by the nature of disease and the type of required care for a special patient , but was due to the fact that care for intensive patients in general wards took more energy and time than care for patients in the icu owing to lack of adequate facilities and equipment . these all are tasks , means time , the simplest case is bed . disproportion between the number of nurses and services labor compared to the number of patients and their expectations and needs . shortage of facilities and equipments was one of the main experiences of nurses , including many sources such as financial aid and the necessary equipments for advanced care . lack of equipment per se could directly increase the workload , and also , waste time , slow work , and increase the complexity of the situation . this merely was not due to increased workload caused by the nature of disease and the type of required care for a special patient , but was due to the fact that care for intensive patients in general wards took more energy and time than care for patients in the icu owing to lack of adequate facilities and equipment . these all are tasks , means time , the simplest case is bed . these all are tasks , means time , the simplest case is bed . experiences of nurses showed that one of the main sources for workload was lack of professional preparedness and assigning responsibility without the required scientific competencies for playing the role and tasks . the experiences of nurses showed that deficiency in educating in terms of necessary scientific and professional competencies start from the university level , and not only has not been compensated during their employment , but also has continued and also intensified due to lack of adequate in - service training . eliminating novice nursing orientation courses , lack of financial support from the training department , and inappropriate quality and insufficient development and in - service training programs were the most important experiences of the participants . 15 ) sated that in - service training and orientation programs used to be in such a way that nurses had a relative familiarity with icu in addition to the information related to their own particular ward ; however , nowadays nursing training programs are not enough and their programs have been eliminated . but now it is a human force shortage , and it is impossible to do it again . but now it is a human force shortage , and it is impossible to do it again . the experiences of nurses showed that the outcome of deficiency in teaching professional competencies was the deficiency of knowledge and inability to take up the assigned responsibilities . in view of some of the participants , assignment with no preparation had similar effects to insufficient resources ; assignment with no preparation was a type of qualitative shortage of workers that increased the workload of other nurses . 5 ) stated the following about lack of competency in some novice nurses and tiredness caused by the workload : one new nurse has recently been added and no one knows how she works ! this was an important source of workload which was confirmed by all the participants . nurses believed that assigning non - medical and non - care tasks was a virtual and unprofessional job descri ption for imposing unnecessary and waste duties . this has been experienced in two forms . changing the nature of health care to competitor of care : one of the important sources for increased workload was overemphasis of managers on indirect cares such as frequent report writing , so that such measures became a competitor of care with increased nursing workload and by wasting time and energy of nurses , caused them lose the care objectives . changing the nature of health care to competitor of care : one of the important sources for increased workload was overemphasis of managers on indirect cares such as frequent report writing , so that such measures became a competitor of care with increased nursing workload and by wasting time and energy of nurses , caused them lose the care objectives . the experience of nurses showed the real needs of patients and families , particularly the need for the presence of a nurse and providing direct care for treatment and supporting their families , as one of the main sources of workload . findings showed that the more complicated the patient condition was , the higher was the workload . in their experiences , in addition to urgent and real needs , the unrealistic expectations also intensified the imbalance between workload and time and increased work pressure of nurses . disproportion between the number of nurses and services labor compared to the number of patients and their expectations and needs . shortage of facilities and equipments was one of the main experiences of nurses , including many sources such as financial aid and the necessary equipments for advanced care . lack of equipment per se could directly increase the workload , and also , waste time , slow work , and increase the complexity of the situation . this merely was not due to increased workload caused by the nature of disease and the type of required care for a special patient , but was due to the fact that care for intensive patients in general wards took more energy and time than care for patients in the icu owing to lack of adequate facilities and equipment . disproportion between the number of nurses and services labor compared to the number of patients and their expectations and needs . shortage of facilities and equipments was one of the main experiences of nurses , including many sources such as financial aid and the necessary equipments for advanced care . lack of equipment per se could directly increase the workload , and also , waste time , slow work , and increase the complexity of the situation . this merely was not due to increased workload caused by the nature of disease and the type of required care for a special patient , but was due to the fact that care for intensive patients in general wards took more energy and time than care for patients in the icu owing to lack of adequate facilities and equipment . these all are tasks , means time , the simplest case is bed . experiences of nurses showed that one of the main sources for workload was lack of professional preparedness and assigning responsibility without the required scientific competencies for playing the role and tasks . the experiences of nurses showed that deficiency in educating in terms of necessary scientific and professional competencies start from the university level , and not only has not been compensated during their employment , but also has continued and also intensified due to lack of adequate in - service training . eliminating novice nursing orientation courses , lack of financial support from the training department , and inappropriate quality and insufficient development and in - service training programs were the most important experiences of the participants . 15 ) sated that in - service training and orientation programs used to be in such a way that nurses had a relative familiarity with icu in addition to the information related to their own particular ward ; however , nowadays nursing training programs are not enough and their programs have been eliminated . but now it is a human force shortage , and it is impossible to do it again . but now it is a human force shortage , and it is impossible to do it again . the experiences of nurses showed that the outcome of deficiency in teaching professional competencies was the deficiency of knowledge and inability to take up the assigned responsibilities . in view of some of the participants , assignment with no preparation had similar effects to insufficient resources ; assignment with no preparation was a type of qualitative shortage of workers that increased the workload of other nurses . 5 ) stated the following about lack of competency in some novice nurses and tiredness caused by the workload : one new nurse has recently been added and no one knows how she works ! nurses believed that assigning non - medical and non - care tasks was a virtual and unprofessional job descri ption for imposing unnecessary and waste duties . this has been experienced in two forms . changing the nature of health care to competitor of care : one of the important sources for increased workload was overemphasis of managers on indirect cares such as frequent report writing , so that such measures became a competitor of care with increased nursing workload and by wasting time and energy of nurses , caused them lose the care objectives . changing the nature of health care to competitor of care : one of the important sources for increased workload was overemphasis of managers on indirect cares such as frequent report writing , so that such measures became a competitor of care with increased nursing workload and by wasting time and energy of nurses , caused them lose the care objectives . the experience of nurses showed the real needs of patients and families , particularly the need for the presence of a nurse and providing direct care for treatment and supporting their families , as one of the main sources of workload . findings showed that the more complicated the patient condition was , the higher was the workload . in their experiences , in addition to urgent and real needs , the unrealistic expectations also intensified the imbalance between workload and time and increased work pressure of nurses . the findings of the study showed that imbalanced facilities and tasks was the main theme of the nurses experiences on the factors influencing increase in nursing workload . the other side of this imbalance is encountering the expectations and demands of the system , patients , and families . in addition to being in a situation with shortcomings and limitations , nurses have to do other irrelevant and non - care tasks , and fulfill the needs and expectations of patients and families . in the present study , insufficient resources indicate shortage of workers , facilities , and equipments for care and nursing duties . shortage of professional and non - professional nurses and its impact on increased workload is one of the main themes in many studies . shortage of nurse is a worldwide concern , causes limitation in worker supply and increased nursing workload , and has negative impacts on health care . changes in demographic population and increased demand due to aging and elderly population , demographic changes in nurses workforce , nursing workforce profile , valued experience , and quality of nursing staff which can transfer them to other industries have major role to play in shortage of nurses . in the present study , shortage of financial resources , facilities , and equipment was one of the other dimensions of insufficiency of resources . inadequate logistics , such as lack of medical equipment and computer and beds in icus , is an important factor that increases nursing workload . other studies reported that physical working environment , inappropriate supply of resources and facilities , and factors related to the technology , tools , and equipments cause increased nursing workload . assignment with no preparation that indicates shortage in education is one of the other resource task imbalances emphasizing on lack of competency . assigning non - medical tasks was one of the sources of nursing workload . also reported that nursing managers did not believe in job description ; therefore , nurses had to do some tasks they were not supposed to do and this increased their workload . needham quoted from barratt ( 1994 ) and reported that as a profession , nurses need to be clear regarding their role and uniqueness of their knowledge and skills that a nurse is able to do . needham also stated that there are many ambiguities concerning the role of nurses both inside the system and in the society . a study regarding the duties of nurses showed that nurses do some affairs at different times , such as cleaner , nutritionist , carrier , office worker , clerk , receptionist , and physician , and it seems that all these professions lead to increased workload for nurses . patients and families expectations and needs was the other major source of workload . according to the workload model , patient is an important determinant for the main nursing workload for clinical set . however , the maximum variation in the sampling from different parts was one of the strengths of this study , which increases the application of the findings . however , the maximum variation in the sampling from different parts was one of the strengths of this study , which increases the application of the findings . findings of this study show that there is a deep and widespread imbalance between resources and ability and competencies of nurses on one hand and tasks and expectations on the other . findings of this study indicate that educational and clinical managers must necessarily consider allocating resources and also adopt approaches for supplying resources and educating qualified nurses . furthermore , the findings of the study can be the basis for the development of tools to measure nursing workload .

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 et al . 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 et al . 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 et al . , 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 et al . 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 .

1. Introduction 2. Central Sympathetic Control and Its Role in Regulation of Metabolism 3. Metabolic Syndrome, Prediabetes, and Diabetes Risk 4. Evidence of Sympathetic Dysfunction in Metabolic Conditions 5. Sympathetic Overactivity as a Consequence of Metabolic Dysfunction 6. Sympathetic Overactivity as a Cause of Metabolic Dysfunction 7. Targeting Central Sympathetic Inhibition to Improve Metabolic Control 8. Summary

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 . , 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 . 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. 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 . 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 . 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 . , 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 . 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 . 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 mets is associated with a 5-fold increased risk for diabetes . 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 . 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 . 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 . 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 . there are several lines of evidence to suggest that insulin exerts sympathoexcitatory effects via actions in the central nervous system . 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 . 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 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 . 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 . 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 . 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 . several studies in human show a positive correlation between circulating plasma nonesterified fatty acids ( nefas ) , obesity , and insulin resistance . 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 . 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 . 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 . 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 . 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 . 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 . 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 ] . 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 . 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 . , 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 . 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 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 ] . 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 . 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 . 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 . 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 . 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 .

after birth , body surfaces transit from complete sterility to the densest microbial ecosystem known on earth . the lower intestine alone is home to nearly 100 trillion microbes , forming a highly complex microbiota with over 1,000 operational taxonomic units ( hooper et al . , 2012 ) . each individual harbors an idiosyncratic microbial consortia ( turnbaugh et al . , 2010 ) that is thought to be shaped by host immunity , environment , and diet ( de filippo et al . , 2010 ; elinav et al . , 2011 ; faith et al . , 2011 ; garrett et al . , 2007 ; muegge et al . , 2011 ) . a sophisticated microbial - host crosstalk shapes immune adaptation and bacterial communities , which underlies mutualism . intestinal microbes shape not only the neighboring intestinal epithelial cells ( cash et al . , 2006 ; chassin et al . , 2010 ) but also the majority of sterile body compartments at mucosal and systemic sites ( cahenzli et al . , 2013 ; ganal et al . , 2012 ; hooper et al . , 2012 ) the pervasive effect of commensal microbes is reflected by its contribution to health and disease , including inflammatory bowel disease , obesity , malnutrition , autoimmunity , and allergic asthma ( herbst et al . , 2011 ; markle et al . , 2013 ; olszak et al . , 2012 ; smith et al . , commensal microbial communities are thought to be a causal link between westernization and increasing immune disorders ( noverr and huffnagle , 2005 ; okada et al . , 2010 ) . over the past few decades , westernized countries have experienced drastic changes in dietary habits and sanitation , including water decontamination , food pasteurization , sterilization , and uninterrupted cold chain delivery , vaccination , and widespread antibiotic use . all these factors have contributed to decreased infectious diseases ( bach , 2002 ) . over this period of improved hygiene , autoimmune and atopic allergic disorders have increased in incidence ; an epidemiologic effect postulated to result from decreased acute infections during early childhood ( holt , 1998 ; martinez and holt , 1999 ; strachan , 1989 ) or from a shift in commensal microbial communities ( braun - fahrlnder et al . , 1999 ; noverr and huffnagle , 2005 ; okada et al . , 2010 ) . certainly , the western lifestyle shapes the composition of commensal bacterial consortia and their development over time ( de filippo et al . , 2010 ; koenig et al . , 2011 ; palmer et al . , 2007 ; walter and ley , 2011 ) . although some mechanistic links between microbiota - induced immune regulation have been experimentally elucidated ( markle et al . , 2013 ; olszak et al . , 2012 ) , most models depend on particular microbes triggering disease ( elinav et al . , 2011 ; ige antibodies play a central role in atopic allergic disease and immunity to parasites ( allen and maizels , 2011 ; gould and sutton , 2008 ; paul and zhu , 2010 ) . healthy individuals maintain serum ige concentrations at basal levels ( < 0.0001% of serum immunoglobulins ) ( sutton and gould , 1993 ) because of an immunoregulatory network that regulates isotype switch to ige . multiple immunodeficiencies , including wiskott - aldrich syndrome , omenn syndrome , or immunodysregulation polyendocrinopathy enteropathy x - linked syndrome , are correlated with elevated serum ige levels despite the absence of allergic reaction or parasite infection ( kotlarz et al . , 2013 ; mouse models of immunodeficiencies also phenocopy the high ige levels ( antn et al . , 2002 ; 2009 ) , and we have previously reported that cd4 , major histocompatibility complex class ii ( mhc ii ) , and athymic nude mice display high ige ( mccoy et al . , 2006 ) . it has been postulated that elevated serum ige in the absence of atopic allergic disease or parasitic infection is a biomarker for immunodeficiencies ( liston et al . , 2008 ) . abnormally high serum ige levels in germ - free mice have been reported previously ( herbst et al . , 2011 ; hill et al . this suggests that immunoregulatory signals stemming from the microbiota are required in order to maintain ige levels at basal levels even in genetically immunocompetent mice . we hypothesized that the proper induction of immune regulation requires adequate microbial exposure during early life . here , we show that only exposure to a diverse microbial community during early life is able to induce functional immune regulation that maintains ige at basal levels and decreases disease severity in a model of antigen - induced oral anaphylaxis . to investigate the role of the microbiota in the inhibition of ige production , we first confirmed the presence of high ige levels in a large cohort of germ - free c57bl/6 mice ( figure 1a ) . as expected , c57bl/6 mice born and raised with a specific pathogen - free ( spf ) microbiota maintained ige levels below the limit of detection ( figure 1a ) . high ige levels were also observed in germ - free balb / c mice ( mean 292 ng / ml ; range 152,393 ng / ml ; n = 19 ) , but not in germ - free swiss webster ( n = 33 ) or nmri ( n = 21 ) mice ( < 0.8 ng / ml ) , possibly because of compensatory mechanisms frequently associated with outbred strains . as reported previously ( hill et al . , 2012 ) , we confirmed that elevated ige levels correlated with an increased frequency of fscssccd19thy1.2 blood basophils ( lantz et al . , 1997 ; voehringer et al . , high ige levels did not reflect a general hyperimmunoglobulinemia , given that all isotypes except ige ( iga , igm , igg1 , igg2b , igg2c , and igg3 ) were reduced in germ - free and not spf mice ( figure 1c ) , supporting the view of an immature immune system in germ - free mice ( macpherson and harris , 2004 ) . from these initial observations , we hypothesized that a diversified microbiota is required in order to maintain serum ige levels at baseline . although culture - dependent and -independent quality control methods were employed rigorously in order to confirm the absence of microbes , helminths , or viruses , we still faced the remote possibility that an undetected organism might trigger ige induction . therefore , we either cohoused spf mice with undetectable serum ige levels with germ - free mice ( with high ige ) or left them with their spf littermates . serum ige levels remained below the limit of detection in both groups of spf mice , confirming that hygiene - mediated ige was not due to the presence of a horizontally transferrable causing agent ( figure 2a ) . ige levels in spf mice remained below the limit of detection at all ages ( figure 2b ) . in contrast , ige levels in germ - free mice became readily detectable at around day 30 of age and increased onward ( figure 2b ) . because the appearance of serum ige coincided with the transition from lactation to solid food , we addressed the possibility of whether allergens in the sterilized chow might provoke increased serum ige levels . spf mice fed the same autoclaved chow did not develop increased ige , and germ - free mice fed and raised on an antigen - free elemental diet also developed high ige levels ( figure 2b ) . therefore , and in agreement with the polyclonal and germline - encoded germ - free ige ( mccoy et al . , 2006 ) , a heightened allergic reaction to dietary antigens in the absence of intestinal microbes was not the cause of elevated ige . collectively , these observations indicated that , in the absence of commensal microbes , the immune regulatory pathways that normally maintain ige at baseline levels were disrupted . hyper - ige is generated in genetically immunocompetent germ- and antigen - free mice , indicating that class - switch recombination to ige reflects immune dysregulation in the absence of commensal microbiota . next , we determined which lymphoid tissues foster ige isotype switch in the absence of microbial exposure . isotype switch is a dna deletion - recombination process that exchanges the constant region exon of the immunoglobulin heavy chain with one of several sets of downstream exons , c in the case of isotype switch to ige . prior to productive class - switch recombination to c , transient transcription is initiated from an intronic ( i ) promoter ( i ) , resulting in the generation of sterile germ - line transcripts ( glts ) that do not encode for protein ( bacharier and geha , 2000 ; geha et al . , 2003 ) . therefore , the expression of glts is a hallmark of active isotype switch to ige . sterile glts were readily detected via quantitative pcr ( qpcr ) in splenic b cells stimulated with anti - cd40 and il-4 and b cells from the spleen and mesenteric lymph nodes ( mlns ) of mice infected with the helminth heligmosomoides polygyrus but not in splenic b cells isolated from naive spf mice ( figure 3a ) . then , we assessed the expression of glts in germ - free mice during neonatal and adult life . whereas low levels of glts were found in the spleen of a few of the germ - free animals , glts were readily detectable in the peyer s patches ( pps ) and mlns of all germ - free mice starting at day 35 , peaking at day 42 , and remaining detectable throughout adulthood ( figure 3b ) . the expression of glts was not detected in the bone marrow , peripheral lymph nodes , or peritoneal cavity , the latter suggesting that peritoneal b-1 cells did not contribute significantly to high ige levels . therefore , in the absence of microbial exposure , ige isotype switch is fostered at mucosal sites , where microbially triggered immune adaptations , such as iga isotype switch , normally take place . given that the efferent lymphatics within the pps drain into the mlns ( macpherson and smith , 2006 ) , we tested whether ige induction could be abrogated in mice lacking pps . after the inhibition of pp ontogeny via il-7r signaling blockade at embryonic stage e14.5 ( yoshida et al . , 1999 ) , germ - free ige induction was fully abrogated ( figure 3c ) . these results suggest that pps foster initial ige isotype switch of b cells , which then rapidly egress to the mlns . although classic ige isotype switch requires cognate cd4 t cell help coupled with il-4 signals ( finkelman et al . , 1988 ) , numerous in vitro and in vivo experiments demonstrate alternative pathways for ige isotype switch that can bypass some of these requirements . we have previously shown that cognate t cell help can be circumvented in immunodeficient cd4 , mhc ii , and athymic nude spf mice ( mccoy et al . , 2006 ) . however , this study addressed ige generation in genetically immunodeficient hosts harboring a diverse microbiota with the possibility that dysbiosis , as a consequence of immunodeficiency , was the cause for the increased ige levels . the administration of a cd4 t cell - depleting antibody to germ - free mice starting at 28 days of age totally abrogated the induction of ige ( figure 4a ) . ige levels were also maintained below the limit of detection in germ - free mice genetically deficient in t cells ( tcr ; figure 4b ) . these results indicate that ige generated in the absence of commensal microbes is dependent on the presence of cd4 t cells . cd4 t cells potentially provide help through cytokine production and/or mhc ii cognate interactions . we assessed the cytokine expression profile of cd4 t cells from the spleen , mlns , pps , and colon lamina propria ( clp ) in germ - free and spf mice . as expected , il10 expression was higher in the clp of spf than that of germ - free mice ( atarashi et al . il4 expression in germ - free , but not spf , mice was upregulated from day 36 of age onward , specifically in the mlns and pps . thus , the expression of il4 and glts shared identical kinetics and occurred within identical immune geography , suggesting that the prototypic ige - inducing cytokine il-4 is also involved in germ - free ige . il13 expression was similar between germ - free and spf mice with the exception of pps , where expression was higher in germ - free than spf mice ( figure 5a ) . administration of an il-4-neutralizing antibody to germ - free mice starting at 28 days of age led to a significant decrease in the concentration of serum ige ( figure 5b ) . failure to completely abrogate the development of detectable ige may be explained by incomplete in vivo neutralization of il-4 or compensation by other cytokines , such as il-13 . to further investigate this pathway , and because performing bone marrow chimeras remains a challenging task under germ - free conditions , we opted for a mixed adoptive transfer of facs - sorted naive b and cd4 t cells into germ - free rag-1 mice . homeostatic proliferation of t cells transferred into lymphopenic recipients is driven by both microbial and self - antigens . rapid homeostatic proliferation is microbially driven , but , given that slow homeostatic proliferation in lymphopenic hosts is mediated exclusively by low - affinity self - antigens , cells transferred into germ - free recipients proliferate relatively slowly ( kieper et al . , 2005 ) . cotransfer of wild - type ( wt ) b and t cells isolated from spf mice into germ - free rag-1 mice led to high ige in the majority of reconstituted mice ( figure 5c ) , indicating that it is the environment within the germ - free host rather than intrinsic defects in germ - free t and b cells that is supportive of hygiene - induced ige . cotransfer of il-4r-deficient b and wt t cells failed to induce ige in seven out of eight mice ( figure 5c ) , suggesting that il-4r - mediated signaling on b cells was crucial for ige isotype switch under germ - free conditions . in contrast , cotransfer of mhc ii - deficient b and wt t cells led to the induction of ige in germ - free rag-1 mice ( figure 5c ) , indicating that cognate interactions between b and t cells mediated by mhc ii and antigenic peptide and t cell receptor ( tcr ) interactions was not required for ige isotype switch under germ - free conditions . flow cytometric analysis confirmed the absence of mhc ii expression on the b cells isolated from rag-1 recipients that received wt t and mhc ii - deficient b cells , confirming there was no cross - contamination of mhc ii - expressing b cells from the wt t cell preparation ( figure s1a available online ) . these results suggest that cd4 t cell dependency may be limited to the production of cytokines , such as il-4 . given that mlns and pp cd4 t cells of germ - free mice express higher levels of il-4 and il-13 in comparison to spf mice ( figure 5a ) and that the proportion of mlns and colon cd4ifn- t cells was lower under germ - free than spf conditions ( figure 5d ) , it is plausible that a default th2 pathway could be favored in the absence of microbes ( zhu et al . thymic stromal lymphopoietin ( tslp ) has been shown to be crucial for the initiation of th2 responses and ige isotype switch in some helminth infections and atopic allergic diseases ( paul and zhu , 2010 ) . therefore , we investigated the role of tslp in hygiene - mediated ige in germ - free mice . the administration of a tslp - neutralizing antibody to germ - free mice starting at 28 days of age failed to prevent the induction of ige ( figure s1b ) . ige levels were also not abrogated in germ - free tslp receptor - deficient mice ( figure s1c ) . these data indicate that tslp and signaling through its receptor are not required for ige induction in the absence of commensal microbes . altogether , these observations suggest that excessive levels of ige in germ - free mice are dependent on cd4 t cells , il-4 , and possibly il-13 . furthermore , this implies that similar immune mediators drive high ige levels in germ - free c57bl/6 mice and immunodeficient spf c57bl/6 mice ( mccoy et al . , 2006 ) and supports the notion that high ige levels in the absence of microbes reflect immune dysregulation caused by the lack of microbial education of the immune system . we have previously shown that intestinal microbial colonization with a limited diversity microbiota leads to the induction of intestinal regulatory t ( treg ) cells and il-10 , which are required in order to limit the induction of proinflammatory th1 and th17 cells ( geuking et al . , 2011 ) . in an attempt to rescue the immune dysregulation and inhibit ige while preserving the germ - free status , we tested whether adoptive transfer of microbially - instructed treg cells or the administration of recombinant il-10 could inhibit the induction of ige in germ - free mice . total cd4 , cd4cd25 naive , or cd4cd25 treg cells were isolated from the spleen or clp of spf donor mice and adoptively transferred into 28-day - old germ - free recipients . although this protocol successfully limits th1 and th17 subset induction upon the colonization of treg - cell - deficient mice ( geuking et al . , 2011 ) , it was not sufficient to limit ige induction under germ - free conditions ( figure s2a ) . similarly , the administration of recombinant il-10 also failed to modulate ige titers even when repetitively administered for the duration of the experiment ( figure s2b ) . our interpretation that ige induction is a default pathway of mucosal immunity in the absence of commensal intestinal microbes predicts that ige levels should be normalized in mice that are born germ free and later colonized by commensal microbes ; the interesting question was whether there was a time window in which this could take place . cohousing of adult germ - free mice ( with high ige ) with spf mice ( with undetectable ige and a complex intestinal microbiota ) did not reduce serum ige levels over an 8-week period ( figure 6a ) . in contrast , exposure to an spf flora at the time of birth completely inhibited the induction of ige ( figure 6a ; day 0 ) . to further refine the window of time whereby spf colonization provides protection , germ - free pups were colonized at days 2 , 7 , 14 , 21 , 28 , and 35 of age . in contrast to colonization during adulthood , the colonization of pups until 1 week postweaning ( when serum ige levels were still low ) was able to fully protect them from the induction of elevated ige during adulthood ( figure 6a ) , indicating that microbial signals shape ige induction pathways early in life during a critical window of early - life development . spf mice contain a highly diverse and undefined microbiota ranging from symbionts to potentially opportunistic pathogens ( round and mazmanian , 2009 ) , making it difficult to use spf mice to determine the minimal microbial diversity or composition required to inhibit ige . to investigate the role of the level of diversity early in life in preventing the induction of high ige later in life , we made use of four additional defined model microbiotas ( figure 6b ) . first , we exposed germ - free c57bl/6 pups three to six times between the ages of 2 and 4 weeks to the single bacterium e. coli ha107 by oral gavage . we used the auxotrophic ha107 strain because we have previously described that this protocol induces iga responses ( hapfelmeier et al . , 2010 ) . despite exposing the pups to multiple high doses ( 2 1010 cfu per gavage ) before weaning , the mice developed hyper - ige at adult age ( figure 6b ) next , we generated mice bicolonized from birth with the two altered schaedler flora ( asf ) species ( dewhirst et al . , 1999 ) , parabacteroides distasonis asf519 and lactobacillus murinus asf361 . then , we performed the same experiment using the full asf , a mutualistic benign microflora composed of eight commensal bacterial species ( dewhirst et al . , 1999 ) . we found that , even when housed within the same flexible film isolator , asf mice clustered into two groups with some animals displaying hyper - ige , whereas others were protected with undetectable ige ( figure 6b ) . in order to investigate whether increasing the diversity further will protect from hyper - ige , we also tested c57bl/6 mice colonized from birth with a low complexity microbiota ( lcm ) consisting of approximately 40 phylotypes ( stecher et al . this indicates that increasing diversity is an important factor in providing protection from the induction of hyper - ige . however , we can not rule out the impact of individual microbial species in mediating protection . in asf - colonized mice , the presence of mice with both high and low ige levels was intriguing and cage specific ( figure 6c ) , and we hypothesized that early - life colonization dynamics were responsible for this segregation . to test this , the compositions of the asf early ( 3040 days of age ) and late ( 77295 days of age ) in life were analyzed with qpcr and bacterial - specific primer pairs . bacterial diversity during early life ranged from two to seven bacterial species , and the observed diversity inversely correlated with ige levels ; association with two to three detectable bacterial species ( p. distasonis and l. murinus ) correlated with high ige , whereas association with a greater detectable bacterial diversity ( four to seven bacterial species ) correlated with low ige levels ( figure 6d ) . in contrast , bacterial diversity in adult life ranged from three to seven bacterial species and was not predictive of ige levels ( figure 6d ) . in our asf colony , p. distasonis asf519 ( > 90% ) and l. murinus asf361 ( 4% ) are the two most abundant species present ( high - throughput 16s amplicon sequencing ) ( data not shown ) . this suggests that increasing early - life diversity by adding less abundant additional species may already be sufficient to mediate protection from hyper - ige . given that ige is known to regulate mast cell homeostasis ( kalesnikoff et al . 2003 ) , we investigated whether the elevated levels of ige in germ - free mice could alter mast - cell - mediated pathologies . we found that the amount of surface - bound ige was significantly increased in peritoneal mast cells from germ - free and bicolonized mice in comparison to spf mice ( figure 7a ) . interestingly , cell - surface levels of cd117 ( c - kit ) were also increased on mast cells from germ - free and bicolonized mice in comparison to spf mice , which may hint to a more immature phenotype ( boyce , 2004 ) . next , we evaluated whether tissue mast cell numbers were increased because of the elevated ige levels in germ - free mice . the number of cutaneous mast cells , as identified by chloroacetate esterase ( cae ) staining in the ear tissue of germ - free mice , was slightly , although not significantly , elevated under steady - state germ - free conditions in comparison to spf conditions ( figure 7b ) . in addition , the reduced number of mast cells in the ear skin of spf animals showed weaker cae staining ( figure 7b ) . we investigated whether elevated ige levels in germ - free mice have an impact on the severity of active antigen - induced anaphylaxis . it has been reported that gastrointestinal , cutaneous , and cardiovascular symptoms in oral antigen - induced anaphylaxis are dependent on ige and mast cells ( ahrens et al . , 2012 ) , whereas , in systemic antigen - induced anaphylaxis , these adverse symptoms are mediated by both ige and igg pathways ( osterfeld et al . , 2010 ) . germ - free and spf balb / c mice were systemically primed with ova and aluminum potassium sulfate ( aik(so4)2 - 12h2o ) ( alum ) or alum alone . mice were challenged 2 weeks later by intravenous injection of ovalbumin ( ova ; active systemic anaphylaxis ) or by oral gavage of ova ( active oral anaphylaxis ) . in response to systemic antigen challenge , germ - free mice developed hypothermia to the same level as spf animals ( figure 7c ) . in contrast , germ - free mice showed significantly increased susceptibility to oral antigen - induced anaphylaxis ( figure 7c ) . bicolonized c57bl/6 mice also displayed increased susceptibility to active oral anaphylaxis in comparison to spf c57bl/6 mice ( figure s3 ) . these data suggest that increased serum ige levels in germ - free mice impact on mast cell homeostasis , which , in turn , leads to greatly increased sensitivity to oral - induced anaphylaxis . the hygiene hypothesis postulated that the quality and/or quantity of microbial exposure early in life might have an important impact on how the immune system behaves later in life ( strachan , 1989 ) . although the original hygiene hypothesis did not suggest a role for the intestinal commensal bacteria , in the past years , it has become clear that our intestinal inhabitants have a powerful influence on immune maturation at both mucosal and systemic sites . systematic characterization of intestinal microbial communities in human babies has indicated that incidental environmental exposure plays a major role in determining the microbial community in each baby ( koenig et al . , 2011 ; palmer et al . , 2007 ) . along with westernization , major changes in the environment have occurred that are likely to heavily influence the composition of the intestinal microbiota . recent studies have also shown that children living in developing countries harbor greater intestinal bacterial diversity than children in developed countries ( de filippo et al . , 2010 ; lin et al . , 2013 ) . decreased bacterial diversity and/or altered composition of the intestinal microbiota early in life are likely to contribute to the increased susceptibility to immune - mediated diseases in westernized countries . using gnotobiotic mouse models , we provide experimental evidence that limited microbial association below a certain threshold of complexity or devoid of putative bacterial species or bacterial consortia with immunoregulatory properties during early life has profound negative and lasting effects on the induction of immune regulation , even when microbial diversity is increased above this threshold later in life . these data demonstrate the presence of a critical time window early in life during which appropriate microbial exposure has to occur in order to induce functional , life - long immune regulation that maintains serum ige levels at baseline . in addition to allergic disorders and certain helminth infections , elevated ige has been suggested to be a biomarker for primary immunodeficiencies ( liston et al . , 2008 ) . data from numerous mouse models of genetic immunodeficiency suggest that reduced functional t cell populations may lead to immune dysregulation and elevated ige levels as a consequence . there are most likely many pathways involved in the immune regulatory network that function to maintain ige at low levels . the development of hyper - ige in genetically immunocompetent germ - free mice indicates that exposure to commensal bacteria is a key factor in the development of a functional immune regulatory network . germ - free mice also show elevated invariant natural killer t cells at mucosal sites ( olszak et al . , 2012 ) , which trigger heightened pathology in models of inflammatory bowel disease and allergic asthma . in this study , we show that elevated serum ige titers leads to increased levels of surface - bound ige on mast cells , and germ - free mice displayed exacerbated antigen - induced oral anaphylaxis in comparison to spf mice , implying that dysregulated immune reactions such as food allergies may be highly dependent on adequate acquisition of bacterial consortia at early age . the fact that hygiene - mediated ige does not require an underlying genetic immunodeficiency suggests that exposure to a diverse microbial population during a critical time window early in life is absolutely required in order to set the baseline immune regulatory state for life . it is interesting that isotype switch to ige has been recently shown to be favored in immature b cells ( wesemann et al . , 2011 ) . although the maturation state of the b cells undergoing ige class switch was not investigated in the current study , b cells were found to switch to ige in mucosal tissues , especially pp , and not in b cell developmental sites , such as bone marrow or spleen . additional studies are required to carefully investigate the impact of microbial exposure on lymphocyte development and function . although ige alone does not constitute allergy and the simplicity of a bicolonization does not necessarily represent the restricted microbiota resulting from improved human hygiene , our results do show that diverse microbial colonization during a critical time window in neonatal life is required in order to limit default immune pathways , resulting in excessive ige levels , mirroring one possible immune component for rationalizing the hygiene hypothesis . c57bl/6 , tcr , jh , rag1 , tslpr ( a kind gift from n. harris [ cole polytechnique fdrale de lausanne ] ) ( all on a c57bl/6 background ) , nih - swiss , balb / c , swiss webster , and nmri mice were rederived to germ - free status via two - cell embryo transfer and bred and maintained in flexible - film isolators as described previously ( smith et al . , 2007 ) . germ - free mice were fed an irradiated ( 5 m rad co-60 for 20 hr ) elemental antigen - free diet consisting of extensively hydrolyzed proteins supplemented with fats , vitamins , and minerals ( pregestimil , enfamil ) . bedding consisted of endotoxin - free sand ( baked at 250c for 30 min ) . gnotobiotic mice associated with altered schaedler flora ( asf ) ( dewhirst et al . , 1999 ) were originally obtained from germ - free mice cohoused with an asf colonizer . for the generation of a bicolonized colony , germ - free c57bl/6 breeding pairs were orally gavaged with pure cultures of p. distasonis and l. murinus . serum samples from low - complexity microbiota mice ( stecher et al . , 2010 ; endt et al . , 2010 ) were kindly provided by w .- d . e.coli ha107 was cultured and prepared for gavage of germ - free mice as described previously ( hapfelmeier et al . , 2010 ) , and 2 1010 cfu ha107 was administered by oral gavage . c57bl/6 , balb / c , and tcr mice were purchased from taconic or maintained in the central animal facilities at mcmaster university or the university of bern . for spf colonization experiments , germ - free mice were cohoused with an spf mouse in individually ventilated cages . all animal experiments were carried out in accordance with the mcmaster university animal utilization protocols and the canadian council on animal care guidelines or in accordance with swiss federal regulations . blood was collected in serum - separating tubes and total serum ige , iga , igm , igg1 , igg2a , igg2c , and igg3 concentrations determined by sandwich elisa as described in the supplemental information . in order to obtain single - cell suspensions , lymph nodes ( plns , pps , and mlns ) were digested with 0.14 wnsch u / ml liberase c1 or liberase tl ( roche applied science ) . then , lymphoid tissues ( spleen , plns , mlns , and pps ) were homogenized and filtered through a 40 m cell strainer . lymphocytes from clp were isolated as described previously ( geuking et al . , 2011 ) . cells were resuspended in trizol reagent ( invitrogen ) and rna isolated according to the manufacturer s instructions . qpcr was performed with sybr green supermix or ssofast evagreen supermix ( bio - rad ) with specific primer pairs ( see table s1 and the supplemental information ) . genomic dna was isolated from cecal contents or fecal pellets with the qiaamp dna stool mini kit ( qiagen ) and analyzed with ssofast evagreen supermix ( bio - rad ) with the use of primers specific for the 16s rrna genes of the individual asf species ( see table s2 ) . pcr and analysis were performed on an iq5 or cfx384 ( bio - rad ) platform and software . dead cells were excluded with live / dead fixable dye efluor 506 ( ebioscience ) and fc receptors blocked with cd16/cd32 fc blocking antibody . data were acquired on a facscalibur ( bd biosciences ) or lsrii ( bd biosciences ) and analyzed with flowjo ( tree star ) . for facs sorting , splenocytes were enriched for cd4 t cells or cd19 b cells ( > 80% of lymphocytes ) with cd19 or cd4 magnetic beads ( miltenyi biotec ) and sorted for igmcd19b220cd3 ( naive b cells ) or cd3cd25cd45rb ( naive cd4 t cells ) populations on a facsaria ( bd biosciences ) . twice a week , germ - free mice ( 28 days old ) were administered ( 200 g intraperitoneal injected ) one of the following sterile mabs : anti - cd4 ( clone yts191.1.2 ) , anti - il-4 ( clone 11b11 ) , anti - tslp ( clone 28f12 , a kind gift from a. farr [ university of washington ] ) , or isotype control ( 35.61 ) . in order to abrogate pp ontogeny in vivo , anti - il7r ( 3 mg , clone a7r34 , bio x cell ) was administered ( intravenous [ i.v . ] the absence of pp and germ - free status was confirmed at the end of each experiment . for cotransfer of purified naive b and t cells , 1 10 facs - sorted splenic b and t cells were i.v . injected into germ - free rag1 recipients . b cells were purified from wt c57bl/6 , il-4r , or mhc ii - deficient mice . samples were fixed in 10% formalin and processed by standard histological techniques prior to paraffin embedding . then , 6 m sections were deparaffinized and stained with an -naphthyl chloroacetate esterase kit ( sigma - aldrich ) according to the manufacturer s instructions . the sections were counterstained with gill s no.3 hematoxylin solution for 30 s. germ - free , bicolonized , or spf mice were subcutaneously injected with 50 g of ovalbumin ( sigma - aldrich ) in the presence of 2 mg of alum adjuvant ( sigma- aldrich ) in sterile pbs or with alum alone . two weeks later , mice received either an oral ( 50 mg/250 l pbs ) or systemic ( 100 g ovalbumin in 500 l pbs ) ovalbumin challenge . for the oral challenge rectal temperatures were monitored every 510 min for 90 min with a tcat-2lv animal temperature controller ( physitemp instruments ) .

summarymicrobial exposure following birth profoundly impacts mammalian immune system development . microbiota alterations are associated with increased incidence of allergic and autoimmune disorders with elevated serum ige as a hallmark . the previously reported abnormally high serum ige levels in germ - free mice suggests that immunoregulatory signals from microbiota are required to control basal ige levels . we report that germ - free mice and those with low - diversity microbiota develop elevated serum ige levels in early life . b cells in neonatal germ - free mice undergo isotype switching to ige at mucosal sites in a cd4 t - cell- and il-4-dependent manner . a critical level of microbial diversity following birth is required in order to inhibit ige induction . elevated ige levels in germ - free mice lead to increased mast - cell - surface - bound ige and exaggerated oral - induced systemic anaphylaxis . thus , appropriate intestinal microbial stimuli during early life are critical for inducing an immunoregulatory network that protects from induction of ige at mucosal sites .

Introduction Results Discussion Experimental Procedures

healthy individuals maintain serum ige concentrations at basal levels ( < 0.0001% of serum immunoglobulins ) ( sutton and gould , 1993 ) because of an immunoregulatory network that regulates isotype switch to ige . multiple immunodeficiencies , including wiskott - aldrich syndrome , omenn syndrome , or immunodysregulation polyendocrinopathy enteropathy x - linked syndrome , are correlated with elevated serum ige levels despite the absence of allergic reaction or parasite infection ( kotlarz et al . abnormally high serum ige levels in germ - free mice have been reported previously ( herbst et al . this suggests that immunoregulatory signals stemming from the microbiota are required in order to maintain ige levels at basal levels even in genetically immunocompetent mice . we hypothesized that the proper induction of immune regulation requires adequate microbial exposure during early life . here , we show that only exposure to a diverse microbial community during early life is able to induce functional immune regulation that maintains ige at basal levels and decreases disease severity in a model of antigen - induced oral anaphylaxis . to investigate the role of the microbiota in the inhibition of ige production , we first confirmed the presence of high ige levels in a large cohort of germ - free c57bl/6 mice ( figure 1a ) . high ige levels were also observed in germ - free balb / c mice ( mean 292 ng / ml ; range 152,393 ng / ml ; n = 19 ) , but not in germ - free swiss webster ( n = 33 ) or nmri ( n = 21 ) mice ( < 0.8 ng / ml ) , possibly because of compensatory mechanisms frequently associated with outbred strains . , high ige levels did not reflect a general hyperimmunoglobulinemia , given that all isotypes except ige ( iga , igm , igg1 , igg2b , igg2c , and igg3 ) were reduced in germ - free and not spf mice ( figure 1c ) , supporting the view of an immature immune system in germ - free mice ( macpherson and harris , 2004 ) . from these initial observations , we hypothesized that a diversified microbiota is required in order to maintain serum ige levels at baseline . although culture - dependent and -independent quality control methods were employed rigorously in order to confirm the absence of microbes , helminths , or viruses , we still faced the remote possibility that an undetected organism might trigger ige induction . therefore , we either cohoused spf mice with undetectable serum ige levels with germ - free mice ( with high ige ) or left them with their spf littermates . ige levels in spf mice remained below the limit of detection at all ages ( figure 2b ) . in contrast , ige levels in germ - free mice became readily detectable at around day 30 of age and increased onward ( figure 2b ) . spf mice fed the same autoclaved chow did not develop increased ige , and germ - free mice fed and raised on an antigen - free elemental diet also developed high ige levels ( figure 2b ) . hyper - ige is generated in genetically immunocompetent germ- and antigen - free mice , indicating that class - switch recombination to ige reflects immune dysregulation in the absence of commensal microbiota . therefore , the expression of glts is a hallmark of active isotype switch to ige . then , we assessed the expression of glts in germ - free mice during neonatal and adult life . whereas low levels of glts were found in the spleen of a few of the germ - free animals , glts were readily detectable in the peyer s patches ( pps ) and mlns of all germ - free mice starting at day 35 , peaking at day 42 , and remaining detectable throughout adulthood ( figure 3b ) . therefore , in the absence of microbial exposure , ige isotype switch is fostered at mucosal sites , where microbially triggered immune adaptations , such as iga isotype switch , normally take place . , 1999 ) , germ - free ige induction was fully abrogated ( figure 3c ) . the administration of a cd4 t cell - depleting antibody to germ - free mice starting at 28 days of age totally abrogated the induction of ige ( figure 4a ) . ige levels were also maintained below the limit of detection in germ - free mice genetically deficient in t cells ( tcr ; figure 4b ) . we assessed the cytokine expression profile of cd4 t cells from the spleen , mlns , pps , and colon lamina propria ( clp ) in germ - free and spf mice . as expected , il10 expression was higher in the clp of spf than that of germ - free mice ( atarashi et al . il4 expression in germ - free , but not spf , mice was upregulated from day 36 of age onward , specifically in the mlns and pps . thus , the expression of il4 and glts shared identical kinetics and occurred within identical immune geography , suggesting that the prototypic ige - inducing cytokine il-4 is also involved in germ - free ige . il13 expression was similar between germ - free and spf mice with the exception of pps , where expression was higher in germ - free than spf mice ( figure 5a ) . administration of an il-4-neutralizing antibody to germ - free mice starting at 28 days of age led to a significant decrease in the concentration of serum ige ( figure 5b ) . to further investigate this pathway , and because performing bone marrow chimeras remains a challenging task under germ - free conditions , we opted for a mixed adoptive transfer of facs - sorted naive b and cd4 t cells into germ - free rag-1 mice . rapid homeostatic proliferation is microbially driven , but , given that slow homeostatic proliferation in lymphopenic hosts is mediated exclusively by low - affinity self - antigens , cells transferred into germ - free recipients proliferate relatively slowly ( kieper et al . cotransfer of wild - type ( wt ) b and t cells isolated from spf mice into germ - free rag-1 mice led to high ige in the majority of reconstituted mice ( figure 5c ) , indicating that it is the environment within the germ - free host rather than intrinsic defects in germ - free t and b cells that is supportive of hygiene - induced ige . cotransfer of il-4r-deficient b and wt t cells failed to induce ige in seven out of eight mice ( figure 5c ) , suggesting that il-4r - mediated signaling on b cells was crucial for ige isotype switch under germ - free conditions . in contrast , cotransfer of mhc ii - deficient b and wt t cells led to the induction of ige in germ - free rag-1 mice ( figure 5c ) , indicating that cognate interactions between b and t cells mediated by mhc ii and antigenic peptide and t cell receptor ( tcr ) interactions was not required for ige isotype switch under germ - free conditions . given that mlns and pp cd4 t cells of germ - free mice express higher levels of il-4 and il-13 in comparison to spf mice ( figure 5a ) and that the proportion of mlns and colon cd4ifn- t cells was lower under germ - free than spf conditions ( figure 5d ) , it is plausible that a default th2 pathway could be favored in the absence of microbes ( zhu et al . therefore , we investigated the role of tslp in hygiene - mediated ige in germ - free mice . the administration of a tslp - neutralizing antibody to germ - free mice starting at 28 days of age failed to prevent the induction of ige ( figure s1b ) . ige levels were also not abrogated in germ - free tslp receptor - deficient mice ( figure s1c ) . altogether , these observations suggest that excessive levels of ige in germ - free mice are dependent on cd4 t cells , il-4 , and possibly il-13 . furthermore , this implies that similar immune mediators drive high ige levels in germ - free c57bl/6 mice and immunodeficient spf c57bl/6 mice ( mccoy et al . , 2006 ) and supports the notion that high ige levels in the absence of microbes reflect immune dysregulation caused by the lack of microbial education of the immune system . we have previously shown that intestinal microbial colonization with a limited diversity microbiota leads to the induction of intestinal regulatory t ( treg ) cells and il-10 , which are required in order to limit the induction of proinflammatory th1 and th17 cells ( geuking et al . in an attempt to rescue the immune dysregulation and inhibit ige while preserving the germ - free status , we tested whether adoptive transfer of microbially - instructed treg cells or the administration of recombinant il-10 could inhibit the induction of ige in germ - free mice . total cd4 , cd4cd25 naive , or cd4cd25 treg cells were isolated from the spleen or clp of spf donor mice and adoptively transferred into 28-day - old germ - free recipients . , 2011 ) , it was not sufficient to limit ige induction under germ - free conditions ( figure s2a ) . our interpretation that ige induction is a default pathway of mucosal immunity in the absence of commensal intestinal microbes predicts that ige levels should be normalized in mice that are born germ free and later colonized by commensal microbes ; the interesting question was whether there was a time window in which this could take place . cohousing of adult germ - free mice ( with high ige ) with spf mice ( with undetectable ige and a complex intestinal microbiota ) did not reduce serum ige levels over an 8-week period ( figure 6a ) . in contrast , exposure to an spf flora at the time of birth completely inhibited the induction of ige ( figure 6a ; day 0 ) . to further refine the window of time whereby spf colonization provides protection , germ - free pups were colonized at days 2 , 7 , 14 , 21 , 28 , and 35 of age . in contrast to colonization during adulthood , the colonization of pups until 1 week postweaning ( when serum ige levels were still low ) was able to fully protect them from the induction of elevated ige during adulthood ( figure 6a ) , indicating that microbial signals shape ige induction pathways early in life during a critical window of early - life development . spf mice contain a highly diverse and undefined microbiota ranging from symbionts to potentially opportunistic pathogens ( round and mazmanian , 2009 ) , making it difficult to use spf mice to determine the minimal microbial diversity or composition required to inhibit ige . in order to investigate whether increasing the diversity further will protect from hyper - ige , we also tested c57bl/6 mice colonized from birth with a low complexity microbiota ( lcm ) consisting of approximately 40 phylotypes ( stecher et al . bacterial diversity during early life ranged from two to seven bacterial species , and the observed diversity inversely correlated with ige levels ; association with two to three detectable bacterial species ( p. distasonis and l. murinus ) correlated with high ige , whereas association with a greater detectable bacterial diversity ( four to seven bacterial species ) correlated with low ige levels ( figure 6d ) . 2003 ) , we investigated whether the elevated levels of ige in germ - free mice could alter mast - cell - mediated pathologies . we found that the amount of surface - bound ige was significantly increased in peritoneal mast cells from germ - free and bicolonized mice in comparison to spf mice ( figure 7a ) . interestingly , cell - surface levels of cd117 ( c - kit ) were also increased on mast cells from germ - free and bicolonized mice in comparison to spf mice , which may hint to a more immature phenotype ( boyce , 2004 ) . next , we evaluated whether tissue mast cell numbers were increased because of the elevated ige levels in germ - free mice . the number of cutaneous mast cells , as identified by chloroacetate esterase ( cae ) staining in the ear tissue of germ - free mice , was slightly , although not significantly , elevated under steady - state germ - free conditions in comparison to spf conditions ( figure 7b ) . we investigated whether elevated ige levels in germ - free mice have an impact on the severity of active antigen - induced anaphylaxis . it has been reported that gastrointestinal , cutaneous , and cardiovascular symptoms in oral antigen - induced anaphylaxis are dependent on ige and mast cells ( ahrens et al . , 2012 ) , whereas , in systemic antigen - induced anaphylaxis , these adverse symptoms are mediated by both ige and igg pathways ( osterfeld et al . germ - free and spf balb / c mice were systemically primed with ova and aluminum potassium sulfate ( aik(so4)2 - 12h2o ) ( alum ) or alum alone . in response to systemic antigen challenge , germ - free mice developed hypothermia to the same level as spf animals ( figure 7c ) . in contrast , germ - free mice showed significantly increased susceptibility to oral antigen - induced anaphylaxis ( figure 7c ) . these data suggest that increased serum ige levels in germ - free mice impact on mast cell homeostasis , which , in turn , leads to greatly increased sensitivity to oral - induced anaphylaxis . the hygiene hypothesis postulated that the quality and/or quantity of microbial exposure early in life might have an important impact on how the immune system behaves later in life ( strachan , 1989 ) . using gnotobiotic mouse models , we provide experimental evidence that limited microbial association below a certain threshold of complexity or devoid of putative bacterial species or bacterial consortia with immunoregulatory properties during early life has profound negative and lasting effects on the induction of immune regulation , even when microbial diversity is increased above this threshold later in life . these data demonstrate the presence of a critical time window early in life during which appropriate microbial exposure has to occur in order to induce functional , life - long immune regulation that maintains serum ige levels at baseline . data from numerous mouse models of genetic immunodeficiency suggest that reduced functional t cell populations may lead to immune dysregulation and elevated ige levels as a consequence . there are most likely many pathways involved in the immune regulatory network that function to maintain ige at low levels . the development of hyper - ige in genetically immunocompetent germ - free mice indicates that exposure to commensal bacteria is a key factor in the development of a functional immune regulatory network . germ - free mice also show elevated invariant natural killer t cells at mucosal sites ( olszak et al . in this study , we show that elevated serum ige titers leads to increased levels of surface - bound ige on mast cells , and germ - free mice displayed exacerbated antigen - induced oral anaphylaxis in comparison to spf mice , implying that dysregulated immune reactions such as food allergies may be highly dependent on adequate acquisition of bacterial consortia at early age . the fact that hygiene - mediated ige does not require an underlying genetic immunodeficiency suggests that exposure to a diverse microbial population during a critical time window early in life is absolutely required in order to set the baseline immune regulatory state for life . it is interesting that isotype switch to ige has been recently shown to be favored in immature b cells ( wesemann et al . although the maturation state of the b cells undergoing ige class switch was not investigated in the current study , b cells were found to switch to ige in mucosal tissues , especially pp , and not in b cell developmental sites , such as bone marrow or spleen . additional studies are required to carefully investigate the impact of microbial exposure on lymphocyte development and function . although ige alone does not constitute allergy and the simplicity of a bicolonization does not necessarily represent the restricted microbiota resulting from improved human hygiene , our results do show that diverse microbial colonization during a critical time window in neonatal life is required in order to limit default immune pathways , resulting in excessive ige levels , mirroring one possible immune component for rationalizing the hygiene hypothesis . c57bl/6 , tcr , jh , rag1 , tslpr ( a kind gift from n. harris [ cole polytechnique fdrale de lausanne ] ) ( all on a c57bl/6 background ) , nih - swiss , balb / c , swiss webster , and nmri mice were rederived to germ - free status via two - cell embryo transfer and bred and maintained in flexible - film isolators as described previously ( smith et al . germ - free mice were fed an irradiated ( 5 m rad co-60 for 20 hr ) elemental antigen - free diet consisting of extensively hydrolyzed proteins supplemented with fats , vitamins , and minerals ( pregestimil , enfamil ) . , 1999 ) were originally obtained from germ - free mice cohoused with an asf colonizer . for the generation of a bicolonized colony , germ - free c57bl/6 breeding pairs were orally gavaged with pure cultures of p. distasonis and l. murinus . e.coli ha107 was cultured and prepared for gavage of germ - free mice as described previously ( hapfelmeier et al . for spf colonization experiments , germ - free mice were cohoused with an spf mouse in individually ventilated cages . in order to obtain single - cell suspensions , lymph nodes ( plns , pps , and mlns ) were digested with 0.14 wnsch u / ml liberase c1 or liberase tl ( roche applied science ) . twice a week , germ - free mice ( 28 days old ) were administered ( 200 g intraperitoneal injected ) one of the following sterile mabs : anti - cd4 ( clone yts191.1.2 ) , anti - il-4 ( clone 11b11 ) , anti - tslp ( clone 28f12 , a kind gift from a. farr [ university of washington ] ) , or isotype control ( 35.61 ) . in order to abrogate pp ontogeny in vivo , anti - il7r ( 3 mg , clone a7r34 , bio x cell ) was administered ( intravenous [ i.v . ] the absence of pp and germ - free status was confirmed at the end of each experiment . the sections were counterstained with gill s no.3 hematoxylin solution for 30 s. germ - free , bicolonized , or spf mice were subcutaneously injected with 50 g of ovalbumin ( sigma - aldrich ) in the presence of 2 mg of alum adjuvant ( sigma- aldrich ) in sterile pbs or with alum alone .

the goal of periodontal therapy is to eliminate disease and restore the periodontium to a state of health which includes comfort , function , and esthetics that can be maintained adequately by both the patient and dental professional . after thorough scaling and root planing , a profound reduction in motile rods , spirochetes , and putative pathogens , such as porphyromonas gingivalis , prevotella intermedia , and aggregatibacter actinomycetemcomitans , and an increase in coccoid cells occur . although ultrasonic scalers are used routinely on patients , there are a few deleterious effects or hazards associated with its use . for example , it may produce thermal damage to the tissues , aerosol production contaminated with blood , and there is a high electromagnetic radiation emitted with these devices that can interfere with electrical devices such as cardiac pacemakers , neurologic disturbances of the hand caused by vibration such as white finger and hearing loss . aerosols are liquid and solid particles , 50 m or less in diameter , suspended in air . aerosol from ultrasonic instrumentation always contains blood and lingers in the air for 30 min or longer in the entire operatory and areas of the dental office outside the operatory . the aerosol produced by sonic and ultrasonic instrumentation may contain potentially infectious blood- and air - borne pathogens . pneumococci , staphylococci , alpha hemolytic , streptococci , and mycobacterium tuberculosis are among the bacteria that have been found in dental aerosols . aerosols also subject dental personnel and patients to many viruses including herpes simplex virus , hepatitis virus , influenza virus , common cold viruses , epstein additional concerns are pathogens that do not originate from patients but are from the contaminated water lines of the dental unit or the ultrasonic device . putative pathogens such as pseudomonas species and legionella pneumophilia have been isolated from dental unit water and can become aerosolized by an ultrasonic scaler . the microorganisms which have been isolated in dental aerosols are associated with various diseases such as staphylococcal infection , tuberculosis , viral infections , conjunctivitis , and other skin infections . culture plates opened for 1 min during use of high - speed handpieces on five tuberculosis patients with positive sputum were all positive up to 48 inches away . serratia marcescens placed in dental unit water supplies as a tracer microorganism have been recovered over 6 ft away from an operating high - speed handpiece . bacterial aerosols are an important consideration for infection control and occupational health hazards in the dental clinic since infective agents can be transmitted via aerosols to patients or dental staff . to prevent this transfer of infection , it is recommended that patients with infectious disease be treated only with hand scalers . unprotected patients may be more susceptible to infection from the aerosol than dental personnel who are wearing protective barriers such as masks , gloves , eyewear , and clinical clothing . this study is the first of its kind as per our knowledge where we investigated the contamination of contact lenses after scaling and root planing with piezoelectric ultrasonic scalers . the contact lens wearers are at great risk of developing ocular infections because of incorrect usages and unhygienic maintenance of contact lenses . in most of the cases of corneal ulcers or bacterial keratitis , many of these adverse responses are produced as a result of bacterial colonization of the contact lenses . concerns about microbial contamination of contact lenses and the adverse responses associated with its use have been expressed since their inception . however , there are no reported cases or studies of microbial contamination of contact lenses after dental treatment procedures such as scaling and root planing with ultrasonic scalers which produce aerosol or splatter . keeping the above facts in mind , the present study was performed to evaluate aerosol contamination of contact lenses of the dentist after scaling and root planing with ultrasonic scalers , qualitative analysis of microorganisms on contact lenses of the dentist , to determine the effectiveness of protective eyewear . the participants for this study were selected from the outpatient section in the department of periodontics , the oxford dental college , hospital and research centre , bangalore , india . thirty subjects ( ten males , twenty females aged between 35 and 50 years ) diagnosed with moderate to severe chronic periodontitis were enrolled in this study . the study protocol was approved by the ethical committee of the institution and informed consent was obtained from the participants . subjects aged between 35 and 50 yearsmoderate to severe chronic periodontitis ( as per the american academy of periodontology classification)minimum of twenty teeth presentno history of periodontal or antibiotic therapy in the preceding 6 monthsthirty dentists who wear contact lens without any history of eye infection in the last 6 months . subjects aged between 35 and 50 years moderate to severe chronic periodontitis ( as per the american academy of periodontology classification ) minimum of twenty teeth present no history of periodontal or antibiotic therapy in the preceding 6 months thirty dentists who wear contact lens without any history of eye infection in the last 6 months . subjects with definite contraindications for the use of ultrasonic and sonic scaling devices , for example , patients with known communicable diseases that can be transmitted by aerosolssubjects with a cardiac pacemakersubjects with respiratory diseases such as chronic pulmonary disordersimmunocompromised subjectssubjects with titanium implants , which can be etched or gougedpregnant or lactating mothers . subjects with definite contraindications for the use of ultrasonic and sonic scaling devices , for example , patients with known communicable diseases that can be transmitted by aerosols subjects with a cardiac pacemaker subjects with respiratory diseases such as chronic pulmonary disorders immunocompromised subjects subjects with titanium implants , which can be etched or gouged pregnant or lactating mothers . after subject selection , 15 subjects were randomly assigned to each treatment group ( using a computer - generated system- the randomization scheme was generated by using the web site randomization.com , http://www.randomization.com ) , group a 15 dentists wearing contact lenses and protective eyewear , group b 15 dentists wearing contact lenses only . the lenses were aseptically worn after wearing a fresh sterile particle free glove ( fisher scientific particle - free gloves ) . scaling and root planing were rendered with piezoelectric ultrasonic scalers ( piezoelectric scaler [ biosonic ] ) in combination with high volume evacuation ( hve ) for about 30 min in both the groups . after scaling and root planing with ultrasonic scalers , the lenses were aseptically collected wearing a fresh sterile particle free glove and transferred into a sterile test tube containing brain heart infusion broth m210 ( composition - ingredients gm / l , calf brain , infusion from 200.000 beef heart , infusion from 250.000 proteose peptone 10.000 dextrose 2.000 sodium chloride 5.000 disodium phosphate 2.500 final ph at 25c the samples were then subjected to microbiological analysis ( culture and 16s rrna [ 16s ribosomal rna ] gene sequencing ) . all the samples were cultured on brain heart infusion broth m210 and incubated in aerobic and anaerobic conditions for 2448 h at 37c . a variety of molecular techniques are used in identification and estimating the phylogenetic relatedness of prokaryotes , the comparison of 16s rrna isolated from thousands of strains is of particular importance . 16s rrnas are the best studied molecular markers since ribosomes have a central role in gene expression . 16s rrnas of bacteria and archaea are used to determine the phylogenetic relationship among these organisms . the advantage of using 16s rrna is that it is found in all organisms and large enough molecules provide a significant number of nucleotides to compare sequence . the statistical analysis was performed using commercially available software ( spss version 10.5 , ibm , chicago , il , usa ) . statistical significance was defined as p < 0.01 , which is highly significant . contingency coefficient test the participants for this study were selected from the outpatient section in the department of periodontics , the oxford dental college , hospital and research centre , bangalore , india . thirty subjects ( ten males , twenty females aged between 35 and 50 years ) diagnosed with moderate to severe chronic periodontitis were enrolled in this study . the study protocol was approved by the ethical committee of the institution and informed consent was obtained from the participants . subjects aged between 35 and 50 yearsmoderate to severe chronic periodontitis ( as per the american academy of periodontology classification)minimum of twenty teeth presentno history of periodontal or antibiotic therapy in the preceding 6 monthsthirty dentists who wear contact lens without any history of eye infection in the last 6 months . subjects aged between 35 and 50 years moderate to severe chronic periodontitis ( as per the american academy of periodontology classification ) minimum of twenty teeth present no history of periodontal or antibiotic therapy in the preceding 6 months thirty dentists who wear contact lens without any history of eye infection in the last 6 months . subjects with definite contraindications for the use of ultrasonic and sonic scaling devices , for example , patients with known communicable diseases that can be transmitted by aerosolssubjects with a cardiac pacemakersubjects with respiratory diseases such as chronic pulmonary disordersimmunocompromised subjectssubjects with titanium implants , which can be etched or gougedpregnant or lactating mothers . subjects with definite contraindications for the use of ultrasonic and sonic scaling devices , for example , patients with known communicable diseases that can be transmitted by aerosols subjects with a cardiac pacemaker subjects with respiratory diseases such as chronic pulmonary disorders immunocompromised subjects subjects with titanium implants , which can be etched or gouged pregnant or lactating mothers . after subject selection , 15 subjects were randomly assigned to each treatment group ( using a computer - generated system- the randomization scheme was generated by using the web site randomization.com , http://www.randomization.com ) , group a 15 dentists wearing contact lenses and protective eyewear , group b 15 dentists wearing contact lenses only . the lenses were aseptically worn after wearing a fresh sterile particle free glove ( fisher scientific particle - free gloves ) . scaling and root planing were rendered with piezoelectric ultrasonic scalers ( piezoelectric scaler [ biosonic ] ) in combination with high volume evacuation ( hve ) for about 30 min in both the groups . after scaling and root planing with ultrasonic scalers , the lenses were aseptically collected wearing a fresh sterile particle free glove and transferred into a sterile test tube containing brain heart infusion broth m210 ( composition - ingredients gm / l , calf brain , infusion from 200.000 beef heart , infusion from 250.000 proteose peptone 10.000 dextrose 2.000 sodium chloride 5.000 disodium phosphate 2.500 final ph at 25c the samples were then subjected to microbiological analysis ( culture and 16s rrna [ 16s ribosomal rna ] gene sequencing ) . all the samples were cultured on brain heart infusion broth m210 and incubated in aerobic and anaerobic conditions for 2448 h at 37c . a variety of molecular techniques are used in identification and estimating the phylogenetic relatedness of prokaryotes , the comparison of 16s rrna isolated from thousands of strains is of particular importance . 16s rrnas are the best studied molecular markers since ribosomes have a central role in gene expression . 16s rrnas of bacteria and archaea are used to determine the phylogenetic relationship among these organisms . the advantage of using 16s rrna is that it is found in all organisms and large enough molecules provide a significant number of nucleotides to compare sequence . subjects aged between 35 and 50 yearsmoderate to severe chronic periodontitis ( as per the american academy of periodontology classification)minimum of twenty teeth presentno history of periodontal or antibiotic therapy in the preceding 6 monthsthirty dentists who wear contact lens without any history of eye infection in the last 6 months . subjects aged between 35 and 50 years moderate to severe chronic periodontitis ( as per the american academy of periodontology classification ) minimum of twenty teeth present no history of periodontal or antibiotic therapy in the preceding 6 months thirty dentists who wear contact lens without any history of eye infection in the last 6 months . subjects with definite contraindications for the use of ultrasonic and sonic scaling devices , for example , patients with known communicable diseases that can be transmitted by aerosolssubjects with a cardiac pacemakersubjects with respiratory diseases such as chronic pulmonary disordersimmunocompromised subjectssubjects with titanium implants , which can be etched or gougedpregnant or lactating mothers . subjects with definite contraindications for the use of ultrasonic and sonic scaling devices , for example , patients with known communicable diseases that can be transmitted by aerosols subjects with a cardiac pacemaker subjects with respiratory diseases such as chronic pulmonary disorders immunocompromised subjects subjects with titanium implants , which can be etched or gouged pregnant or lactating mothers . after subject selection , 15 subjects were randomly assigned to each treatment group ( using a computer - generated system- the randomization scheme was generated by using the web site randomization.com , http://www.randomization.com ) , group a 15 dentists wearing contact lenses and protective eyewear , group b 15 dentists wearing contact lenses only . the lenses were aseptically worn after wearing a fresh sterile particle free glove ( fisher scientific particle - free gloves ) . scaling and root planing were rendered with piezoelectric ultrasonic scalers ( piezoelectric scaler [ biosonic ] ) in combination with high volume evacuation ( hve ) for about 30 min in both the groups . after scaling and root planing with ultrasonic scalers , the lenses were aseptically collected wearing a fresh sterile particle free glove and transferred into a sterile test tube containing brain heart infusion broth m210 ( composition - ingredients gm / l , calf brain , infusion from 200.000 beef heart , infusion from 250.000 proteose peptone 10.000 dextrose 2.000 sodium chloride 5.000 disodium phosphate 2.500 final ph at 25c the samples were then subjected to microbiological analysis ( culture and 16s rrna [ 16s ribosomal rna ] gene sequencing ) . all the samples were cultured on brain heart infusion broth m210 and incubated in aerobic and anaerobic conditions for 2448 h at 37c . a variety of molecular techniques are used in identification and estimating the phylogenetic relatedness of prokaryotes , the comparison of 16s rrna isolated from thousands of strains is of particular importance . 16s rrnas are the best studied molecular markers since ribosomes have a central role in gene expression . 16s rrnas of bacteria and archaea are used to determine the phylogenetic relationship among these organisms . the advantage of using 16s rrna is that it is found in all organisms and large enough molecules provide a significant number of nucleotides to compare sequence . the statistical analysis was performed using commercially available software ( spss version 10.5 , ibm , chicago , il , usa ) . statistical significance was defined as p < 0.01 , which is highly significant . contingency coefficient test all the samples were cultured on brain heart infusion broth m210 and incubated in aerobic and anaerobic conditions for 2448 h at 37c . after 48 h of incubation , in group a ( contact lens + with protective eyewear ) 15 out of thirty samples [ figure 2 ] and in group b ( contact lens + without protective eyewear ) all the thirty samples were contaminated [ figure 3 and table 1 ] . out of sixty samples , 45 were contaminated and of 45 samples , five samples showed fungal growth ( two samples in group a and three in group b [ figures 4 , 5 and table 1 ] ) . group a showing fungal and other bacterial colonies group b showing bacterial growth around the lens contamination groups cross tabulation group a showing fungal growth group b showing fungal growth around the periphery of the contact lens grams ' staining was done for forty contaminated samples which revealed most of the samples were gram - positive bacteria [ figures 6 and 7 ] . group a and group b showing gram positive bacteria at 4x group a and group b showing gram positive bacteria at 20x out of sixty samples , 45 were contaminated . in 45 samples , ten samples were contaminated with streptococcus mutans , three with staphylococcus aureus , and two with fungi . in group 21 samples were contaminated with s. mutans , six with s. aureus , and three with fungi . ( in total 31 samples were contaminated with s. mutans strain , nine with s. aureus and five with fungi . ) overall , the results of this study indicate low microbial contamination of contact lens in group a ( contact lens with protective eyewear ) when compared to group b ( contact lens without protective eyewear ) which is statistically significant ( p < 0.01 ) [ table 2 ] . however , even after wearing protective eyewear , there is contamination of the contact lens which can be very hazardous to the eyes [ flowchart 1 ] . all the samples were cultured on brain heart infusion broth m210 and incubated in aerobic and anaerobic conditions for 2448 h at 37c . after 48 h of incubation , in group a ( contact lens + with protective eyewear ) 15 out of thirty samples [ figure 2 ] and in group b ( contact lens + without protective eyewear ) all the thirty samples were contaminated [ figure 3 and table 1 ] . out of sixty samples , 45 were contaminated and of 45 samples , five samples showed fungal growth ( two samples in group a and three in group b [ figures 4 , 5 and table 1 ] ) . group a showing fungal and other bacterial colonies group b showing bacterial growth around the lens contamination groups cross tabulation group a showing fungal growth group b showing fungal growth around the periphery of the contact lens grams ' staining was done for forty contaminated samples which revealed most of the samples were gram - positive bacteria [ figures 6 and 7 ] . group a and group b showing gram positive bacteria at 4x group a and group b showing gram positive bacteria at 20x ten samples were contaminated with streptococcus mutans , three with staphylococcus aureus , and two with fungi . in group 21 samples were contaminated with s. mutans , six with s. aureus , and three with fungi . ( in total 31 samples were contaminated with s. mutans strain , nine with s. aureus and five with fungi . ) overall , the results of this study indicate low microbial contamination of contact lens in group a ( contact lens with protective eyewear ) when compared to group b ( contact lens without protective eyewear ) which is statistically significant ( p < 0.01 ) [ table 2 ] . however , even after wearing protective eyewear , there is contamination of the contact lens which can be very hazardous to the eyes [ flowchart 1 ] . disease transmission and barrier techniques during dental treatment have been areas of recent concern in dentistry . the interest in dental aerobiology has been stimulated by concern with the obvious splatter caused by the visible particles emanating from the patient 's mouth during dental procedures . the driving force to conduct this study is an observation that dentists do not wear protective eyewear while rendering treatment which is very important in preventing damage to the eyes . the interest of the present study was to determine aerosol contamination of contact lenses of the dentist after scaling and root planing using a piezoelectric scaler in combination with hve , qualitative analysis of microorganisms on contact lenses of the dentist , and to determine the effectiveness of protective eyewear in preventing the contamination of the contact lens . the oral cavity harbors numerous bacteria and viruses from the respiratory tract , dental plaque , and oral fluids . it is a unique environment which provides an ideal medium for the growth of bacteria . any dental procedure that has a potential to aerosolize the saliva will cause airborne contamination with organisms from some or all of these sources . any cross infection can result even in the death of the person in case of an immunosuppressed patient . ultrasonic scalers are known to produce a fine spray called aerosol or splatter , and these aerosols are often produced from areas of significant disease activity including bleeding . aerosols may be heavily contaminated with oral organisms and present a considerable microbial challenge to the dentist , patients , and the nursing staff . it contains infected droplets which remain suspended in the environment for about 30 min or longer and can be inhaled into the lungs . the contaminated water in dental chair waterlines is yet another source for transmission of infection . this may potentially contribute to the transmission of infections through the dental office , which has become a source of increased concern to the dental profession . this study is the first of its kind as per our knowledge where we investigated the contamination of contact lenses of the dentists after scaling and root planing with ultrasonic scalers . scaling and root planing was rendered for 30 min after which the lenses were aseptically collected and cultured in both aerobic and anaerobic conditions . the microbial contamination of contact lens in group a was less when compared to group b , ( statistical significance was defined as p < 0.01 , which is highly significant ) proving the importance of wearing protective eyewear . however , even after wearing protective eyewear , there was contamination of the contact lenses which can be very hazardous to the eyes . in the present clinical and microbiological comparative study , ultrasonic scalers generate aerosols with bacteria peaking at over 300 cfu / cu ft of dental operatory volume . demonstrated that the aerosol produced by the in vivo use of an ultrasonic scaler on periodontally involved teeth was contaminated with blood and that the contamination occurred regardless of the level of inflammation . in a study by bhat , sterile swabs were used to collect samples from the chest area and pocket area of the aprons / overcoats of dental students which were then cultured , the results showed that there was microbial contamination of overcoats of dental students . many studies have confirmed that an aerosolized bacterial contamination is produced during the use of ultrasonic scalers and other dental equipment that produce an aerosol spray . miller et al . demonstrated that dental operations involving air and water sprays in combination with rotating instruments may cause levels of contamination exceeding those produced by common oral activities . they showed that a sneeze and the use of the air turbine handpiece produced comparable aerosols and splatter . a four - fold increase of airborne bacteria has been observed in areas where aerosol producing equipment was used . a considerable bacterial challenge exists in the aerosol produced by ultrasonic scalers , and it is probable that viruses and bacteria may be spread via this route . the traditional view of this bacterial contamination in the dental office is that these are nonpathogenic bacteria . however , studies have linked an increase in respiratory illness to the use of ultrasonic scalers . with the reported resurgence in bacterial diseases and the presence of other pathologic organisms with the potential for airborne transmission , there is an increased concern about aerosol contamination and decreased air quality in the dental office . bacterial contamination from ultrasonic scaler due to aerosol has been noted in the past . according to the center for disease control guidelines for infection control in dental health - care settings ( 2003 ) , preventive measures to control dental office air contamination include universal precautions , which consist of dental staff protective equipment ( gown , mask , gloves , eyeglasses ) , preprocedural patient mouth - rinsing with antimicrobial products ( such as chlorhexidine gluconate ) , operatory isolation ( rubber dam ) , vacuum and electrostatic extraction of aerosols during dental procedures , air circulation methods ( ventilation and air conditioning systems ) , air filtration systems for solid particles and mercury , ultraviolet lamps , disinfectants , and microbial controls for instruments and surfaces . people choose to wear contact lenses for many reasons such as visual , esthetics , and cosmetics which are often motivating factors for people who would like to avoid wearing glasses or change the appearance of their eyes . occasionally , adverse responses to contact lens wear occur such as microbial keratitis , contact lens - induced acute red eye , contact lens - induced peripheral ulcers , and infiltrative keratitis . one of the initial steps in the development of the bacterially driven adverse responses is the binding of bacteria to a contact lens . contact lenses do not protect the eyes and can increase the risk of exposure to microorganisms if contaminated fluids gain access beneath the lens . eyes are particularly vulnerable to injury by high - velocity particles / debris generated during use of high - speed handpieces and ultrasonic scalers . it has been reported that the environment , the type of contact lens , the duration of wear , and the type of contact lens cleansing solution determined the microbial load on the contact lenses . the normal ocular microbiota in the absence of contact lens wear is composed almost exclusively of bacterial types such as corynebacterium sp . and propionibacterium sp . martins et al . observed the presence of fungi , parasites and bacteria in contact lens swabs cultures . subsequent to adhesion , it is likely that bacteria further colonize the lens surface by growing on that lens surface . hume and willcox demonstrated that s. marcescens was able to grow on a contact lens after adhesion to contact lenses coated with an artificial tear film . the unique structure of the human eye , the use of contact lenses and the constant exposure of the eye directly to the environment renders it vulnerable to a number of uncommon infectious diseases caused by microorganisms . protective measures recommended to reduce the risk of infection from aerosols may be classified as physical , chemical , and personal safeguards . this study highlights the importance of protective eyewear in the prevention of contamination of the contact lenses . going by the results of this study , we would like to conclude that dental practitioners should better avoid contact lenses in a dental setup because of the risk of contamination of the contact lenses from the various dental procedures which can produce aerosol / splatter and if worn it is recommended to wear protective eyewear . therefore , similar studies with a larger sample size and a negative control are required to affirm the observations of our study .

background : ultrasonic scaler is a preferential treatment modality among the clinicians . however , the aerosol / splatter generated is a concern for patients and practitioners . therefore , the purpose of this study was to evaluate contamination of contact lenses of the dentist after scaling and root planing using ultrasonic scalers with and without protective eyewear.materials and methods : thirty patients were randomly selected for scaling and root planing and divided into 2 groups of 15 each . group a dentist wearing contact lenses and protective eyewear . group b - dentist wearing only contact lenses . after scaling and root planing using ultrasonic scalers , the lenses were subjected to culture and 16s rrna ( 16s ribosomal rna ) gene sequencing.results:in group a 15 out of thirty samples were contaminated , in group b all the thirty samples were contaminated . most of the samples showed gram - positive bacteria and 5 samples were contaminated with fungi . 16s rrna gene sequencing of forty contaminated samples showed that 31 were contaminated with streptococcus mutans and 9 with staphylococcus aureus.conclusion:keeping in mind the limitation of the study for the absence of negative control , we would like to conclude that dental practitioners should better avoid contact lenses in a dental setup because of the risk of contamination of the contact lenses from the various dental procedures which can produce aerosol / splatter and if worn , it is recommended to wear protective eyewear .

INTRODUCTION MATERIALS AND METHODS Source of data Selection criteria Inclusion criteria Exclusion criteria Statistical analysis RESULTS Culture Histopathological examination 16S ribosomal RNA sequencing DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

after thorough scaling and root planing , a profound reduction in motile rods , spirochetes , and putative pathogens , such as porphyromonas gingivalis , prevotella intermedia , and aggregatibacter actinomycetemcomitans , and an increase in coccoid cells occur . although ultrasonic scalers are used routinely on patients , there are a few deleterious effects or hazards associated with its use . for example , it may produce thermal damage to the tissues , aerosol production contaminated with blood , and there is a high electromagnetic radiation emitted with these devices that can interfere with electrical devices such as cardiac pacemakers , neurologic disturbances of the hand caused by vibration such as white finger and hearing loss . the aerosol produced by sonic and ultrasonic instrumentation may contain potentially infectious blood- and air - borne pathogens . aerosols also subject dental personnel and patients to many viruses including herpes simplex virus , hepatitis virus , influenza virus , common cold viruses , epstein additional concerns are pathogens that do not originate from patients but are from the contaminated water lines of the dental unit or the ultrasonic device . to prevent this transfer of infection , it is recommended that patients with infectious disease be treated only with hand scalers . unprotected patients may be more susceptible to infection from the aerosol than dental personnel who are wearing protective barriers such as masks , gloves , eyewear , and clinical clothing . this study is the first of its kind as per our knowledge where we investigated the contamination of contact lenses after scaling and root planing with piezoelectric ultrasonic scalers . the contact lens wearers are at great risk of developing ocular infections because of incorrect usages and unhygienic maintenance of contact lenses . in most of the cases of corneal ulcers or bacterial keratitis , many of these adverse responses are produced as a result of bacterial colonization of the contact lenses . concerns about microbial contamination of contact lenses and the adverse responses associated with its use have been expressed since their inception . however , there are no reported cases or studies of microbial contamination of contact lenses after dental treatment procedures such as scaling and root planing with ultrasonic scalers which produce aerosol or splatter . keeping the above facts in mind , the present study was performed to evaluate aerosol contamination of contact lenses of the dentist after scaling and root planing with ultrasonic scalers , qualitative analysis of microorganisms on contact lenses of the dentist , to determine the effectiveness of protective eyewear . the participants for this study were selected from the outpatient section in the department of periodontics , the oxford dental college , hospital and research centre , bangalore , india . thirty subjects ( ten males , twenty females aged between 35 and 50 years ) diagnosed with moderate to severe chronic periodontitis were enrolled in this study . the study protocol was approved by the ethical committee of the institution and informed consent was obtained from the participants . subjects with definite contraindications for the use of ultrasonic and sonic scaling devices , for example , patients with known communicable diseases that can be transmitted by aerosolssubjects with a cardiac pacemakersubjects with respiratory diseases such as chronic pulmonary disordersimmunocompromised subjectssubjects with titanium implants , which can be etched or gougedpregnant or lactating mothers . subjects with definite contraindications for the use of ultrasonic and sonic scaling devices , for example , patients with known communicable diseases that can be transmitted by aerosols subjects with a cardiac pacemaker subjects with respiratory diseases such as chronic pulmonary disorders immunocompromised subjects subjects with titanium implants , which can be etched or gouged pregnant or lactating mothers . after subject selection , 15 subjects were randomly assigned to each treatment group ( using a computer - generated system- the randomization scheme was generated by using the web site randomization.com , http://www.randomization.com ) , group a 15 dentists wearing contact lenses and protective eyewear , group b 15 dentists wearing contact lenses only . the lenses were aseptically worn after wearing a fresh sterile particle free glove ( fisher scientific particle - free gloves ) . scaling and root planing were rendered with piezoelectric ultrasonic scalers ( piezoelectric scaler [ biosonic ] ) in combination with high volume evacuation ( hve ) for about 30 min in both the groups . after scaling and root planing with ultrasonic scalers , the lenses were aseptically collected wearing a fresh sterile particle free glove and transferred into a sterile test tube containing brain heart infusion broth m210 ( composition - ingredients gm / l , calf brain , infusion from 200.000 beef heart , infusion from 250.000 proteose peptone 10.000 dextrose 2.000 sodium chloride 5.000 disodium phosphate 2.500 final ph at 25c the samples were then subjected to microbiological analysis ( culture and 16s rrna [ 16s ribosomal rna ] gene sequencing ) . all the samples were cultured on brain heart infusion broth m210 and incubated in aerobic and anaerobic conditions for 2448 h at 37c . a variety of molecular techniques are used in identification and estimating the phylogenetic relatedness of prokaryotes , the comparison of 16s rrna isolated from thousands of strains is of particular importance . the advantage of using 16s rrna is that it is found in all organisms and large enough molecules provide a significant number of nucleotides to compare sequence . contingency coefficient test the participants for this study were selected from the outpatient section in the department of periodontics , the oxford dental college , hospital and research centre , bangalore , india . the study protocol was approved by the ethical committee of the institution and informed consent was obtained from the participants . subjects with definite contraindications for the use of ultrasonic and sonic scaling devices , for example , patients with known communicable diseases that can be transmitted by aerosolssubjects with a cardiac pacemakersubjects with respiratory diseases such as chronic pulmonary disordersimmunocompromised subjectssubjects with titanium implants , which can be etched or gougedpregnant or lactating mothers . subjects with definite contraindications for the use of ultrasonic and sonic scaling devices , for example , patients with known communicable diseases that can be transmitted by aerosols subjects with a cardiac pacemaker subjects with respiratory diseases such as chronic pulmonary disorders immunocompromised subjects subjects with titanium implants , which can be etched or gouged pregnant or lactating mothers . after subject selection , 15 subjects were randomly assigned to each treatment group ( using a computer - generated system- the randomization scheme was generated by using the web site randomization.com , http://www.randomization.com ) , group a 15 dentists wearing contact lenses and protective eyewear , group b 15 dentists wearing contact lenses only . the lenses were aseptically worn after wearing a fresh sterile particle free glove ( fisher scientific particle - free gloves ) . scaling and root planing were rendered with piezoelectric ultrasonic scalers ( piezoelectric scaler [ biosonic ] ) in combination with high volume evacuation ( hve ) for about 30 min in both the groups . after scaling and root planing with ultrasonic scalers , the lenses were aseptically collected wearing a fresh sterile particle free glove and transferred into a sterile test tube containing brain heart infusion broth m210 ( composition - ingredients gm / l , calf brain , infusion from 200.000 beef heart , infusion from 250.000 proteose peptone 10.000 dextrose 2.000 sodium chloride 5.000 disodium phosphate 2.500 final ph at 25c the samples were then subjected to microbiological analysis ( culture and 16s rrna [ 16s ribosomal rna ] gene sequencing ) . all the samples were cultured on brain heart infusion broth m210 and incubated in aerobic and anaerobic conditions for 2448 h at 37c . a variety of molecular techniques are used in identification and estimating the phylogenetic relatedness of prokaryotes , the comparison of 16s rrna isolated from thousands of strains is of particular importance . 16s rrnas of bacteria and archaea are used to determine the phylogenetic relationship among these organisms . the advantage of using 16s rrna is that it is found in all organisms and large enough molecules provide a significant number of nucleotides to compare sequence . subjects with definite contraindications for the use of ultrasonic and sonic scaling devices , for example , patients with known communicable diseases that can be transmitted by aerosolssubjects with a cardiac pacemakersubjects with respiratory diseases such as chronic pulmonary disordersimmunocompromised subjectssubjects with titanium implants , which can be etched or gougedpregnant or lactating mothers . subjects with definite contraindications for the use of ultrasonic and sonic scaling devices , for example , patients with known communicable diseases that can be transmitted by aerosols subjects with a cardiac pacemaker subjects with respiratory diseases such as chronic pulmonary disorders immunocompromised subjects subjects with titanium implants , which can be etched or gouged pregnant or lactating mothers . after subject selection , 15 subjects were randomly assigned to each treatment group ( using a computer - generated system- the randomization scheme was generated by using the web site randomization.com , http://www.randomization.com ) , group a 15 dentists wearing contact lenses and protective eyewear , group b 15 dentists wearing contact lenses only . the lenses were aseptically worn after wearing a fresh sterile particle free glove ( fisher scientific particle - free gloves ) . scaling and root planing were rendered with piezoelectric ultrasonic scalers ( piezoelectric scaler [ biosonic ] ) in combination with high volume evacuation ( hve ) for about 30 min in both the groups . after scaling and root planing with ultrasonic scalers , the lenses were aseptically collected wearing a fresh sterile particle free glove and transferred into a sterile test tube containing brain heart infusion broth m210 ( composition - ingredients gm / l , calf brain , infusion from 200.000 beef heart , infusion from 250.000 proteose peptone 10.000 dextrose 2.000 sodium chloride 5.000 disodium phosphate 2.500 final ph at 25c the samples were then subjected to microbiological analysis ( culture and 16s rrna [ 16s ribosomal rna ] gene sequencing ) . all the samples were cultured on brain heart infusion broth m210 and incubated in aerobic and anaerobic conditions for 2448 h at 37c . a variety of molecular techniques are used in identification and estimating the phylogenetic relatedness of prokaryotes , the comparison of 16s rrna isolated from thousands of strains is of particular importance . the advantage of using 16s rrna is that it is found in all organisms and large enough molecules provide a significant number of nucleotides to compare sequence . contingency coefficient test all the samples were cultured on brain heart infusion broth m210 and incubated in aerobic and anaerobic conditions for 2448 h at 37c . after 48 h of incubation , in group a ( contact lens + with protective eyewear ) 15 out of thirty samples [ figure 2 ] and in group b ( contact lens + without protective eyewear ) all the thirty samples were contaminated [ figure 3 and table 1 ] . out of sixty samples , 45 were contaminated and of 45 samples , five samples showed fungal growth ( two samples in group a and three in group b [ figures 4 , 5 and table 1 ] ) . group a showing fungal and other bacterial colonies group b showing bacterial growth around the lens contamination groups cross tabulation group a showing fungal growth group b showing fungal growth around the periphery of the contact lens grams ' staining was done for forty contaminated samples which revealed most of the samples were gram - positive bacteria [ figures 6 and 7 ] . group a and group b showing gram positive bacteria at 4x group a and group b showing gram positive bacteria at 20x out of sixty samples , 45 were contaminated . in 45 samples , ten samples were contaminated with streptococcus mutans , three with staphylococcus aureus , and two with fungi . in group 21 samples were contaminated with s. mutans , six with s. aureus , and three with fungi . ( in total 31 samples were contaminated with s. mutans strain , nine with s. aureus and five with fungi . ) overall , the results of this study indicate low microbial contamination of contact lens in group a ( contact lens with protective eyewear ) when compared to group b ( contact lens without protective eyewear ) which is statistically significant ( p < 0.01 ) [ table 2 ] . however , even after wearing protective eyewear , there is contamination of the contact lens which can be very hazardous to the eyes [ flowchart 1 ] . all the samples were cultured on brain heart infusion broth m210 and incubated in aerobic and anaerobic conditions for 2448 h at 37c . after 48 h of incubation , in group a ( contact lens + with protective eyewear ) 15 out of thirty samples [ figure 2 ] and in group b ( contact lens + without protective eyewear ) all the thirty samples were contaminated [ figure 3 and table 1 ] . out of sixty samples , 45 were contaminated and of 45 samples , five samples showed fungal growth ( two samples in group a and three in group b [ figures 4 , 5 and table 1 ] ) . group a showing fungal and other bacterial colonies group b showing bacterial growth around the lens contamination groups cross tabulation group a showing fungal growth group b showing fungal growth around the periphery of the contact lens grams ' staining was done for forty contaminated samples which revealed most of the samples were gram - positive bacteria [ figures 6 and 7 ] . group a and group b showing gram positive bacteria at 4x group a and group b showing gram positive bacteria at 20x ten samples were contaminated with streptococcus mutans , three with staphylococcus aureus , and two with fungi . in group 21 samples were contaminated with s. mutans , six with s. aureus , and three with fungi . ( in total 31 samples were contaminated with s. mutans strain , nine with s. aureus and five with fungi . ) overall , the results of this study indicate low microbial contamination of contact lens in group a ( contact lens with protective eyewear ) when compared to group b ( contact lens without protective eyewear ) which is statistically significant ( p < 0.01 ) [ table 2 ] . however , even after wearing protective eyewear , there is contamination of the contact lens which can be very hazardous to the eyes [ flowchart 1 ] . the interest in dental aerobiology has been stimulated by concern with the obvious splatter caused by the visible particles emanating from the patient 's mouth during dental procedures . the driving force to conduct this study is an observation that dentists do not wear protective eyewear while rendering treatment which is very important in preventing damage to the eyes . the interest of the present study was to determine aerosol contamination of contact lenses of the dentist after scaling and root planing using a piezoelectric scaler in combination with hve , qualitative analysis of microorganisms on contact lenses of the dentist , and to determine the effectiveness of protective eyewear in preventing the contamination of the contact lens . the oral cavity harbors numerous bacteria and viruses from the respiratory tract , dental plaque , and oral fluids . it is a unique environment which provides an ideal medium for the growth of bacteria . any cross infection can result even in the death of the person in case of an immunosuppressed patient . aerosols may be heavily contaminated with oral organisms and present a considerable microbial challenge to the dentist , patients , and the nursing staff . this study is the first of its kind as per our knowledge where we investigated the contamination of contact lenses of the dentists after scaling and root planing with ultrasonic scalers . scaling and root planing was rendered for 30 min after which the lenses were aseptically collected and cultured in both aerobic and anaerobic conditions . the microbial contamination of contact lens in group a was less when compared to group b , ( statistical significance was defined as p < 0.01 , which is highly significant ) proving the importance of wearing protective eyewear . however , even after wearing protective eyewear , there was contamination of the contact lenses which can be very hazardous to the eyes . demonstrated that the aerosol produced by the in vivo use of an ultrasonic scaler on periodontally involved teeth was contaminated with blood and that the contamination occurred regardless of the level of inflammation . in a study by bhat , sterile swabs were used to collect samples from the chest area and pocket area of the aprons / overcoats of dental students which were then cultured , the results showed that there was microbial contamination of overcoats of dental students . many studies have confirmed that an aerosolized bacterial contamination is produced during the use of ultrasonic scalers and other dental equipment that produce an aerosol spray . demonstrated that dental operations involving air and water sprays in combination with rotating instruments may cause levels of contamination exceeding those produced by common oral activities . they showed that a sneeze and the use of the air turbine handpiece produced comparable aerosols and splatter . a considerable bacterial challenge exists in the aerosol produced by ultrasonic scalers , and it is probable that viruses and bacteria may be spread via this route . however , studies have linked an increase in respiratory illness to the use of ultrasonic scalers . people choose to wear contact lenses for many reasons such as visual , esthetics , and cosmetics which are often motivating factors for people who would like to avoid wearing glasses or change the appearance of their eyes . contact lenses do not protect the eyes and can increase the risk of exposure to microorganisms if contaminated fluids gain access beneath the lens . eyes are particularly vulnerable to injury by high - velocity particles / debris generated during use of high - speed handpieces and ultrasonic scalers . it has been reported that the environment , the type of contact lens , the duration of wear , and the type of contact lens cleansing solution determined the microbial load on the contact lenses . the normal ocular microbiota in the absence of contact lens wear is composed almost exclusively of bacterial types such as corynebacterium sp . subsequent to adhesion , it is likely that bacteria further colonize the lens surface by growing on that lens surface . hume and willcox demonstrated that s. marcescens was able to grow on a contact lens after adhesion to contact lenses coated with an artificial tear film . the unique structure of the human eye , the use of contact lenses and the constant exposure of the eye directly to the environment renders it vulnerable to a number of uncommon infectious diseases caused by microorganisms . protective measures recommended to reduce the risk of infection from aerosols may be classified as physical , chemical , and personal safeguards . this study highlights the importance of protective eyewear in the prevention of contamination of the contact lenses . going by the results of this study , we would like to conclude that dental practitioners should better avoid contact lenses in a dental setup because of the risk of contamination of the contact lenses from the various dental procedures which can produce aerosol / splatter and if worn it is recommended to wear protective eyewear . therefore , similar studies with a larger sample size and a negative control are required to affirm the observations of our study .

in the mammalian brain the cerebrospinal fluid ( csf ) is produced by the choroid plexus ( cp ) , which not only regulates homeostasis in the central nervous system ( cns ) , but also participates in neurohumoral brain modulation as well as neuroimmune interaction [ 1 , 2 ] . the cp is a highly perfused organ ; the endothelial and epithelial cells of the cp separate the blood from the csf . whereas the endothelial cells are fenestrated , the epithelial cells of the cp are closely connected to each other by tight junctions ( tjs ) and constitute the structural basis of the blood - csf barrier . the barrier function of the tjs can be subject to modulation and thereby regulates the entry of physiologically important substances as well as immune modulatory components and cells during inflammatory events [ 3 , 4 ] . over the course of neuroinflammatory diseases , including meningitis after infection with bacterial pathogens , the composition of the csf undergoes significant changes including the accumulation of certain cytokines [ 5 , 6 ] . in agreement , expression of the proinflammatory cytokine tnf is markedly increased in porcine choroid plexus epithelial cells after infection with the gram - positive bacterium streptococcus suis . subsequently , injury of the cp due to inflammatory responses may cause further impairment of the blood - csf barrier and allow enhanced entry of immune system cells into the cns [ 1 , 2 ] . tnf exhibits pleiotropic functions and is involved in several cellular processes including inflammation , immune responses , cell death and cell proliferation . tnfr1 is expressed on all cells , whereas tnfr2 is found on endothelial cells and cells of the immune system [ 810 ] . signalling through tnfr ligation can initiate ( i ) programmed cell death ( apoptosis ) , ( ii ) antiapoptotic and proinflammatory responses through nf-b , and ( iii ) activation of the mitogen activated protein kinase ( mapk ) jnk signalling pathway . these signal transduction mechanisms are tightly interconnected with each other through a complex network and their modulation allows to shifting the cellular state either towards survival or cell death [ 11 , 12 ] . employing an in vitro model of the blood - csf barrier we have previously shown that cell death by apoptosis and necrosis participates in barrier loss of porcine choroid plexus epithelial cells ( pcpec ) after infection with streptococcus suis . furthermore , treatment of pcpec with tnf caused distinct inflammatory responses including tight junction and actin cytoskeleton disorganisation , the upregulation of cell adhesion molecules and an increased activity of matrix metalloproteases in pcpec supernatants . we now investigated in detail the involvement of apoptotic and non - apoptotic mechanisms in the modulation of pcpec barrier function in response to treatment with tnf. epithelial cells from porcine choroid plexus were obtained by a modified preparation as basically described by gath et al . . briefly , brains from freshly slaughtered pigs were dissected and the choroid plexus tissue from the lateral and the fourth ventricles was removed and treated with consecutive cold and warm trypsinisation ( 0.2% solution , biochrom , berlin , germany , 45 minutes at 4c , 20 minutes at 37c ) . the cells were centrifuged at 20 g for 10 minutes and resuspended in dmem / ham 's f12 1 : 1 supplemented with 4 mm l - glutamine , 10% heat inactivated fetal calf serum , 5 g / ml insuline , and penicillin ( 100 u / ml)/streptomycin ( 100 g / ml ) . in order to suppress the growth of contaminating fibroblast like cells , 20 m of cytosine - arabinoside were added . the cells were either plated on 6- , 24- , or 96-well plates ( falcon , bd , le pont de claix , france ) using a seeding density of 50 cm / g wet weight of choroid plexus tissue or on laminin coated permeable transwell filter membranes ( costar , cambridge , usa ) with a diameter of 12 mm . upon confluence , pcpec had a seeding density of approximately 1 10 cells / cm . after reaching confluence they were cultivated in serum - free medium and were used for the experiments 35 days later . all experiments were performed with cells that had been cultured in sfm for at least 3 - 4 days and exhibited high teer values of at least 1000 ohm cm on transwell filter membranes . to investigate pcpec barrier function recombinant porcine tnf ( r&d systems , minneapolis , usa ) was applied at 1 , 10 , or 100 ng / ml apically or basolaterally as indicated in the respective experiments . the protein synthesis inhibitor cycloheximide was used at a concentration of 1 g / ml , the nf-b inhibitor caffeic acid phenethyl ester ( cape ) at 5 m , the jnk - inhibitor sp600125 at 10 m ( all products were purchased from merck , darmstadt , germany ) , and the pan - caspase - inhibitor n - benzyloxycarbonyl - val - ala - asp - fluoromethyl - ketone ( zvad - fmk ; icn , heidelberg , germany ) was used at 10 m . staurosporine ( calbiochem , germany ) was added at a concentration of 1 m . confluence of the pcpec monolayers and barrier properties were documented by measuring teer using an epithelial tissue voltohmmeter ( evom , world precision instruments , sarasota , fl , usa ) and the stx-2 electrode system . pcpec inverted cultures were used when teer values reached more than 1000 cm . in tnf assays teer resistance values of cells in medium alone were used as negative control values and stayed above 1000 cm during all experiments . teer changes reflect variation in tight junction integrity , although they may also indicate changes in permeability at the membrane level . as an independent measure of paracellular permeability of choroid plexus epithelium monolayers , we examined the passage of a membrane - impermeable water - soluble compound fitc - inulin ( sigma , deisenhofen , germany ) across cell monolayers . the fitc - inulin flux was determined by measuring its paracellular passage from the apical to the basolateral compartment of the transwell filter . the tracer solution ( 100 g / ml ) was loaded into the apical transwell filter compartment during the incubation period . after stimulation with tnf medium samples were collected from the basolateral transwell compartment over a range of poststimulation intervals of 24 hours and the fluorescence was determined by measurement in a tecan infinite m200 multiwell reader ( tecan , switzerland ) . for identification of apoptotic cells an elisa was used measuring intracellular histone - associated dna fragments according to the manufacturer 's protocol ( cell death detection elisa plus ; roche molecular biochemicals , mannheim , germany ) . this quantitative sandwich enzyme immunoassay specifically detects the histone region of mono- and oligonucleosomes that are released during apoptosis . pcpecs were stimulated with tnf for 8 hours , 24 hours , and 48 hours with or without the indicated inhibitors . after stimulation the cells were lysed and the supernatant containing the nuclear derived histone - associated dna fragments was used for elisa . absorption was monitored by reading the plate at 405 nm using a flow titertek multiscan plus ( icn , heidelberg , germany ) . apoptosis was measured in duplicate from each group and expressed as the absorbance ( optical density ) of the experimental cell lysates after 30 minutes of development . caspase activity was measured using a fluorometric assay as described in . in brief , of tnf stimulated cells pcpecs were first lysed in 75 l lysis buffer to collect their intracellular contents . for the fluorometric cleavage assay , 50 g of the cell extracts were directly dissolved in 200 l substrate buffer ( 50 mm hepes ph 7.3 , 100 mm nacl , 10% sucrose , 0.1% chaps , 10 mm dtt ) supplemented with 50 m of the fluorogenic substrates ac - devd - amc for caspase-3 , ac - ietd - amc for caspase-8 , or ac - lehd - amc for caspase-9 ( mp biomedicals , eschwege , germany ) . the release of aminomethyl - coumarin was measured fluorometrically over 5 hours at 37c using a lambda fluro 320 plus fluorometer ( biotek , bad friedrichsall , germany ; excitation : 360 nm , emission : 475 nm ) . vitality of the cells was measured using life / dead assay ( molecular probes , gttingen , germany ) . calcein penetrates the membrane of vital cells , and esterases in the cytoplasm render it fluorescent ( green ) . ethidium homodimer is not able to penetrate intact cell membranes but adheres to nucleic acids of damaged cells ( red fluorescent cells ) . for cell staining a 2 m solution ethidium homodimer and a 4 m solution of calcein - am were used . cell integrity was determined after treatment by measuring the lactate dehydrogenase activity in the culture supernatant using a commercially available kit ( roche , mannheim , germany ) . ldh release into the medium was expressed as the percentage of total ldh ( i.e. , ldh present in medium and the cells ) . after stimulation with tnf cells were washed with hbss / hepes , fixed with pbs/4% paraformaldehyde , and permeabilized with pbs/0.5% hmgb1 was detected by indirect immunofluorescence using an anti - hmgb1 antibody ( bd biosiences , heidelberg , germany ) at 1 : 500 dilution for 12 hours at 4c and chicken anti - rabbit antibody coupled to alexa fluor 594 ( molecular probes , oregon , usa ) at 1 : 250 dilution for 15 minutes at 4c . cells were also stained with 4-6-diamidino-2-phenylindole dihydrochloride ( dapi , 100 ng / ml ) dilution for 5 minutes at room temperature and observed on an axioplan 2 microscope with filter set 2 and 31 ( carl zeiss , gttingen , germany ) . the statistical significance between the groups comparisons was determined by student 's t - test . to investigate the influence of tnf on pcpec barrier function we treated pcpec monolayers grown on transwell filters with tnf for 24 hours and measured the transepithelial electrical resistance ( teer ) . addition of increasing amounts of tnf to the apical ( figure 1(a ) ) or basolateral ( figure 1(b ) ) side of pcpec caused a dose - dependent drop of the teer under both conditions at 10 ng / ml and 100 ng / ml of tnf , but not at 1 ng / ml . supression of protein synthesis by cotreatment with the translation elongation inhibitor cycloheximide ( chx ) leads to a strong decrease of the teer under all conditions investigated . treatment with chx alone also leads to a significant decrease of the teer ( figures 1(a ) and 1(b ) ) . since apical and basolateral tnf application with or without chx caused similar effects on tnf signaling is mediated by tnfrs and involves induction of apoptosis as well as activation of nf-b and jnk signaling pathways [ 911 ] . to dissect the contribution of the three above - mentioned pathways to modulation of pcpec barrier function by apically added tnf , we preincubated pcpec grown on transwell filters with specific inhibitors prior to treatment with tnf in absence or presence of chx . to inhibit apoptosis , the cells were preincubated for 2 hours with 10 m of the pan - caspase inhibitor zvad - fmk . for attenuation of either nf-b or jnk signaling , a 2 hours preincubation step with a low ( 5 m ) concentration of the nf-b inhibitor caffeic acid phenethyl ester ( cape ) or with 10 m of the jnk inhibitor sp600125 , respectively , was conducted . as can be seen in figure 2(a ) pretreatment with all three inhibitors caused a slight but significant attenuation of the teer drop caused by subsequent treatment with tnf alone , whereas none of the inhibitors by itself caused a significant change in teer . when cells where stimulated with a combination of tnf and chx , pretreatment with both zvad - fmk and cape significantly inhibited the observed strong decrease in teer , with zvad - fmk being a more potent inhibitor then cape ( figure 2(b ) ) . under these conditions sp600125 exhibited no significant effect ( figure 2(b ) ) . in addition to the teer values the paracellular inulin flux levels can be regarded as a measure for the barrier function of epithelia . determination of the inulin flux revealed that apical addition of tnf alone to the pcpec caused a slight but significant increase in permeability ( figure 3(a ) ) . in contrast , coincubation of the pcpec with tnf and chx leads to a strong increase in inulin flux ( figure 3(b ) ) . under both conditions preincubation with zvad - fmk and cape significantly attenuated the gain in inulin - flux , although zvad - fmk demonstrated a stronger effect when pcpec were activated with tnf alone . pretreatment with the jnk inhibitor sp600125 did not reveal an effect on permeability under any of the conditions investigated ( figure 3 ) . one of the hallmarks of apoptosis is the appearance of oligonucleosomal dna fragmentation caused by the caspase - activated dna nuclease , cad [ 17 , 18 ] . to investigate the level of apoptotic cell death in pcpec after apical treatment with tnf in the presence or absence of chx we measured the appearance of cytoplasmic histone - associated dna fragments by elisa . addition of tnf alone resulted in a slight but significant increase in the apoptosis after 24 hours and 48 hours . in contrast , coincubation of pcpec with tnf and chx leads . to a strong induction of programmed cell death already after 8 hours , which further increased after 24 hours and 48 hours ( figure 4(a ) ) . treatment of pcpec with different doses of tnf ( 1 , 10 , 100 ng / ml ) for 24 hours caused only low levels of cell death in absence of chx . in contrast significant amounts of apoptosis were observed when pcpec were preincubated with chx ( figure 4(b ) ) . levels of apoptosis correlated to some extend with the loss of pcpec barrier function after treatment with tnf in presence and absence of chx . additionally to the induction of apoptosis , tnf activates signalling by nf-b and jnk , which are both known to contribute to inhibition of apoptosis due to the activation of antiapoptotic gene expression . therefore , we next analyzed the effect of zvad - fmk , cape , and sp600125 on the appearance of oligonucleosomal dna fragmentation in pcpec . inhibition of caspases by zvad - fmk attenuated dna fragmentation of pcpec treated apically with tnf in the absence ( figure 5(a ) ) or presence ( figure 5(b ) ) of chx . in contrast , cape and sp600125 had no inhibitory effect under all conditions analysed ( figure 5 ) . sp600125 did even lead to a slight increase in dna fragmentation when compared to tnf alone ( figure 5(a ) ) . the three inhibitors zvad , cape and sp600125 alone had no effect on dna fragmentation ( data not shown ) . to evaluate apoptosis in pcpec in more detail we measured activation of caspase-3 by a fluorometric assay ( figure 6 ) . noteworthy , active caspase-3 levels in cells treated with tnf for 24 hours were similar in absence ( figure 6(a ) ) and presence ( figure 6(b ) ) of cotreatment with chx . in agreement with the appearance of dna fragmentation , of the three inhibitors only zvad - fmk was able to reduce the level of caspase-3 activation . again , preincubation with sp600125 might cause rather a slight increase in activation of caspase-3 in cells treated with tnf alone . zvad - fmk , cape or sp600125 did not cause caspase-3 activation in absence of tnf treatment ( figure 6(a ) ) . treatment of cells with cytotoxic stimuli can result in both apoptosis and necrosis . in vivo , in contrast , necrosis is characterized by the release of intracellular components leading to a proinflammatory response in surrounding tissues [ 19 , 20 ] . to analyze the cytotoxic effects of tnf on pcpec in more detail , we measured the release of lactate dehydrogenase ( ldh ) , which is more closely associated with necrosis than with apoptosis . tnf alone caused a slight but significant ldh - release , which could only be attenuated by cape , but not by zvad - fmk or sp600125 ( figure 7(a ) ) . when pcpec were treated with the combination of tnf and chx a stronger ldh - release this release was inhibited by both zvad - fmk and cape ( figure 7(b ) ) . treatment of pcpec with zvad - fmk , cape or sp600125 ( figure 7(a ) ) or with chx ( figure 7(b ) ) alone had no cytotoxic effect . the high mobility group 1 ( hmgb1 ) protein is known to be associated with condensed chromatin or to be released from cells undergoing either apoptosis or necrosis , respectively , . pcpec treated with tnf in presence or absence of chx were stained with dapi and analyzed by immunofluorescence with an antibody against hmgb1 . apoptotic cells resulting from the different treatments contained hmgb1 , confirming programmed cell death caused by either tnf or tnf in combination with chx . cotreatment of pcpec with tnf and chx as well as addition of staurosporine produced also cells with their hmgb1 released indicative of post - apoptotic necrosis ( figure 8(b ) ) . increased amounts of dead cells after incubation with the combination of tnf and chx compared to treatment with tnf alone were confirmed by a live / dead assay ( figure 8(a ) ) . the participation of proinflammatory cytokines in the pathophysiology of inflammatory cns disease as bacterial meningitis is well recognized . such cytokines have been implicated in epithelial barrier dysfunction [ 22 , 23 ] and in inducing epithelial cell apoptosis . our study was designed to provide a detailed insight into the mechanism(s ) by which proinflammatory cytokines like tnf compromise pcpec barrier function , and whether or not these are causally linked to apoptosis . significant amount of tnf ( 45 ng / ml ) has been observed in the csf of rabbits with experimental meningitis caused by haemophilus influenzae type b lipooligosaccharide . additionally , concentrations reaching up to 40 ng / ml ( mean ~15 ng / ml ) have been described in serum for a porcine model of meningococcal shock . the epithelial cells of the choroid plexus constitute the structural basis of the blood - csf barrier and have been shown to respond to inflammatory challenge by the pleiotropic cytokine tnf in an in vitro model employing primary porcine choroid plexus epithelial cells . during these studies tnf was shown to alter tight junction function in part via matrix - metalloproteases . interestingly , the gene encoding for tnf , among other inflammatory response genes , is upregulated in the same cellular system after infection with the gram - positive bacterium streptococcus suis . infection with s. suis affects the barrier function of the pcpec in part by inducing cell death of pcpec by apoptotic and necrotic mechanisms [ 7 , 13 ] . tnf is known to exert its functions mainly via two tnfrs , tnfr1 and tnfr2 [ 810 ] . although to our knowledge the distribution of tnfrs in the porcine brain has not been investigated , data from an in vivo rat model have indicated that in the choroid plexus tnfr1 is basally expressed and upregulated after challenge with lipopolysaccharide ( lps ) or tnf , whereas low tnfr2 expression could only be detected after lps or tnf treatment . we tested several antibodies against tnfr1 and tnfr2 , but none of them recognized porcine tnfrs and therefore we could neither determine potential inhibitory effects ( data not shown ) . tnf induces distinct downstream regulatory pathways , notably tnfr - mediated apoptosis and induction of the nf-b and jnk signaling pathways . in the present study we investigated the downstream effects contributing to choroid plexus epithelial cell barrier loss caused by tnf in further detail . treatment of pcpec with tnf for 24 hours caused low levels of apoptosis as evidenced by dna fragmentation , chromatin condensation and activation of caspase-3 , which could be overcome by the addition of zvad - fmk . under the same conditions a significant drop of the transepithelial membrane potential concomitant with an increase in paracellular permeability occurred indicating an impairment of barrier function . in ht-29/b6 epithelial cells leaks in epithelial barrier due to single - cell apoptosis , particularly following incubation with tnf , have been reported . however , inhibition of tnf-induced pulmonary epithelial cell apoptosis only partially restored barrier function , which underscores the complexity of the mechanisms that regulate the biological response to this cytokine in inflammatory injury cascades . in our experiments , also a significant ldh release , which could be attenuated by the nf-b inhibitor cape but not zvad - fmk , was observed pointing to additional necrotic events . however , even pretreatment with cape or zvad - fmk could not completely inhibit loss of pcpec barrier function . these data support the hypothesis that not only cell death by apoptosis and necrosis , but also cell death independent effects contribute to blood - csf barrier breakdown during inflammation in the cns [ 13 , 14 , 2831 ] . hallmarks of apoptosis ( dna fragmentation , chromatin condensation ) were strongly increased when protein synthesis was inhibited by chx . this is most likely caused by the inhibition of nf-b mediated induction of survival and/or antiapoptotic proteins . in parallel necrosis markers like ldh release and the detachment of hmgb1 from chromatin were strongly enhanced . these phenomena are most likely caused by post - apoptotic events or secondary necrosis occurring at late time points after the onset of apoptosis , which was initiated 24 hours prior to analysis of the cells . this is supported by the observation that caspase-3 activity in cells treated with the combination of tnf and chx was similar to that of cells treated with tnf only . it is likely that the cells had already passed maximum apoptosis and caspase-3 activity at this time point and went into post - apoptotic necrosis . cape has been described to inhibit the activation of nf-b and therefore it prevents the increased expression of antiapoptotic proteins , which explains antiinflammatory and proapoptotic effects of cape [ 3436 ] . noteworthy , in our analyses preincubation of tnf-induced cells with cape did not lead to increased apoptosis as might be expected . this is presumably due to the rather low concentrations ( 5 m ) of cape applied , which might not have a strong enough impact on regulation of antiapoptotic gene expression mediated by nf-b . low concentrations of cape as used by others [ 37 , 38 ] were employed since higher amounts displayed cytotoxicity in pcpec ( data not shown ) . still , cape was added in active concentrations , because preincubation with cape attenuated loss of barrier function caused by tnf or the combination of tnf with chx . tnf was shown to increase tight junction permeability via nf-b activation in brain microvascular endothelial cells and caco2 intestinal epithelial cells [ 37 , 39 ] . in line with our experiments , in the study by ma and coworkers tnf alone did not lead to a substantial amount of apoptosis and cotreatment with nf-b inhibitors prevented loss of barrier function . cape has been shown to exhibit antioxidant , antiinflammatory and immunomodulatory characteristics and can alter the intracellular redox state . possibly , cape inhibits additional downstream effects of nf-b , which are involved in barrier breakdown . since cape did not inhibit the apoptotic hallmarks caused at low levels by tnf and in higher amounts by tnf in combination with chx , these downstream effects apparently do not involve apoptotic phenomena . we speculate that this effect is due to the inhibition of late proapoptotic functions mediated by jnk after prolonged induction with tnf [ 12 , 41 , 42 ] . otherwise , we observed no significant effects of the jnk inhibitor , indicating that jnk signalling plays a rather minor role in tnf mediated barrier breakdown . in summary , our data point to a role of apoptotic and necrotic phenomena as well as cell death independent mechanisms during the loss of pcpec barrier function after treatment with tnf , mediated via tnfr downstream pathways involving activation of caspases and signaling by nf-b .

the choroid plexus epithelium constitutes the structural basis of the blood - cerebrospinal fluid barrier . since the cytokine tnf is markedly increased during inflammatory diseases in the blood and the central nervous system , we investigated by which mechanisms tnf induces barrier alteration in porcine choroid plexus epithelial cells . we found a dose - dependent decrease of transepithelial electrical resistance , increase of paracellular inulin - flux , and induction of histone - associated dna fragmentation and caspase-3 activation after tnf stimulation . this response was strongly aggravated by the addition of cycloheximide and could partially be inhibited by the nf-b inhibitor cape , but most effectively by the pan - caspase - inhibitor zvad - fmk and not by the jnk inhibitor sp600125 . partial loss of cell viability could also be attenuated by cape . immunostaining showed cell condensation and nuclear binding of high - mobility group box 1 protein as a sign of apoptosis after tnf stimulation . taken together our findings indicate that tnf compromises pcpec barrier function by caspase and nf-b dependent mechanisms .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

in the mammalian brain the cerebrospinal fluid ( csf ) is produced by the choroid plexus ( cp ) , which not only regulates homeostasis in the central nervous system ( cns ) , but also participates in neurohumoral brain modulation as well as neuroimmune interaction [ 1 , 2 ] . the cp is a highly perfused organ ; the endothelial and epithelial cells of the cp separate the blood from the csf . whereas the endothelial cells are fenestrated , the epithelial cells of the cp are closely connected to each other by tight junctions ( tjs ) and constitute the structural basis of the blood - csf barrier . the barrier function of the tjs can be subject to modulation and thereby regulates the entry of physiologically important substances as well as immune modulatory components and cells during inflammatory events [ 3 , 4 ] . over the course of neuroinflammatory diseases , including meningitis after infection with bacterial pathogens , the composition of the csf undergoes significant changes including the accumulation of certain cytokines [ 5 , 6 ] . in agreement , expression of the proinflammatory cytokine tnf is markedly increased in porcine choroid plexus epithelial cells after infection with the gram - positive bacterium streptococcus suis . subsequently , injury of the cp due to inflammatory responses may cause further impairment of the blood - csf barrier and allow enhanced entry of immune system cells into the cns [ 1 , 2 ] . tnfr1 is expressed on all cells , whereas tnfr2 is found on endothelial cells and cells of the immune system [ 810 ] . signalling through tnfr ligation can initiate ( i ) programmed cell death ( apoptosis ) , ( ii ) antiapoptotic and proinflammatory responses through nf-b , and ( iii ) activation of the mitogen activated protein kinase ( mapk ) jnk signalling pathway . employing an in vitro model of the blood - csf barrier we have previously shown that cell death by apoptosis and necrosis participates in barrier loss of porcine choroid plexus epithelial cells ( pcpec ) after infection with streptococcus suis . furthermore , treatment of pcpec with tnf caused distinct inflammatory responses including tight junction and actin cytoskeleton disorganisation , the upregulation of cell adhesion molecules and an increased activity of matrix metalloproteases in pcpec supernatants . we now investigated in detail the involvement of apoptotic and non - apoptotic mechanisms in the modulation of pcpec barrier function in response to treatment with tnf. epithelial cells from porcine choroid plexus were obtained by a modified preparation as basically described by gath et al . briefly , brains from freshly slaughtered pigs were dissected and the choroid plexus tissue from the lateral and the fourth ventricles was removed and treated with consecutive cold and warm trypsinisation ( 0.2% solution , biochrom , berlin , germany , 45 minutes at 4c , 20 minutes at 37c ) . the cells were centrifuged at 20 g for 10 minutes and resuspended in dmem / ham 's f12 1 : 1 supplemented with 4 mm l - glutamine , 10% heat inactivated fetal calf serum , 5 g / ml insuline , and penicillin ( 100 u / ml)/streptomycin ( 100 g / ml ) . the cells were either plated on 6- , 24- , or 96-well plates ( falcon , bd , le pont de claix , france ) using a seeding density of 50 cm / g wet weight of choroid plexus tissue or on laminin coated permeable transwell filter membranes ( costar , cambridge , usa ) with a diameter of 12 mm . to investigate pcpec barrier function recombinant porcine tnf ( r&d systems , minneapolis , usa ) was applied at 1 , 10 , or 100 ng / ml apically or basolaterally as indicated in the respective experiments . the protein synthesis inhibitor cycloheximide was used at a concentration of 1 g / ml , the nf-b inhibitor caffeic acid phenethyl ester ( cape ) at 5 m , the jnk - inhibitor sp600125 at 10 m ( all products were purchased from merck , darmstadt , germany ) , and the pan - caspase - inhibitor n - benzyloxycarbonyl - val - ala - asp - fluoromethyl - ketone ( zvad - fmk ; icn , heidelberg , germany ) was used at 10 m . confluence of the pcpec monolayers and barrier properties were documented by measuring teer using an epithelial tissue voltohmmeter ( evom , world precision instruments , sarasota , fl , usa ) and the stx-2 electrode system . as an independent measure of paracellular permeability of choroid plexus epithelium monolayers , we examined the passage of a membrane - impermeable water - soluble compound fitc - inulin ( sigma , deisenhofen , germany ) across cell monolayers . the fitc - inulin flux was determined by measuring its paracellular passage from the apical to the basolateral compartment of the transwell filter . after stimulation with tnf medium samples were collected from the basolateral transwell compartment over a range of poststimulation intervals of 24 hours and the fluorescence was determined by measurement in a tecan infinite m200 multiwell reader ( tecan , switzerland ) . for identification of apoptotic cells an elisa was used measuring intracellular histone - associated dna fragments according to the manufacturer 's protocol ( cell death detection elisa plus ; roche molecular biochemicals , mannheim , germany ) . pcpecs were stimulated with tnf for 8 hours , 24 hours , and 48 hours with or without the indicated inhibitors . after stimulation the cells were lysed and the supernatant containing the nuclear derived histone - associated dna fragments was used for elisa . apoptosis was measured in duplicate from each group and expressed as the absorbance ( optical density ) of the experimental cell lysates after 30 minutes of development . vitality of the cells was measured using life / dead assay ( molecular probes , gttingen , germany ) . calcein penetrates the membrane of vital cells , and esterases in the cytoplasm render it fluorescent ( green ) . cell integrity was determined after treatment by measuring the lactate dehydrogenase activity in the culture supernatant using a commercially available kit ( roche , mannheim , germany ) . , ldh present in medium and the cells ) . after stimulation with tnf cells were washed with hbss / hepes , fixed with pbs/4% paraformaldehyde , and permeabilized with pbs/0.5% hmgb1 was detected by indirect immunofluorescence using an anti - hmgb1 antibody ( bd biosiences , heidelberg , germany ) at 1 : 500 dilution for 12 hours at 4c and chicken anti - rabbit antibody coupled to alexa fluor 594 ( molecular probes , oregon , usa ) at 1 : 250 dilution for 15 minutes at 4c . to investigate the influence of tnf on pcpec barrier function we treated pcpec monolayers grown on transwell filters with tnf for 24 hours and measured the transepithelial electrical resistance ( teer ) . addition of increasing amounts of tnf to the apical ( figure 1(a ) ) or basolateral ( figure 1(b ) ) side of pcpec caused a dose - dependent drop of the teer under both conditions at 10 ng / ml and 100 ng / ml of tnf , but not at 1 ng / ml . supression of protein synthesis by cotreatment with the translation elongation inhibitor cycloheximide ( chx ) leads to a strong decrease of the teer under all conditions investigated . treatment with chx alone also leads to a significant decrease of the teer ( figures 1(a ) and 1(b ) ) . since apical and basolateral tnf application with or without chx caused similar effects on tnf signaling is mediated by tnfrs and involves induction of apoptosis as well as activation of nf-b and jnk signaling pathways [ 911 ] . to dissect the contribution of the three above - mentioned pathways to modulation of pcpec barrier function by apically added tnf , we preincubated pcpec grown on transwell filters with specific inhibitors prior to treatment with tnf in absence or presence of chx . to inhibit apoptosis , the cells were preincubated for 2 hours with 10 m of the pan - caspase inhibitor zvad - fmk . for attenuation of either nf-b or jnk signaling , a 2 hours preincubation step with a low ( 5 m ) concentration of the nf-b inhibitor caffeic acid phenethyl ester ( cape ) or with 10 m of the jnk inhibitor sp600125 , respectively , was conducted . as can be seen in figure 2(a ) pretreatment with all three inhibitors caused a slight but significant attenuation of the teer drop caused by subsequent treatment with tnf alone , whereas none of the inhibitors by itself caused a significant change in teer . when cells where stimulated with a combination of tnf and chx , pretreatment with both zvad - fmk and cape significantly inhibited the observed strong decrease in teer , with zvad - fmk being a more potent inhibitor then cape ( figure 2(b ) ) . in addition to the teer values the paracellular inulin flux levels can be regarded as a measure for the barrier function of epithelia . determination of the inulin flux revealed that apical addition of tnf alone to the pcpec caused a slight but significant increase in permeability ( figure 3(a ) ) . in contrast , coincubation of the pcpec with tnf and chx leads to a strong increase in inulin flux ( figure 3(b ) ) . under both conditions preincubation with zvad - fmk and cape significantly attenuated the gain in inulin - flux , although zvad - fmk demonstrated a stronger effect when pcpec were activated with tnf alone . pretreatment with the jnk inhibitor sp600125 did not reveal an effect on permeability under any of the conditions investigated ( figure 3 ) . one of the hallmarks of apoptosis is the appearance of oligonucleosomal dna fragmentation caused by the caspase - activated dna nuclease , cad [ 17 , 18 ] . to investigate the level of apoptotic cell death in pcpec after apical treatment with tnf in the presence or absence of chx we measured the appearance of cytoplasmic histone - associated dna fragments by elisa . addition of tnf alone resulted in a slight but significant increase in the apoptosis after 24 hours and 48 hours . to a strong induction of programmed cell death already after 8 hours , which further increased after 24 hours and 48 hours ( figure 4(a ) ) . treatment of pcpec with different doses of tnf ( 1 , 10 , 100 ng / ml ) for 24 hours caused only low levels of cell death in absence of chx . in contrast significant amounts of apoptosis were observed when pcpec were preincubated with chx ( figure 4(b ) ) . levels of apoptosis correlated to some extend with the loss of pcpec barrier function after treatment with tnf in presence and absence of chx . additionally to the induction of apoptosis , tnf activates signalling by nf-b and jnk , which are both known to contribute to inhibition of apoptosis due to the activation of antiapoptotic gene expression . therefore , we next analyzed the effect of zvad - fmk , cape , and sp600125 on the appearance of oligonucleosomal dna fragmentation in pcpec . inhibition of caspases by zvad - fmk attenuated dna fragmentation of pcpec treated apically with tnf in the absence ( figure 5(a ) ) or presence ( figure 5(b ) ) of chx . sp600125 did even lead to a slight increase in dna fragmentation when compared to tnf alone ( figure 5(a ) ) . the three inhibitors zvad , cape and sp600125 alone had no effect on dna fragmentation ( data not shown ) . in agreement with the appearance of dna fragmentation , of the three inhibitors only zvad - fmk was able to reduce the level of caspase-3 activation . zvad - fmk , cape or sp600125 did not cause caspase-3 activation in absence of tnf treatment ( figure 6(a ) ) . tnf alone caused a slight but significant ldh - release , which could only be attenuated by cape , but not by zvad - fmk or sp600125 ( figure 7(a ) ) . when pcpec were treated with the combination of tnf and chx a stronger ldh - release this release was inhibited by both zvad - fmk and cape ( figure 7(b ) ) . treatment of pcpec with zvad - fmk , cape or sp600125 ( figure 7(a ) ) or with chx ( figure 7(b ) ) alone had no cytotoxic effect . cotreatment of pcpec with tnf and chx as well as addition of staurosporine produced also cells with their hmgb1 released indicative of post - apoptotic necrosis ( figure 8(b ) ) . the participation of proinflammatory cytokines in the pathophysiology of inflammatory cns disease as bacterial meningitis is well recognized . our study was designed to provide a detailed insight into the mechanism(s ) by which proinflammatory cytokines like tnf compromise pcpec barrier function , and whether or not these are causally linked to apoptosis . significant amount of tnf ( 45 ng / ml ) has been observed in the csf of rabbits with experimental meningitis caused by haemophilus influenzae type b lipooligosaccharide . the epithelial cells of the choroid plexus constitute the structural basis of the blood - csf barrier and have been shown to respond to inflammatory challenge by the pleiotropic cytokine tnf in an in vitro model employing primary porcine choroid plexus epithelial cells . interestingly , the gene encoding for tnf , among other inflammatory response genes , is upregulated in the same cellular system after infection with the gram - positive bacterium streptococcus suis . infection with s. suis affects the barrier function of the pcpec in part by inducing cell death of pcpec by apoptotic and necrotic mechanisms [ 7 , 13 ] . tnf is known to exert its functions mainly via two tnfrs , tnfr1 and tnfr2 [ 810 ] . although to our knowledge the distribution of tnfrs in the porcine brain has not been investigated , data from an in vivo rat model have indicated that in the choroid plexus tnfr1 is basally expressed and upregulated after challenge with lipopolysaccharide ( lps ) or tnf , whereas low tnfr2 expression could only be detected after lps or tnf treatment . tnf induces distinct downstream regulatory pathways , notably tnfr - mediated apoptosis and induction of the nf-b and jnk signaling pathways . in the present study we investigated the downstream effects contributing to choroid plexus epithelial cell barrier loss caused by tnf in further detail . treatment of pcpec with tnf for 24 hours caused low levels of apoptosis as evidenced by dna fragmentation , chromatin condensation and activation of caspase-3 , which could be overcome by the addition of zvad - fmk . under the same conditions a significant drop of the transepithelial membrane potential concomitant with an increase in paracellular permeability occurred indicating an impairment of barrier function . in ht-29/b6 epithelial cells leaks in epithelial barrier due to single - cell apoptosis , particularly following incubation with tnf , have been reported . however , inhibition of tnf-induced pulmonary epithelial cell apoptosis only partially restored barrier function , which underscores the complexity of the mechanisms that regulate the biological response to this cytokine in inflammatory injury cascades . in our experiments , also a significant ldh release , which could be attenuated by the nf-b inhibitor cape but not zvad - fmk , was observed pointing to additional necrotic events . however , even pretreatment with cape or zvad - fmk could not completely inhibit loss of pcpec barrier function . these data support the hypothesis that not only cell death by apoptosis and necrosis , but also cell death independent effects contribute to blood - csf barrier breakdown during inflammation in the cns [ 13 , 14 , 2831 ] . hallmarks of apoptosis ( dna fragmentation , chromatin condensation ) were strongly increased when protein synthesis was inhibited by chx . this is most likely caused by the inhibition of nf-b mediated induction of survival and/or antiapoptotic proteins . these phenomena are most likely caused by post - apoptotic events or secondary necrosis occurring at late time points after the onset of apoptosis , which was initiated 24 hours prior to analysis of the cells . this is supported by the observation that caspase-3 activity in cells treated with the combination of tnf and chx was similar to that of cells treated with tnf only . it is likely that the cells had already passed maximum apoptosis and caspase-3 activity at this time point and went into post - apoptotic necrosis . still , cape was added in active concentrations , because preincubation with cape attenuated loss of barrier function caused by tnf or the combination of tnf with chx . tnf was shown to increase tight junction permeability via nf-b activation in brain microvascular endothelial cells and caco2 intestinal epithelial cells [ 37 , 39 ] . in line with our experiments , in the study by ma and coworkers tnf alone did not lead to a substantial amount of apoptosis and cotreatment with nf-b inhibitors prevented loss of barrier function . otherwise , we observed no significant effects of the jnk inhibitor , indicating that jnk signalling plays a rather minor role in tnf mediated barrier breakdown . in summary , our data point to a role of apoptotic and necrotic phenomena as well as cell death independent mechanisms during the loss of pcpec barrier function after treatment with tnf , mediated via tnfr downstream pathways involving activation of caspases and signaling by nf-b .

controlled ovarian hyperstimulation ( coh ) with gonadotropins has improved success rates of in vitro fertilization ( ivf ) by increasing the number and opportunity for selection of embryos before transfer [ 13 ] as well as permitting the cryopreservation of supernumerary embryos for further fertility treatment [ 4 , 5 ] . the basis of coh is to support the growth of multiple follicles to the preovulatory stage , a process achieved by bypassing physiological regulatory mechanisms . urinary - derived or recombinant follicle stimulating hormone ( fsh ) is administered to increase serum concentrations above the threshold required for dominant follicle selection , thus enabling the entire cohort of recruited follicles to develop and attain preovulatory status . luteinising hormone ( lh ) is often coadministered although , following pituitary downregulation , this is not essential for follicular development as remnant basal lh levels are sufficient to stimulate the theca cells . administration of a gnrh analogue ( long protocol ) or an antagonist ( short protocol ) that desensitizes the pituitary is primarily used to prevent premature lh surge as a consequence of supraphysiological serum oestradiol ( e2 ) levels which , if it occurs , can lead to premature luteinisation and/or ovulation . with few exceptions , the last two decades have witnessed a mounting body of evidence indicating that ovarian stimulation has a detrimental effect on oogenesis , embryo quality , and endometrial receptivity [ 713 ] . demonstrated that retrieval of > 10 oocytes per woman adversely affected their quality based on oocyte / embryo morphology , fertilization , and implantation rates . more recently van der gaast et al . found 13 oocytes to be the optimum number retrieved in order to achieve a pregnancy using a long protocol , above which there was a fall in pregnancy rates . an extreme example of the negative impact of coh is the excessively high number of poor quality oocytes seen in ovarian hyperstimulation syndrome ( ohss ) , which is putatively attributable to detrimental supraphysiological e2 levels . these observations in humans are supported by a number of rodent studies that investigated the impact of exogenous gonadotropin stimulation on oocytes and demonstrated a delay in embryo development [ 17 , 18 ] . it has been suggested that gonadotropin stimulation may affect oocyte maturation and the completion of meiosis , thus leading to an increased risk of having aneuploid oocytes and/or embryos [ 10 , 19 ] . as such , in vitro maturation ( ivm ) has been proposed as an alternative strategy since it reduces exposure to exogenous gonadotropin stimulation , but the process itself introduces a host of other variables / complications ( e.g. , disruption of the meiotic spindle ) that do not allow a fair comparison of these approaches to be made . von wolff et al . recently demonstrated a varying endocrine follicular milieu together with the concentration of putative markers of oocyte quality , specifically anti - mllerian hormone ( amh ) between nc and coh ff , and suggest that this may be the cause for the lower oocyte quality following coh compared with naturally matured oocytes . there has also been some concern that suppressed lh concentrations in the late follicular phase may be detrimental through downstream perturbations in follicular steroid synthesis . consequently , stimulation protocols incorporating exogenous lh were developed , resulting in an increase in the percentage of diploid and good quality embryos obtained [ 22 , 23 ] . by contrast , other investigators have reported a reduction in fertility and increased risk of miscarriage when incorporating exogenous lh into protocols [ 24 , 25 ] . lh window below which e2 production is inadequate and above which lh may begin premature luteinisation and be detrimental to follicular development . postulate that the reduced levels of intrafollicular amh they demonstrated following coh compared with nc may be attributed to lh suppression , resulting initially in low follicular androgen concentrations , and subsequently to low amh production which in turn may be responsible for lower oocyte quality . natural cycle ivf ( nc - ivf ) has been proposed as an alternative treatment for older women and poor responders . indeed , there has been a resurgence of interest in nc - ivf for all patients in recent years because it avoids coh and its potential sequelae . moreover , this also supports the international drive to reduce multiple pregnancies rates with elective single embryo transfer and to minimise complications such as ohss [ 2830 ] . conducted a systematic review of 1,800 natural ivf cycles reported between 1989 and 2000 and concluded that nc - ivf has a pregnancy rate of less than 10% per cycle . more recent reports concur , presenting a similar 15.2% live birth rate per initiated cycle in all reported unstimulated ncs in women < 35 years ( n = 795 ) in the united states ( 2006 - 2007 ) . this method incorporates the use of low dose gonadotropin stimulation together with a gonadotropin releasing hormone ( gnrh ) antagonist aimed at generating fewer than eight oocytes per cycle . the term modified natural cycle ivf ( mnc - ivf ) is applied when drugs ( e.g. , human chorionic gonadotropin ( hcg ) ) are used to induce final oocyte maturation whereby a gnrh antagonist is administered during a spontaneous cycle to reduce the risk of cancellation and/or where luteal support is provided . during folliculogenesis , follicular fluid ( ff ) composition exhibits dynamic changes as individual follicular cell types respond to gonadotropins by secreting different hormones and cytokines [ 34 , 35 ] . as growth factors regulating all stages of folliculogenesis , cytokines have been shown to govern the development / function of somatic cells and the oocyte as well as the composition of ff [ 3642 ] . given that oocyte quality influences subsequent embryo viability , it has been suggested that the disruption in the balance of these intrafollicular mediators following coh may influence cycle outcome [ 4452 ] . the correct regulation of cytokine networks is essential to support normal physiology and this central role is underscored by the fact that inflammatory / immune dysfunctions underpin many pathological reproductive conditions , resulting in both local and systemic changes in cytokine profiles [ 5355 ] . for example , the levels of interleukin- ( il- ) 8 , a chemotactic and angiogenic cytokine essential to folliculogenesis , have been found to rise from the midfollicular to the late follicular phase . these levels are comparable to those found during a coh cycle , implying that granulosa cell ( gc ) and theca cell ( tc ) il-8 secretion is a true physiological phenomenon associated with follicular growth / maturation rather than resulting from gonadotropin stimulation . in vitro enhancement of il-8 secretion by cultured gcs and tcs was evident following exposure to il-1 and il-1 , but not tumour necrosis factor- ( tnf- ) , suggesting that il-8 is both gonadotropin and cytokine - induced and may thus be involved in the hormonally regulated stages of folliculogenesis and ovulation . although cytokines are readily detected in ff , the complexity of their network regulation makes their study in isolation difficult to interpret . in view of their biological properties ( pleiotropism , synergy , antagonism , functional redundancy , and differential sensitivity ) [ 5860 ] , cytokines should ideally be investigated in terms of their interrelationships as much as in terms of their absolute concentrations . whilst a recent study by bersinger et al . failed to demonstrate a difference in 13 ff cytokines between women undergoing nc and coh ivf , no specific attention was paid towards the complex interrelations within the cytokine networks . to date , there has been a paucity of studies focusing on minimal stimulation regimens and mncs , and comparisons of isolated cytokine concentrations ( e.g. , vascular endothelial growth factor ( vegf ) in coh and nc - ivf ) have been inconsistent [ 62 , 63 ] . therefore , this pilot study aimed at examining the impact of gonadotropins on the intrafollicular cytokine milieu in mnc and following coh cycles . from november 2008 to march 2009 , ten women who required treatment with ivf / icsi due to unexplained or male factor infertility aged 2535 years with a body mass index ( bmi ) 1930 were selected to undergo mnc - ivf / intracytoplasmic sperm injection ( icsi ) at the assisted conception unit , st james 's university hospital , leeds , uk . they were required to be ovulatory ( confirmed by transvaginal ultrasound scan ( tvuss ) , progesterone levels , or commercial lh surge kits within the preceding three months ) and have a normal endocrine profile ( early follicular phase fsh < 8.0 iu / l and e2 50200 pmol / l ) , a negative infection screen ( including negative serum chlamydia antigens ) , and normal pelvic anatomy confirmed by tvuss and laparoscopy . furthermore , they were required to have no risk factors for pelvic pathology ( e.g. , history of pelvic inflammatory disease , incomplete miscarriage , ectopic pregnancy , cervical dyskaryosis , and abdominal / pelvic / cervical surgery ) . women with coexisting morbidity ( e.g. , autoimmune diseases , inflammatory conditions , and diabetes mellitus ) and those taking regular medications were also excluded . the study protocol was approved by national research ethics service , leeds ( east ) research ethics committee , and all participants provided written informed consent . all women underwent a baseline tvuss ( aloka sssd 1700 ) in the early follicular phase . in the mnc cohort , a tvuss assessment was performed on alternate days from day 8 of the cycle , until the mean maximal diameter ( mmd ) measured in two planes ( sagittal and transverse ) of the dominant follicle measured 14 mm , after which they were performed daily . women were asked to use urinary lh kits twice daily ( 06:0008:00 and 18:0020:00 ) in order to identify the onset of lh surge prior to spontaneous ovulation . an injection of 5,000 iu hcg ( pregnyl ( organon , cambridge , uk ) ) was given when the mmd of the lead follicle measured 17 mm ( 17.018.1 mm ) . if the urinary lh kit was positive , ultrasound directed oocyte retrieval ( udor ) was performed the day after the surge was detected ; otherwise it was planned for 36 h after hcg . for subsequent analysis , women who had a spontaneous lh surge and women who were administered exogenous hcg were grouped together as the mnc cohort . pituitary downregulation was attained using leuprorelin acetate sr 3.75 mg ( prostap ( wyeth , maidenhead , berkshire , uk ) ) administered on the first day of the menstrual cycle . coh was achieved with 225 iu human menopausal gonadotropin ( hmg ) daily ( menopur , ferring , slough , berkshire , uk ) . as with the mnc - ivf / icsi cycle , when one or more follicles had an mmd of 17 mm , 5,000 iu hcg ( pregnyl ( organon , cambridge , uk ) ) was administered 36 h prior to udor . all udors were performed between 09:00 and 11:00 to accommodate putative circadian variations in ovarian physiology . dead space ( containing 1.5 ml 0.9% sodium chloride solution ) within the oocyte harvesting needle and tubing was constant / uniform throughout such that the first 1.5 ml aspirated was checked and discarded . subsequent aspirate was considered to contain ff and , following oocyte assessment and retrieval , was subsequently labelled to ensure longitudinal tracking of the corresponding oocyte to its fate ( for multifollicular cycles ) . follicles were flushed up to four times with culture medium ( enhance htf culture medium with hepes ; conception technologies , san diego , california , usa ) if no oocyte was obtained in the initial aspirate to minimise the risk of inadvertently aspirating a second follicle and collecting the oocyte from the previous one due to being contained within the dead space . all ultrasonically visible follicles were individually aspirated irrespective of their size . the aspiration pressure applied was uniform on all follicles ( 183185 mm / hg ) . whole blood and ff samples were centrifuged ( 2,000 rpm at 4c for 10 minutes ) to isolate plasma and remove cell debris , respectively . all samples were frozen at 80c within one hour of retrieval until required for analysis . in the mnc cohort , only the ff from the single dominant follicle was analysed . in the coh cohort , ff analysis was performed on the follicles yielding the oocytes that generated transferred embryos ( since double ets were performed in these cycles , the follicle selected for analysis was the one yielding the embryo with the highest morphological grading ) . in both cycles , routine procedures for fertilization with ivf / icsi were performed as previously described . a single et was performed in the mnc cohort , whilst a double et was performed in the coh cohort ( as per acu protocols at the time ) . all women in the mnc cohort who underwent an et received 2,500 iu hcg ( pregnyl ) on the day of et and again 72 h later for luteal support . women in the coh cohort who developed < 15 follicles had an identical luteal support regimen , whereas those women with 15 follicles following coh were given daily intramuscular injection of 100 mg progesterone ( gestone , nordic pharma , reading , berkshire , uk ) , which was continued throughout the first trimester of pregnancy . pregnancy tests were performed on first void urine 14 days after et with a commercial urinary kit . a clinical pregnancy was defined as one demonstrating a gestational sac with a fetal pole and a fetal heart or an ectopic pregnancy by tvuss at 6 - 7 weeks ' gestation . cytokine levels in both ff and plasma were measured by fluid - phase cytometric multiplex immunoassay ( bio - rad laboratories , hercules , ca , usa ) ( bio - rad assays : human cytokine 27-plex assay m50 - 0kcaf0y ; human cytokine 21-plex assay mf0 - 005kmii ) using a luminex 100 cytometer ( luminex corporation , austin , texas , usa ) equipped with bioplex 4.0 manager software ( bio - rad laboratories , ltd . target cytokines included interleukin- ( il- ) 1 receptor antagonist ( ra ) , il-2ra , il-3 , il-6 , il-7 , il-8 , il-9 , il-10 , il-12 ( p40 ) , il-12 ( p70 ) , il-13 , il-15 , il-16 , il-18 , leukaemia inhibitory factor ( lif ) , granulocyte macrophage - colony stimulating factor ( gm - csf ) , macrophage- ( m- ) csf , granulocyte- ( g- ) csf , stem cell factor ( scf ) , interferon- ( ifn- ) , ifn- , ifn- inducible protein- ( ip-10 ) , tnf- , tnf- , tnf related apoptosis inducing ligand ( trail ) , vegf , platelet derived growth factor ( pdgf ) , basic fibroblast growth factor ( b - fgf ) , nerve growth factor- ( ngf- ) , stem cell growth factor- ( scgf- ) , growth regulated oncogene- ( gro- ) , macrophage inflammatory protein- ( mip- ) 1 , monocyte chemoattractant protein- ( mcp- ) 1 , mcp-3 , eotaxin , regulated upon activation of normal t cell expressed and secreted ( rantes ) , stromal cell - derived factor- ( sdf- ) 1 , cutaneous t - cell attracting chemokine ( ctack ) , monokine induced by ifn- ( mig ) , and macrophage migration inhibitory factor ( mif ) . oocyte retrieval frequently results in disruption of the intraovarian vasculature such that blood ( macroscopic or microscopic ) contaminates the ff retrieved . furthermore , although the needle and tubing were primed with normal saline at the commencement of aspiration of each ovary , in between subsequent follicular aspirations , protein - free flush medium formulated with gentamicin ( enhanced htf culture medium with hepes ; conception technologies , san diego , california , usa ) was used , with potential dilution of the ff . this entailed cytokine , total protein ( by lowry assay , bio - rad ) , and von willebrand factor ( vwf ; by enzyme - linked immunosorbent assay ; r&d systems , abingdon , uk ) measurement in both plasma and ff . since vwf is a large plasma multimeric glycoprotein that does not pass through the basement membrane and is not produced by follicular cells , it enabled accurate quantification of ff blood ( and therefore circulatory cytokine ) contamination ( present authors , manuscript under review ) . the dilutional effect of the flush medium was instead accounted for by standardising both ff and plasma ( the latter to enable a valid comparison with the former ) cytokine concentrations to total protein ( pg cytokine / mg protein ) . chi - squared , independent samples t - tests , or mann - whitney u tests were used to compare mnc and coh patient demographics , cycle details , ff , and plasma cytokines following tests for normal distribution by shapiro - wilk test ( stata / se 11.1 , texas , usa ) . in order to address the interrelationships between multiple cytokines and the impact that coh has upon these , heat maps were generated using r 2.7.0 software ( r foundation for statistical computing , vienna , austria ) . correlations between the different cytokines were determined for mnc and coh data using kendall 's tau as a measure of correlation ( stata / se 11.1 ) . resulting p values were adjusted for multiple comparisons with holm 's correction ( p < 0.05 was considered significant ) . in the mnc cohort , one patient had a positive lh surge and therefore did not receive exogenous hcg . at the time of udor 12 hours after the positive surge , spontaneous ovulation had occurred such that no ff was retrieved and an oocyte was not obtained . no statistically significant differences were noted in patient demographics ( age , bmi , baseline endocrine profile ( fsh , lh , and e2 ) , and ethnicity ) , day of udor , follicular aspirate volume , oocyte maturity , and cycle outcome between the two groups ( table 1 ) . most ff cytokines ( 29 out of 40 ) were found to be at higher concentrations in the mnc group compared to the coh group ( binomial test : p < 0.001 ) . this relationship was statistically significant for lif ( p < 0.01 ) and sdf-1 ( p < 0.05 ) ( figure 1 ) . as with ff , the majority of circulatory cytokines in the mnc cohort were present at higher concentrations than in the coh group ( figure 2 ) , a relationship which was significant for 12 of these : il-2ra , il-3 , il-12 ( p40 ) , lif , m - csf , ifn- , trail , ngf- , gro- , mcp-3 , rantes , and sdf-1 ( p < 0.05 ) . conversely , plasma il-12 ( p70 ) levels were present at significantly higher levels following coh ( p < 0.05 ) ( figure 2 ) . heat maps were generated following correlation analysis using kendall 's tau to demonstrate ff cytokine interrelationships ( figure 3 ) . significantly more pairs of cytokines exhibited strong correlations in the mnc data compared to the coh data ( binomial test ) ( p < 0.001 ) . various relationship alterations were also noted ; for example , lif and tnf- demonstrated a weak negative correlation in the mnc group ( kendall 's tau : 0.08 ) compared with a strong positive correlation following coh ( kendall 's tau : 0.46 ) . when plasma : ff cytokine ratios were compared between the mnc and coh cohorts , significantly higher concentrations of various cytokines including il-1 , il-5 , il-10 , il-12 , il-17 , tnf- , and eotaxin have been recorded in endometrial secretions from stimulated cycles compared to ncs . similarly , in the ovary , it has been suggested that exogenous gonadotropins may influence the levels of cytokines such as il-1 , il-6 , and tnf- following earlier studies on ff . . demonstrated altered cumulus cell gene expression for leukocyte differentiation and t - cell activation and regulation following coh , which may in turn influence follicular cytokine profiles as highlighted by the present findings . to the best of our knowledge , ff has not previously been analysed for such an extensive range of cytokines in nc / mnc and coh cycles . in the present pilot study , ff concentrations of 29 out of the 40 cytokines analysed were found to be higher in the mnc cohort , significantly so for lif and sdf-1. lif is an embryotrophic cytokine whose secretion by gcs and stromal cells into ff has previously been shown to be stimulated by hcg [ 7173 ] . since both the mnc and coh cohorts in this study received identical doses of exogenous hcg , the elevated levels of lif in mnc ff may represent an enhanced response to hcg , whereas the likely perturbed cytokine response allied to coh may reflect a reduced sensitivity to hcg resulting in lower lif concentrations . coh also appeared to perturb relationships between cytokines , highlighted by the relative changes in intrafollicular lif and tnf- ( where the latter modulates ovarian stromal cell secretion of the former ) . the heat map displays a strong positive correlation between lif and tnf- in the mnc cohort whereas this relationship is weakened following coh . interestingly , ff lif concentrations have also been correlated with e2 concentrations ( possibly through its role in enhancing aromatase expression ) which , in turn , relate to follicular maturity [ 74 , 75 ] . although a comparable causal association has not been identified to date in the ovary , 17- oestradiol is known to induce lif synthesis in bovine oviduct epithelial cells . it is tempting to speculate that an analogous mechanism is at play in the ovary , where the supraphysiological e2 levels associated with coh may impair the induction of follicular lif synthesis , with a consequent impact on the ff milieu and oocyte quality . sdf-1 is a chemokine secreted by oocytes , which acts in a paracrine manner to prevent follicular activation , thereby controlling the entry of primordial follicles into the growing pool in ncs . furthermore , ff sdf-1 has previously been positively correlated with ff vegf levels in coh cycles , where it is believed to play a proangiogenic role in supporting follicular growth . our findings corroborate this correlation in the coh group ( figure 3 ) . by contrast , this relationship was much weaker in the mnc cohort , suggesting that it may thus in part be gonadotropin dependent . akin to what was noted for ff , plasma cytokine levels in the mnc group were higher than in coh cycles . the exception was circulatory il-12 ( p70 ) , which was measured at significantly higher concentrations following coh . furthermore , correlations between ff il-12 ( p70 ) and other cytokines were markedly altered following stimulation . in the mnc cohort , most ff cytokines were positively correlated with il-12 ( p70 ) , whilst this relationship was reversed following coh . conversely , ifn- and tnf- were negatively correlated with il-12 ( p70 ) in the mnc group and positively in the coh cohort . interestingly , an intricate triumvirate relationship between these particular cytokines has been identified in the regulation of inflammatory responses . il-12 ( p70 ) induces ifn- production and , in turn , ifn- markedly augments il-12 ( p70 ) production , thereby providing a key inflammatory amplifying mechanism . by contrast , tnf- is thought to inhibit ifn--induced il-12 ( p70 ) production , as demonstrated by hodge - dufour et al . , whose tnf mice exhibited a prompt inflammatory response which resolved spontaneously compared with a delayed , more vigorous , inflammatory response leading to death associated with elevated il-12 levels in tnf mice when injected with corynebacterium parvum . thus , tnf- is thought to contribute to the resolution of il-12- ( p70- ) driven inflammatory processes . in the mnc cohort , tnf- was negatively correlated with both il-12 ( p70 ) and ifn- , thus suggesting that , in the absence of stimulatory gonadotropins , tnf- of follicular cell origin may play an analogous role via its capacity to regulate il-12 ( p70 ) production . a positive correlation between these ff cytokines in combination with significantly elevated systemic il-12 ( p70 ) levels suggests a disruption of this mechanism following coh . an analogous disruption in the ovary following coh could thus contribute to the detrimental sequelae of increased il-12 ( p70 ) and ifn- levels despite the observed compensatory rise in modulatory tnf- levels . fluctuations in systemic white cell populations have been attributed to stimulation with exogenous gonadotropins such that the total number of circulatory leukocytes and neutrophils was increased on the day of hcg administration in coh compared to ncs . other studies have reported an increase in plasma white cell count and g - csf , but not in m - csf or il-6 , during coh . furthermore , various cytokines have been found to be elevated in ff following coh , including il-1 , il-6 , and tnf- . the main difference between the present study and those cited is our administration of exogenous hcg in the mnc cohort . both lh and hcg induce il-1 and tnf- , both of which subsequently upregulate gm - csf expression . despite identical doses of hcg being used to trigger ovulation in both groups in the current study , gm - csf in particular was measured at higher ( although not statistically significant ) levels following coh in both ff and plasma . furthermore , following coh , there was a trend towards several other proinflammatory cytokines in both ff and plasma to be found at higher concentration , with il-12 ( p70 ) levels in particular being significantly higher in coh plasma samples . this provides further evidence that coh can induce both local and systemic inflammatory network deregulations featuring perturbations in cytokine interrelationships which , speculatively , may potentially also impact oocyte viability and treatment outcome . unfortunately , the present pilot study was not powered to answer this particular question . in conclusion , this investigation demonstrated that coh not only alters ff cytokine profiles compared to mncs but also perturbs their circulatory levels and disrupts their interrelationships . given their central role in orchestrating normal follicular physiology , these changes have the potential to adversely affect follicular function and compromise oocyte viability .

background . the natural cycle is the prototype to which we aspire to emulate in assisted reproduction techniques . increasing evidence is emerging that controlled ovarian hyperstimulation ( coh ) with exogenous gonadotropins may be detrimental to oogenesis , embryo quality , and endometrial receptivity . this research aimed at assessing the impact of coh on the intrafollicular milieu by comparing follicular fluid ( ff ) cytokine profiles during stimulated in vitro fertilization ( ivf ) and modified natural cycle ( mnc ) ivf . methods . ten women undergoing coh ivf and 10 matched women undergoing mnc ivf were recruited for this pilot study . 40 ff cytokine concentrations from individual follicles and plasma were measured by fluid - phase multiplex immunoassay . demographic / cycle / cytokine data were compared and correlations between cytokines were computed . results . no significant differences were found between coh and mnc groups for patient and cycle demographics , including outcome . overall mean ff cytokine levels were higher in the mnc group for 29/40 cytokines , significantly so for leukaemia inhibitory factor and stromal cell - derived factor-1. furthermore , ff mnc cytokine correlations were significantly stronger than for coh data . conclusions . these findings suggest that coh perturbs intrafollicular cytokine networks , in terms of both cytokine levels and their interrelationships . this may impact oocyte maturation / fertilization and embryo developmental competence .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusions

controlled ovarian hyperstimulation ( coh ) with gonadotropins has improved success rates of in vitro fertilization ( ivf ) by increasing the number and opportunity for selection of embryos before transfer [ 13 ] as well as permitting the cryopreservation of supernumerary embryos for further fertility treatment [ 4 , 5 ] . the basis of coh is to support the growth of multiple follicles to the preovulatory stage , a process achieved by bypassing physiological regulatory mechanisms . with few exceptions , the last two decades have witnessed a mounting body of evidence indicating that ovarian stimulation has a detrimental effect on oogenesis , embryo quality , and endometrial receptivity [ 713 ] . an extreme example of the negative impact of coh is the excessively high number of poor quality oocytes seen in ovarian hyperstimulation syndrome ( ohss ) , which is putatively attributable to detrimental supraphysiological e2 levels . these observations in humans are supported by a number of rodent studies that investigated the impact of exogenous gonadotropin stimulation on oocytes and demonstrated a delay in embryo development [ 17 , 18 ] . it has been suggested that gonadotropin stimulation may affect oocyte maturation and the completion of meiosis , thus leading to an increased risk of having aneuploid oocytes and/or embryos [ 10 , 19 ] . as such , in vitro maturation ( ivm ) has been proposed as an alternative strategy since it reduces exposure to exogenous gonadotropin stimulation , but the process itself introduces a host of other variables / complications ( e.g. recently demonstrated a varying endocrine follicular milieu together with the concentration of putative markers of oocyte quality , specifically anti - mllerian hormone ( amh ) between nc and coh ff , and suggest that this may be the cause for the lower oocyte quality following coh compared with naturally matured oocytes . there has also been some concern that suppressed lh concentrations in the late follicular phase may be detrimental through downstream perturbations in follicular steroid synthesis . consequently , stimulation protocols incorporating exogenous lh were developed , resulting in an increase in the percentage of diploid and good quality embryos obtained [ 22 , 23 ] . lh window below which e2 production is inadequate and above which lh may begin premature luteinisation and be detrimental to follicular development . postulate that the reduced levels of intrafollicular amh they demonstrated following coh compared with nc may be attributed to lh suppression , resulting initially in low follicular androgen concentrations , and subsequently to low amh production which in turn may be responsible for lower oocyte quality . natural cycle ivf ( nc - ivf ) has been proposed as an alternative treatment for older women and poor responders . more recent reports concur , presenting a similar 15.2% live birth rate per initiated cycle in all reported unstimulated ncs in women < 35 years ( n = 795 ) in the united states ( 2006 - 2007 ) . this method incorporates the use of low dose gonadotropin stimulation together with a gonadotropin releasing hormone ( gnrh ) antagonist aimed at generating fewer than eight oocytes per cycle . the term modified natural cycle ivf ( mnc - ivf ) is applied when drugs ( e.g. during folliculogenesis , follicular fluid ( ff ) composition exhibits dynamic changes as individual follicular cell types respond to gonadotropins by secreting different hormones and cytokines [ 34 , 35 ] . given that oocyte quality influences subsequent embryo viability , it has been suggested that the disruption in the balance of these intrafollicular mediators following coh may influence cycle outcome [ 4452 ] . the correct regulation of cytokine networks is essential to support normal physiology and this central role is underscored by the fact that inflammatory / immune dysfunctions underpin many pathological reproductive conditions , resulting in both local and systemic changes in cytokine profiles [ 5355 ] . these levels are comparable to those found during a coh cycle , implying that granulosa cell ( gc ) and theca cell ( tc ) il-8 secretion is a true physiological phenomenon associated with follicular growth / maturation rather than resulting from gonadotropin stimulation . in vitro enhancement of il-8 secretion by cultured gcs and tcs was evident following exposure to il-1 and il-1 , but not tumour necrosis factor- ( tnf- ) , suggesting that il-8 is both gonadotropin and cytokine - induced and may thus be involved in the hormonally regulated stages of folliculogenesis and ovulation . in view of their biological properties ( pleiotropism , synergy , antagonism , functional redundancy , and differential sensitivity ) [ 5860 ] , cytokines should ideally be investigated in terms of their interrelationships as much as in terms of their absolute concentrations . failed to demonstrate a difference in 13 ff cytokines between women undergoing nc and coh ivf , no specific attention was paid towards the complex interrelations within the cytokine networks . to date , there has been a paucity of studies focusing on minimal stimulation regimens and mncs , and comparisons of isolated cytokine concentrations ( e.g. , vascular endothelial growth factor ( vegf ) in coh and nc - ivf ) have been inconsistent [ 62 , 63 ] . therefore , this pilot study aimed at examining the impact of gonadotropins on the intrafollicular cytokine milieu in mnc and following coh cycles . from november 2008 to march 2009 , ten women who required treatment with ivf / icsi due to unexplained or male factor infertility aged 2535 years with a body mass index ( bmi ) 1930 were selected to undergo mnc - ivf / intracytoplasmic sperm injection ( icsi ) at the assisted conception unit , st james 's university hospital , leeds , uk . they were required to be ovulatory ( confirmed by transvaginal ultrasound scan ( tvuss ) , progesterone levels , or commercial lh surge kits within the preceding three months ) and have a normal endocrine profile ( early follicular phase fsh < 8.0 iu / l and e2 50200 pmol / l ) , a negative infection screen ( including negative serum chlamydia antigens ) , and normal pelvic anatomy confirmed by tvuss and laparoscopy . furthermore , they were required to have no risk factors for pelvic pathology ( e.g. , autoimmune diseases , inflammatory conditions , and diabetes mellitus ) and those taking regular medications were also excluded . the study protocol was approved by national research ethics service , leeds ( east ) research ethics committee , and all participants provided written informed consent . all women underwent a baseline tvuss ( aloka sssd 1700 ) in the early follicular phase . in the mnc cohort , a tvuss assessment was performed on alternate days from day 8 of the cycle , until the mean maximal diameter ( mmd ) measured in two planes ( sagittal and transverse ) of the dominant follicle measured 14 mm , after which they were performed daily . pituitary downregulation was attained using leuprorelin acetate sr 3.75 mg ( prostap ( wyeth , maidenhead , berkshire , uk ) ) administered on the first day of the menstrual cycle . as with the mnc - ivf / icsi cycle , when one or more follicles had an mmd of 17 mm , 5,000 iu hcg ( pregnyl ( organon , cambridge , uk ) ) was administered 36 h prior to udor . follicles were flushed up to four times with culture medium ( enhance htf culture medium with hepes ; conception technologies , san diego , california , usa ) if no oocyte was obtained in the initial aspirate to minimise the risk of inadvertently aspirating a second follicle and collecting the oocyte from the previous one due to being contained within the dead space . in the mnc cohort , only the ff from the single dominant follicle was analysed . in the coh cohort , ff analysis was performed on the follicles yielding the oocytes that generated transferred embryos ( since double ets were performed in these cycles , the follicle selected for analysis was the one yielding the embryo with the highest morphological grading ) . a single et was performed in the mnc cohort , whilst a double et was performed in the coh cohort ( as per acu protocols at the time ) . all women in the mnc cohort who underwent an et received 2,500 iu hcg ( pregnyl ) on the day of et and again 72 h later for luteal support . women in the coh cohort who developed < 15 follicles had an identical luteal support regimen , whereas those women with 15 follicles following coh were given daily intramuscular injection of 100 mg progesterone ( gestone , nordic pharma , reading , berkshire , uk ) , which was continued throughout the first trimester of pregnancy . cytokine levels in both ff and plasma were measured by fluid - phase cytometric multiplex immunoassay ( bio - rad laboratories , hercules , ca , usa ) ( bio - rad assays : human cytokine 27-plex assay m50 - 0kcaf0y ; human cytokine 21-plex assay mf0 - 005kmii ) using a luminex 100 cytometer ( luminex corporation , austin , texas , usa ) equipped with bioplex 4.0 manager software ( bio - rad laboratories , ltd . target cytokines included interleukin- ( il- ) 1 receptor antagonist ( ra ) , il-2ra , il-3 , il-6 , il-7 , il-8 , il-9 , il-10 , il-12 ( p40 ) , il-12 ( p70 ) , il-13 , il-15 , il-16 , il-18 , leukaemia inhibitory factor ( lif ) , granulocyte macrophage - colony stimulating factor ( gm - csf ) , macrophage- ( m- ) csf , granulocyte- ( g- ) csf , stem cell factor ( scf ) , interferon- ( ifn- ) , ifn- , ifn- inducible protein- ( ip-10 ) , tnf- , tnf- , tnf related apoptosis inducing ligand ( trail ) , vegf , platelet derived growth factor ( pdgf ) , basic fibroblast growth factor ( b - fgf ) , nerve growth factor- ( ngf- ) , stem cell growth factor- ( scgf- ) , growth regulated oncogene- ( gro- ) , macrophage inflammatory protein- ( mip- ) 1 , monocyte chemoattractant protein- ( mcp- ) 1 , mcp-3 , eotaxin , regulated upon activation of normal t cell expressed and secreted ( rantes ) , stromal cell - derived factor- ( sdf- ) 1 , cutaneous t - cell attracting chemokine ( ctack ) , monokine induced by ifn- ( mig ) , and macrophage migration inhibitory factor ( mif ) . furthermore , although the needle and tubing were primed with normal saline at the commencement of aspiration of each ovary , in between subsequent follicular aspirations , protein - free flush medium formulated with gentamicin ( enhanced htf culture medium with hepes ; conception technologies , san diego , california , usa ) was used , with potential dilution of the ff . the dilutional effect of the flush medium was instead accounted for by standardising both ff and plasma ( the latter to enable a valid comparison with the former ) cytokine concentrations to total protein ( pg cytokine / mg protein ) . chi - squared , independent samples t - tests , or mann - whitney u tests were used to compare mnc and coh patient demographics , cycle details , ff , and plasma cytokines following tests for normal distribution by shapiro - wilk test ( stata / se 11.1 , texas , usa ) . in order to address the interrelationships between multiple cytokines and the impact that coh has upon these , heat maps were generated using r 2.7.0 software ( r foundation for statistical computing , vienna , austria ) . correlations between the different cytokines were determined for mnc and coh data using kendall 's tau as a measure of correlation ( stata / se 11.1 ) . in the mnc cohort , one patient had a positive lh surge and therefore did not receive exogenous hcg . no statistically significant differences were noted in patient demographics ( age , bmi , baseline endocrine profile ( fsh , lh , and e2 ) , and ethnicity ) , day of udor , follicular aspirate volume , oocyte maturity , and cycle outcome between the two groups ( table 1 ) . most ff cytokines ( 29 out of 40 ) were found to be at higher concentrations in the mnc group compared to the coh group ( binomial test : p < 0.001 ) . this relationship was statistically significant for lif ( p < 0.01 ) and sdf-1 ( p < 0.05 ) ( figure 1 ) . as with ff , the majority of circulatory cytokines in the mnc cohort were present at higher concentrations than in the coh group ( figure 2 ) , a relationship which was significant for 12 of these : il-2ra , il-3 , il-12 ( p40 ) , lif , m - csf , ifn- , trail , ngf- , gro- , mcp-3 , rantes , and sdf-1 ( p < 0.05 ) . conversely , plasma il-12 ( p70 ) levels were present at significantly higher levels following coh ( p < 0.05 ) ( figure 2 ) . significantly more pairs of cytokines exhibited strong correlations in the mnc data compared to the coh data ( binomial test ) ( p < 0.001 ) . various relationship alterations were also noted ; for example , lif and tnf- demonstrated a weak negative correlation in the mnc group ( kendall 's tau : 0.08 ) compared with a strong positive correlation following coh ( kendall 's tau : 0.46 ) . when plasma : ff cytokine ratios were compared between the mnc and coh cohorts , significantly higher concentrations of various cytokines including il-1 , il-5 , il-10 , il-12 , il-17 , tnf- , and eotaxin have been recorded in endometrial secretions from stimulated cycles compared to ncs . similarly , in the ovary , it has been suggested that exogenous gonadotropins may influence the levels of cytokines such as il-1 , il-6 , and tnf- following earlier studies on ff . demonstrated altered cumulus cell gene expression for leukocyte differentiation and t - cell activation and regulation following coh , which may in turn influence follicular cytokine profiles as highlighted by the present findings . to the best of our knowledge , ff has not previously been analysed for such an extensive range of cytokines in nc / mnc and coh cycles . in the present pilot study , ff concentrations of 29 out of the 40 cytokines analysed were found to be higher in the mnc cohort , significantly so for lif and sdf-1. lif is an embryotrophic cytokine whose secretion by gcs and stromal cells into ff has previously been shown to be stimulated by hcg [ 7173 ] . coh also appeared to perturb relationships between cytokines , highlighted by the relative changes in intrafollicular lif and tnf- ( where the latter modulates ovarian stromal cell secretion of the former ) . the heat map displays a strong positive correlation between lif and tnf- in the mnc cohort whereas this relationship is weakened following coh . interestingly , ff lif concentrations have also been correlated with e2 concentrations ( possibly through its role in enhancing aromatase expression ) which , in turn , relate to follicular maturity [ 74 , 75 ] . although a comparable causal association has not been identified to date in the ovary , 17- oestradiol is known to induce lif synthesis in bovine oviduct epithelial cells . it is tempting to speculate that an analogous mechanism is at play in the ovary , where the supraphysiological e2 levels associated with coh may impair the induction of follicular lif synthesis , with a consequent impact on the ff milieu and oocyte quality . furthermore , ff sdf-1 has previously been positively correlated with ff vegf levels in coh cycles , where it is believed to play a proangiogenic role in supporting follicular growth . our findings corroborate this correlation in the coh group ( figure 3 ) . by contrast , this relationship was much weaker in the mnc cohort , suggesting that it may thus in part be gonadotropin dependent . akin to what was noted for ff , plasma cytokine levels in the mnc group were higher than in coh cycles . furthermore , correlations between ff il-12 ( p70 ) and other cytokines were markedly altered following stimulation . in the mnc cohort , most ff cytokines were positively correlated with il-12 ( p70 ) , whilst this relationship was reversed following coh . conversely , ifn- and tnf- were negatively correlated with il-12 ( p70 ) in the mnc group and positively in the coh cohort . il-12 ( p70 ) induces ifn- production and , in turn , ifn- markedly augments il-12 ( p70 ) production , thereby providing a key inflammatory amplifying mechanism . in the mnc cohort , tnf- was negatively correlated with both il-12 ( p70 ) and ifn- , thus suggesting that , in the absence of stimulatory gonadotropins , tnf- of follicular cell origin may play an analogous role via its capacity to regulate il-12 ( p70 ) production . an analogous disruption in the ovary following coh could thus contribute to the detrimental sequelae of increased il-12 ( p70 ) and ifn- levels despite the observed compensatory rise in modulatory tnf- levels . fluctuations in systemic white cell populations have been attributed to stimulation with exogenous gonadotropins such that the total number of circulatory leukocytes and neutrophils was increased on the day of hcg administration in coh compared to ncs . furthermore , various cytokines have been found to be elevated in ff following coh , including il-1 , il-6 , and tnf- . the main difference between the present study and those cited is our administration of exogenous hcg in the mnc cohort . despite identical doses of hcg being used to trigger ovulation in both groups in the current study , gm - csf in particular was measured at higher ( although not statistically significant ) levels following coh in both ff and plasma . furthermore , following coh , there was a trend towards several other proinflammatory cytokines in both ff and plasma to be found at higher concentration , with il-12 ( p70 ) levels in particular being significantly higher in coh plasma samples . this provides further evidence that coh can induce both local and systemic inflammatory network deregulations featuring perturbations in cytokine interrelationships which , speculatively , may potentially also impact oocyte viability and treatment outcome . unfortunately , the present pilot study was not powered to answer this particular question . in conclusion , this investigation demonstrated that coh not only alters ff cytokine profiles compared to mncs but also perturbs their circulatory levels and disrupts their interrelationships .

nigella sativa l. ( ranunculaceae ) ( n. sativa ) is an annual herbaceous plant native to ( and cultivated in ) south west asia , and cultivated and naturalized in europe and north africa . n. sativa seeds are commonly known as black cumin , and have been used as a spice and a condiment . in traditional medicine , n. sativa has been used in different forms to treat many diseases including asthma , hypertension , diabetes , inflammation , cough , bronchitis , headache , eczema , fever , dizziness and influenza ( 1 , 2 ) . recent research reports conducted in muslim countries have shown that n. sativa is very commonly used by cancer patients as dietary supplement ( ds ) in complementary and alternative medicine ( cam ) along with chemotherapy ( 3 , 4 ) . n. sativa seed extract , fixed oil and essential oil showed a wide spectrum of favorable biological activities , the most prominent being antioxidant ( 2 , 5 - 7 ) , anti - inflammatory ( 2 , 8 , 9 ) , antibacterial ( 10 - 12 ) , hepatoprotective ( 13 - 17 ) , antimutagenic ( 18 , 19 ) and antitumor ( 20 - 22 ) activities . the plant attracts the interest of researchers all over the world , and a lot of investigations have reported its importance . searching the database pubmed for the keyword , black cumin , gives 645 results , and searching for the keyword , nigella sativa , gives more than 582 results . in preparing this review article we used the key words , nigella sativa and thymoquinone , and the most recently published articles are cited in this review . n. sativa seeds contain fixed oil , proteins , alkaloids , saponins , and essential oil . the biological effects of n. sativa are attributed to the various characterized constituents ( 1 ) . thymoquinone ( tq ) , the most prominent constituent of n. sativa seeds essential oil has been intensively investigated , 406 research reports have been posted on the pubmed database about tq since 1960 . a selection of these properties will be discussed ( table 1 ) . selected pharmacological effects of thymoquinone to investigate the cytoprotective effects of tq against acetaminophen - induced hepatotoxicity , wistar albino rats were given 500 mg / kg acetaminophen orally , followed by three doses of tq at a total dose of 15 mg / kg within an 18 hr time interval ( three times 5 mg / kg oral thymoquinone for every six hr ) . the levels of serum alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , tissue levels of malondialdehyde ( mda ) , oxidized glutathione ( gssg ) , and superoxide dismutase ( sod ) activity were found to be lower compared to that of rats treated with acetaminophen only . histopathological studies further revealed significant liver necrosis and toxicity with acetaminophen treatment , whereas those of tq treatment significantly lowered liver injury scores ( 23 ) . supplementation of tq ( 2 mg / kg / day ) for 5 days before acetaminophen administration reversed the acetaminophen - induced increase in alt , total nitrate / nitrite and lipid peroxide , and the decrease of reduced gsh and atp . tq was effective in protecting mice against acetaminophen - induced hepatotoxicity possibly via increased resistance to oxidative and nitrosative stress ( 24 ) . treatment with anti - cancer drugs like the alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide ( cb 1954 ) is associated with significant hepatotoxicity . balb / c mice transplanted with the mouse mammary cancer cell line ( 66cl-4-gfp ) were treated in vivo with the antitumor drug cb 1954 ( 141 mg / kg ) , tq ( 10 mg / kg ) , and a combination of cb 1954 and tq . histological examination revealed significant tumor regression and maintenance of the liver enzymes alt and ast in the combined treatment compared to cb 1954 alone ( 25 ) . furthermore , the effects of aqueous extracts of n. sativa seeds ( 50 mg / kg ) or tq ( 5 mg / kg in corn oil ) applied by gavage for 5 days were investigated on detoxifying enzymes and glutathione by comparing healthy and ccl4-challenged ( 1 ml / kg in corn oil , intraperitoneally , a single dose ) rats . both n. sativa and tq reduced the increased levels of serum alt activity , the levels of oxidized glutathione , and the stress ratio caused by ccl4 . both n. sativa and tq also ameliorated the reduced messenger rna ( mrna ) levels of glutathione s - transferase ( gst ) , nad ( p ) h - quinone oxido - reductase ( nqo1 ) , and microsomal epoxide hydrolase ( ephx1 ) , as well as the reductions in reduced glutathione and cysteine levels caused by ccl4 . this protection may be attributed to the increased transcription of chemoprotective enzyme mrnas ( 26 ) . tq supplementation also normalized liver reduced glutathione ( gsh ) and decreased the levels of mda and caspase-3 activity in the liver , and reduced serum tumor necrosis factor - alpha ( tnf - alpha ) , serum total bilirubin and the activities of alkaline phosphatase ( alp ) and gamma - glutamyl transferase ( gamma - gt ) enzymes . histopathological examination revealed that tq administration improved lipopolysaccharide ( lps)-induced pathological abnormalities in liver tissues ( 27 ) . summarizing these investigations revealed a protective effect of tq against the cytotoxicity of different agents in vivo . cyto- and genotoxicity evaluation of tq in primary rat hepatocyte cultures at final concentrations ranging from 1.25 to 20 m and three hr exposure , in contrast to the in vivo studies , revealed cytotoxicity of tq as evidenced by increased levels of necrotic cells at concentrations between 2.5 and 20 m , and gave also evidence for genotoxicity at concentrations 1.25 m using the same assay system ( 28 ) . serum / glucose deprivation - induced dna damage was significantly decreased in pc12 cells pretreated with n. sativa extract and tq ( 29 ) . for the in vivo cytoprotective studies absolute doses ranging from 2 to 10 mg / kg of animal body weight for a period of 15 days were applied ( 23 - 25 ) . taking the pharmacodynamics and pharmacokinetics of the compound into consideration , the effective concentration in vivo is certainly lower than the final concentrations applied directly to hepatocyte primary cultures in vitro . furthermore , an acute treatment like this does not allow any adaptive response , which will gradually establish . there are many reports on the anti - inflammatory activity of tq ( 30 - 50 ) . kundu et al ( 30 ) , stated that the anti - inflammatory effect of tq is caused by the upregulated expression of heme - oxygenase 1 ( ho-1 ) in human keratinocytes ( hacat ) by activating nuclear factor ( nf)-erythroid2-(e2)-related factor-2 ( nrf2 ) via reactive oxygen species ( ros)-mediated phosphorylation of protein kinase b ( pkb / akt ) and cyclic amp - activated protein kinase - alpha ( ampkalpha ) . according to bai et al ( 37 ) , tq attenuated thioacetamide ( taa)-induced liver fibrosis accompanied by reduced protein and mrna expression of of -smooth muscle actin ( -sma ) , collagen - i and tissue inhibitor of toll - like receptor 4 ( tlr4 ) and decreased pro - inflammatory cytokine levels . it also inhibited phosphatidylinositol 3-kinase phosphorylation and enhanced the phosphorylation of adenosine monophosphate - activated protein kinase ( ampk ) and liver kinase b ( lkb ) . tq has also been reported to inhibit the effects of 12-o - tetradecanoylphorbol-13-acetate ( tpa)-induced expression of cyclooxygenase-2 ( cox-2 ) and nuclear factor kappa - light - chain - enhancer of activated b cells ( nf-b ) ( 38 ) . n. sativa and tq treatment also suppressed the expression of the cox-2 enzyme in the pancreatic tissue of streptozotocin ( stz)-induced diabetic rats ( 39 ) . the anti - ulcerative effect of n. sativa and tq was demonstrated by kanter et al ( 40 , 41 ) by investigating ethanol induced mucosal ulceration in rats , which was inhibited by pretreatment with tq and n. sativa . furthermore , oral administration of tq in wistar rats at 5mg / kg body weight for 21 days led to a significant reduction of the levels of different pro - inflammatory mediators ( il-1 , il-6 , tnf , ifn and pge(2 ) ) ( 42 ) . intraperitoneal treatment of mice with thymoquinone ( 6 mg / kg ; ip ) , 24 and 1 hr before intratracheal treatment with diesel exhaust particles ( dep ) ( 30 g / mouse ) , prevented pulmonary inflammation and the increase of airway resistance caused by dep , and inhibited the increase of blood leukocyte numbers and plasma il-6 concentrations ( 43 ) . the effects of tq on airway inflammation in a mouse model of allergic asthma were investigated by intraperitoneal injection of tq before airway challenge of ovalbumin ( ova)-sensitized mice , and caused a marked decrease in lung eosinophilia and elevated th2 cytokines - both in vivo and in vitro - following stimulation of lung cells with ova . histological examination of lung tissue demonstrated that the compound significantly inhibited allergen - induced lung eosinophilic inflammation and mucus - producing goblet cells ( 44 ) . using an asthmatic murine model , tq has also been demonstrated to have a high potential in inhibiting the inflammatory changes associated with asthma , especially the aggregation of inflammatory cells in bronchoalveolar lavage ( bal ) fluid and in lung tissues . in addition it inhibited mrna expression of inducible nitric oxide synthase ( inos ) and transforming growth factor-1 ( tgf-1 ) ( 45 ) . in experiments on ovalbumin - sensitized guinea pigs and sulfur mustard exposed guinea pigs , an outstanding evidence of the preventive anti - inflammatory effects of tq and n. sativa has been reported ( 46 - 50 ) . different extracts , mainly aqueous extracts , from n. sativa seeds proved to possess relaxant ( bronchodilatory ) effects on tracheal chains of guinea pigs ( 51 ) . thymoquinone and thymohydroquinone inhibited in vitro non - enzymatic lipid peroxidation in hippocampal homogenates induced by iron - ascorbate ( 52 ) . pretreatment of male nmri rats with tq and n. sativa oil significantly decreased lipid peroxidation levels measured as mda in hippocampus portion following cerebral ischemia - reperfusion injury ( iri ) ( 53 ) . according to abdel - wahab and aly ( 6 ) , n. sativa oil neutralized the toxicity of aflatoxins , and treatment with n. sativa oil of rats fed an aflatoxin - contaminated diet resulted in significant protection against aflatoxicosis . recent reports further demonstrate that tq at a dose of 9 mg / kg body weight protects liver injury induced by aflatoxin b1 ( afb1 ) as evidenced by a reduction of the serum concentrations of ast , alt and alp as marker enzymes for liver injury . when rats were pretreated with tq followed by afb1 the gsh content of the liver was restored and mda production prevented ( 54 ) . n. sativa oil and its active component , tq have also been shown to protect brain tissue from radiation - induced nitrosative stress ( 55 ) . oral administration of tq in wistar rats at 5 mg / kg body weight for 21 days resulted in a significant reduction of the levels of different antioxidant parameters ( myeloperoxidase mpo , lpo , gsh , catalase ( cat ) , sod and no ) in collagen induced arthritis ( cia ) ( 42 ) , and similarly reduced the fe(iii ) nitrilotriacetic acid ( fe - nta ) induced oxidative stress after oral administration in wistar rats ( 56 ) . furthermore , the glycation of sod by glucose or methylglyoxal ( mg ) and its protection by tq has been investigated . incubation of sod with glucose at 37c resulted in a progressive decrease in the activity of the enzyme due to fragmentation , evidenced by a decrease in the amount of protein on sds - page gels . on the other hand , incubation of sod with mg or both glucose and mg glucose at 37c caused protein cross linking evidenced by the formation of high molecular weight aggregates . tq offered protection against glucose or methylglyoxal ( mg ) induced loss of sod activity and fragmentation or cross - linking ( 57 ) . pretreatment of wistar rats with tq and 1,2-dimethylhydrazine ( dmh ) for 10 weeks prevented the depletion of antioxidant enzymes catalase , glutathione peroxidase , and superoxide dismutase in red blood cells and maintained a similar value as the control group . at the same time , it prevented erythrocyte damage in dmh - induced colon post initiation carcinogenesis in rats ( 58 ) . tq and n. sativa oil possess cytoprotective effects against the anti - cancer drugs cyclophosphamide ( ctx ) via maintenance of hemoglobin and blood sugar levels , and the activities of liver enzymes , bilirubin , urea , creatinine , lipids ( triglyceride , cholesterol and low - density lipoprotein ( ldl)-cholesterol ) and lipid peroxidation in the liver . the cytoprotective effects of n. sativa oil and tq were associated with induction of antioxidant mechanisms ( 59 ) . neuron - protective effects have also been studied in cultured hippocampal and cortical neurons treated with amyloid- peptide ( a1 - 42 ) and tq simultaneously for 72 h. tq efficiently attenuated a1 - 42-induced neurotoxicity by improving cell viability . it has also been shown to inhibit mitochondrial membrane potential depolarization and the generation of reactive oxygen species caused by a1 - 42 , and to restore synaptic vesicle recycling inhibition and to partially reverse the loss of spontaneous firing activity , and a1 - 42 aggregation in vitro ( 60 ) . there has been growing interest in natural compounds with anti - cancer properties because they are presumably non - toxic to healthy cells and are available in a readily digestible form . there is a wide consensus in cancer research that tq has promising anti - cancer activity . thus it may be useful as a dietary supplement to enhance the effects of anti - cancer drugs . there is evidence that tq induces p53-independent apoptosis via the activation of caspase-8 and caspases 9 and 3 in the caspase cascade . it also modulates the bax / bcl2 ratio by upregulation of proapoptotic bax and down - regulation of antiapoptotic bcl2 proteins in p53-null hl-60 cells during apoptosis ( 61 ) . investigating the anti - cancer effects of tq on a549 non - small cell lung cancer cells exposed to benzo(a)pyrene , ulasli et al ( 62 ) found that tq treatment up - regulated bax and down - regulated bcl2 proteins , and increased the bax / bcl2 ratio . it also decreased the expression of cyclin d and increased the expression of p21 , and it up - regulated trail receptor 1 and 2 expression . these molecular events lead to regulatory p53 levels affecting the induction of g2/m cell cycle arrest and apoptosis . in breast cancer cells tq was able to increase peroxisome proliferator - activated receptor gamma ( ppar- ) activity and to down - regulate the expression of the genes for bcl-2 , bcl - xl and survivin . more importantly , the increase in ppar- activity was prevented in the presence of ppar- specific inhibitors and ppar- dominant negative plasmids , suggesting that tq may act as a ligand of ppar- ( 63 ) . treatment of human breast carcinoma in both in vitro and in vivo models demonstrated antiproliferative and proapoptotic effects of tq , which are mediated by its inductive effect on p38 and ros signaling . tq possesses anti - tumor effects in breast tumor xenograft mice and it potentiates the antitumor effect of doxorubicin ( 64 ) . tq has also been shown to inhibit the growth of the human cholangiocarcinoma ( cca ) cell lines tfk-1 and hucct1 in a dose- and time - dependent manner . the mechanism of cca cell line growth inhibition is exerted by down - regulation of pi3k / akt and nf-b , and regulated gene products , including x - linked inhibitor of apoptosis protein ( xiap ) , vascular endothelial growth factor ( vegf ) , p - akt , p65 , bcl-2 and cox-2 ( 65 ) . tq also exerts an inhibitory effect on migration of metastatic human ( a375 ) and mouse ( b16f10 ) melanoma cells by inhibition of nlrp3 inflammasome resulting in a decreased proteolytic cleavage of caspase-1 . thus , it can be a potential immunotherapeutic agent not only in adjuvant therapy for melanoma , but also in the control and prevention of metastatic melanoma ( 66 ) . tq is also a microtubule - targeting agent ( mta ) , and binds to the tubulin - microtubule network , thus preventing microtubule polymerization and causing mitotic arrest and apoptosis of a549 cells but not of normal huvec cells ( 67 ) . investigating the putative anti - cancer activities of tq on / tubulin expression in human astrocytoma cells ( cell line u87 , solid tumor model ) and in jurkat cells ( t lymphoblastic leukaemia cells ) evidence was provided for tq to target the level of / tubulin proteins in cancer cells . the degradation found was associated with the upregulation of the tumor suppressor p73 with subsequent induction of apoptosis . no effect on / tubulin protein expression these data indicate that tq exerts a selective effect on / tubulin in cancer cells ( 68 ) . furthermore , tq effects on human topoisomerase ii were investigated and demonstrated that it enhances enzyme - mediated dna cleavage 5-fold , which is similar to the anti - cancer drug etoposide indicating that tq can be considered as human type ii topoisomerase poison ( 69 ) . the majority of patients with glioblastoma , the most aggressive malignant astrocytic brain tumor in adults , experience a recurrence of the tumor because of these cells ` resistance to apoptotic cell death following ionizing radiation and chemotherapy with temozolomide ( tmz ) , and an increased autophagy , tq proved to induce caspase - dependent apoptosis and to inhibit autophagy of glioblastoma cells ( 70 ) . by studying the mechanisms of cytotoxicity on neuroblastoma ( neuro-2a ) cells it was additionally found that tq induces apoptosis by increasing the bax / bcl-2 ratio , which leads to the release of cytochrome c from mitochondria into the cytoplasm . tq treatment also directs the activation of caspase-3 followed by the cleavage of poly ( adp - ribose ) polymerase ( parp ) and down - regulates the caspase inhibitor xiap ( 71 ) . cytotoxicity of tq was also tested in triple - negative breast cancer ( tnbc ) cells that lack functional tumor suppressor p53 . tq treated cells showed g1 phase cell cycle arrest and apoptosis characterized by the loss of mitochondrial membrane integrity as evidenced by release of cytochrome c and caspase 9 activation ( 72 ) . thymoquinone treatment also inhibits the proliferation of multiple myeloma ( mm ) cells and potentiates the apoptotic effect of bortezomib in various mm cell lines via the activation of caspase-3 , resulting in the cleavage of parp . tq treatment also inhibits chemotaxis and invasion induced by c - x - c motif chemokine 12 ( cxcl12 ) in mm cells in vitro and a xenograft mouse model ( 73 ) . it increases levels of ros and mrnas of the oxidative stress - related genes , nqo1 and ho-1 . pretreatment of hepg2 cells with n - acetylcysteine , a scavenger of ros , prevented tq - induced cell death . tq treatment also stimulated mrna expression of pro - apoptotic bcl - xs and trail death receptors , and inhibited expression of the anti - apoptotic gene bcl-2 . conclusively , tq enhanced trail - induced death of hepg2 cells , in part by upregulating trail death receptors , inhibiting nf-b and il-8 and stimulating apoptosis . these manifold molecular mechanisms of tq - dependent suppression of hcc cell growth underscore the potential of this compound as anti - hcc drug ( 74 ) . n. sativa seeds contain fixed oil , proteins , alkaloids , saponins , and essential oil . the biological effects of n. sativa are attributed to the various characterized constituents ( 1 ) . thymoquinone ( tq ) , the most prominent constituent of n. sativa seeds essential oil has been intensively investigated , 406 research reports have been posted on the pubmed database about tq since 1960 . a selection of these properties will be discussed ( table 1 ) . selected pharmacological effects of thymoquinone to investigate the cytoprotective effects of tq against acetaminophen - induced hepatotoxicity , wistar albino rats were given 500 mg / kg acetaminophen orally , followed by three doses of tq at a total dose of 15 mg / kg within an 18 hr time interval ( three times 5 mg / kg oral thymoquinone for every six hr ) . the levels of serum alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , tissue levels of malondialdehyde ( mda ) , oxidized glutathione ( gssg ) , and superoxide dismutase ( sod ) activity were found to be lower compared to that of rats treated with acetaminophen only . histopathological studies further revealed significant liver necrosis and toxicity with acetaminophen treatment , whereas those of tq treatment significantly lowered liver injury scores ( 23 ) . supplementation of tq ( 2 mg / kg / day ) for 5 days before acetaminophen administration reversed the acetaminophen - induced increase in alt , total nitrate / nitrite and lipid peroxide , and the decrease of reduced gsh and atp . tq was effective in protecting mice against acetaminophen - induced hepatotoxicity possibly via increased resistance to oxidative and nitrosative stress ( 24 ) . treatment with anti - cancer drugs like the alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide ( cb 1954 ) is associated with significant hepatotoxicity . balb / c mice transplanted with the mouse mammary cancer cell line ( 66cl-4-gfp ) were treated in vivo with the antitumor drug cb 1954 ( 141 mg / kg ) , tq ( 10 mg / kg ) , and a combination of cb 1954 and tq . histological examination revealed significant tumor regression and maintenance of the liver enzymes alt and ast in the combined treatment compared to cb 1954 alone ( 25 ) . furthermore , the effects of aqueous extracts of n. sativa seeds ( 50 mg / kg ) or tq ( 5 mg / kg in corn oil ) applied by gavage for 5 days were investigated on detoxifying enzymes and glutathione by comparing healthy and ccl4-challenged ( 1 ml / kg in corn oil , intraperitoneally , a single dose ) rats . both n. sativa and tq reduced the increased levels of serum alt activity , the levels of oxidized glutathione , and the stress ratio caused by ccl4 . both n. sativa and tq also ameliorated the reduced messenger rna ( mrna ) levels of glutathione s - transferase ( gst ) , nad ( p ) h - quinone oxido - reductase ( nqo1 ) , and microsomal epoxide hydrolase ( ephx1 ) , as well as the reductions in reduced glutathione and cysteine levels caused by ccl4 . this protection may be attributed to the increased transcription of chemoprotective enzyme mrnas ( 26 ) . tq supplementation also normalized liver reduced glutathione ( gsh ) and decreased the levels of mda and caspase-3 activity in the liver , and reduced serum tumor necrosis factor - alpha ( tnf - alpha ) , serum total bilirubin and the activities of alkaline phosphatase ( alp ) and gamma - glutamyl transferase ( gamma - gt ) enzymes . histopathological examination revealed that tq administration improved lipopolysaccharide ( lps)-induced pathological abnormalities in liver tissues ( 27 ) . summarizing these investigations revealed a protective effect of tq against the cytotoxicity of different agents in vivo . cyto- and genotoxicity evaluation of tq in primary rat hepatocyte cultures at final concentrations ranging from 1.25 to 20 m and three hr exposure , in contrast to the in vivo studies , revealed cytotoxicity of tq as evidenced by increased levels of necrotic cells at concentrations between 2.5 and 20 m , and gave also evidence for genotoxicity at concentrations 1.25 m using the same assay system ( 28 ) . serum / glucose deprivation - induced dna damage was significantly decreased in pc12 cells pretreated with n. sativa extract and tq ( 29 ) . for the in vivo cytoprotective studies absolute doses ranging from 2 to 10 mg / kg of animal body weight for a period of 15 days were applied ( 23 - 25 ) . taking the pharmacodynamics and pharmacokinetics of the compound into consideration , the effective concentration in vivo is certainly lower than the final concentrations applied directly to hepatocyte primary cultures in vitro . furthermore , an acute treatment like this does not allow any adaptive response , which will gradually establish . there are many reports on the anti - inflammatory activity of tq ( 30 - 50 ) . kundu et al ( 30 ) , stated that the anti - inflammatory effect of tq is caused by the upregulated expression of heme - oxygenase 1 ( ho-1 ) in human keratinocytes ( hacat ) by activating nuclear factor ( nf)-erythroid2-(e2)-related factor-2 ( nrf2 ) via reactive oxygen species ( ros)-mediated phosphorylation of protein kinase b ( pkb / akt ) and cyclic amp - activated protein kinase - alpha ( ampkalpha ) . according to bai et al ( 37 ) , tq attenuated thioacetamide ( taa)-induced liver fibrosis accompanied by reduced protein and mrna expression of of -smooth muscle actin ( -sma ) , collagen - i and tissue inhibitor of toll - like receptor 4 ( tlr4 ) and decreased pro - inflammatory cytokine levels . it also inhibited phosphatidylinositol 3-kinase phosphorylation and enhanced the phosphorylation of adenosine monophosphate - activated protein kinase ( ampk ) and liver kinase b ( lkb ) . tq has also been reported to inhibit the effects of 12-o - tetradecanoylphorbol-13-acetate ( tpa)-induced expression of cyclooxygenase-2 ( cox-2 ) and nuclear factor kappa - light - chain - enhancer of activated b cells ( nf-b ) ( 38 ) . n. sativa and tq treatment also suppressed the expression of the cox-2 enzyme in the pancreatic tissue of streptozotocin ( stz)-induced diabetic rats ( 39 ) . the anti - ulcerative effect of n. sativa and tq was demonstrated by kanter et al ( 40 , 41 ) by investigating ethanol induced mucosal ulceration in rats , which was inhibited by pretreatment with tq and n. sativa . furthermore , oral administration of tq in wistar rats at 5mg / kg body weight for 21 days led to a significant reduction of the levels of different pro - inflammatory mediators ( il-1 , il-6 , tnf , ifn and pge(2 ) ) ( 42 ) . intraperitoneal treatment of mice with thymoquinone ( 6 mg / kg ; ip ) , 24 and 1 hr before intratracheal treatment with diesel exhaust particles ( dep ) ( 30 g / mouse ) , prevented pulmonary inflammation and the increase of airway resistance caused by dep , and inhibited the increase of blood leukocyte numbers and plasma il-6 concentrations ( 43 ) . the effects of tq on airway inflammation in a mouse model of allergic asthma were investigated by intraperitoneal injection of tq before airway challenge of ovalbumin ( ova)-sensitized mice , and caused a marked decrease in lung eosinophilia and elevated th2 cytokines - both in vivo and in vitro - following stimulation of lung cells with ova . histological examination of lung tissue demonstrated that the compound significantly inhibited allergen - induced lung eosinophilic inflammation and mucus - producing goblet cells ( 44 ) . using an asthmatic murine model , tq has also been demonstrated to have a high potential in inhibiting the inflammatory changes associated with asthma , especially the aggregation of inflammatory cells in bronchoalveolar lavage ( bal ) fluid and in lung tissues . in addition it inhibited mrna expression of inducible nitric oxide synthase ( inos ) and transforming growth factor-1 ( tgf-1 ) ( 45 ) . in experiments on ovalbumin - sensitized guinea pigs and sulfur mustard exposed guinea pigs , an outstanding evidence of the preventive anti - inflammatory effects of tq and n. sativa has been reported ( 46 - 50 ) . different extracts , mainly aqueous extracts , from n. sativa seeds proved to possess relaxant ( bronchodilatory ) effects on tracheal chains of guinea pigs ( 51 ) . thymoquinone and thymohydroquinone inhibited in vitro non - enzymatic lipid peroxidation in hippocampal homogenates induced by iron - ascorbate ( 52 ) . pretreatment of male nmri rats with tq and n. sativa oil significantly decreased lipid peroxidation levels measured as mda in hippocampus portion following cerebral ischemia - reperfusion injury ( iri ) ( 53 ) . according to abdel - wahab and aly ( 6 ) , n. sativa oil neutralized the toxicity of aflatoxins , and treatment with n. sativa oil of rats fed an aflatoxin - contaminated diet resulted in significant protection against aflatoxicosis . recent reports further demonstrate that tq at a dose of 9 mg / kg body weight protects liver injury induced by aflatoxin b1 ( afb1 ) as evidenced by a reduction of the serum concentrations of ast , alt and alp as marker enzymes for liver injury . when rats were pretreated with tq followed by afb1 the gsh content of the liver was restored and mda production prevented ( 54 ) . n. sativa oil and its active component , tq have also been shown to protect brain tissue from radiation - induced nitrosative stress ( 55 ) . oral administration of tq in wistar rats at 5 mg / kg body weight for 21 days resulted in a significant reduction of the levels of different antioxidant parameters ( myeloperoxidase mpo , lpo , gsh , catalase ( cat ) , sod and no ) in collagen induced arthritis ( cia ) ( 42 ) , and similarly reduced the fe(iii ) nitrilotriacetic acid ( fe - nta ) induced oxidative stress after oral administration in wistar rats ( 56 ) . furthermore , the glycation of sod by glucose or methylglyoxal ( mg ) and its protection by tq has been investigated . incubation of sod with glucose at 37c resulted in a progressive decrease in the activity of the enzyme due to fragmentation , evidenced by a decrease in the amount of protein on sds - page gels . on the other hand , incubation of sod with mg or both glucose and mg glucose at 37c caused protein cross linking evidenced by the formation of high molecular weight aggregates . tq offered protection against glucose or methylglyoxal ( mg ) induced loss of sod activity and fragmentation or cross - linking ( 57 ) . pretreatment of wistar rats with tq and 1,2-dimethylhydrazine ( dmh ) for 10 weeks prevented the depletion of antioxidant enzymes catalase , glutathione peroxidase , and superoxide dismutase in red blood cells and maintained a similar value as the control group . at the same time , it prevented erythrocyte damage in dmh - induced colon post initiation carcinogenesis in rats ( 58 ) . tq and n. sativa oil possess cytoprotective effects against the anti - cancer drugs cyclophosphamide ( ctx ) via maintenance of hemoglobin and blood sugar levels , and the activities of liver enzymes , bilirubin , urea , creatinine , lipids ( triglyceride , cholesterol and low - density lipoprotein ( ldl)-cholesterol ) and lipid peroxidation in the liver . the cytoprotective effects of n. sativa oil and tq were associated with induction of antioxidant mechanisms ( 59 ) . neuron - protective effects have also been studied in cultured hippocampal and cortical neurons treated with amyloid- peptide ( a1 - 42 ) and tq simultaneously for 72 h. tq efficiently attenuated a1 - 42-induced neurotoxicity by improving cell viability . it has also been shown to inhibit mitochondrial membrane potential depolarization and the generation of reactive oxygen species caused by a1 - 42 , and to restore synaptic vesicle recycling inhibition and to partially reverse the loss of spontaneous firing activity , and a1 - 42 aggregation in vitro ( 60 ) . there has been growing interest in natural compounds with anti - cancer properties because they are presumably non - toxic to healthy cells and are available in a readily digestible form . there is a wide consensus in cancer research that tq has promising anti - cancer activity . thus it may be useful as a dietary supplement to enhance the effects of anti - cancer drugs . there is evidence that tq induces p53-independent apoptosis via the activation of caspase-8 and caspases 9 and 3 in the caspase cascade . it also modulates the bax / bcl2 ratio by upregulation of proapoptotic bax and down - regulation of antiapoptotic bcl2 proteins in p53-null hl-60 cells during apoptosis ( 61 ) . investigating the anti - cancer effects of tq on a549 non - small cell lung cancer cells exposed to benzo(a)pyrene , ulasli et al ( 62 ) found that tq treatment up - regulated bax and down - regulated bcl2 proteins , and increased the bax / bcl2 ratio . it also decreased the expression of cyclin d and increased the expression of p21 , and it up - regulated trail receptor 1 and 2 expression . these molecular events lead to regulatory p53 levels affecting the induction of g2/m cell cycle arrest and apoptosis . in breast cancer cells tq was able to increase peroxisome proliferator - activated receptor gamma ( ppar- ) activity and to down - regulate the expression of the genes for bcl-2 , bcl - xl and survivin . more importantly , the increase in ppar- activity was prevented in the presence of ppar- specific inhibitors and ppar- dominant negative plasmids , suggesting that tq may act as a ligand of ppar- ( 63 ) . treatment of human breast carcinoma in both in vitro and in vivo models demonstrated antiproliferative and proapoptotic effects of tq , which are mediated by its inductive effect on p38 and ros signaling . tq possesses anti - tumor effects in breast tumor xenograft mice and it potentiates the antitumor effect of doxorubicin ( 64 ) . tq has also been shown to inhibit the growth of the human cholangiocarcinoma ( cca ) cell lines tfk-1 and hucct1 in a dose- and time - dependent manner . the mechanism of cca cell line growth inhibition is exerted by down - regulation of pi3k / akt and nf-b , and regulated gene products , including x - linked inhibitor of apoptosis protein ( xiap ) , vascular endothelial growth factor ( vegf ) , p - akt , p65 , bcl-2 and cox-2 ( 65 ) . tq also exerts an inhibitory effect on migration of metastatic human ( a375 ) and mouse ( b16f10 ) melanoma cells by inhibition of nlrp3 inflammasome resulting in a decreased proteolytic cleavage of caspase-1 . thus , it can be a potential immunotherapeutic agent not only in adjuvant therapy for melanoma , but also in the control and prevention of metastatic melanoma ( 66 ) . tq is also a microtubule - targeting agent ( mta ) , and binds to the tubulin - microtubule network , thus preventing microtubule polymerization and causing mitotic arrest and apoptosis of a549 cells but not of normal huvec cells ( 67 ) . investigating the putative anti - cancer activities of tq on / tubulin expression in human astrocytoma cells ( cell line u87 , solid tumor model ) and in jurkat cells ( t lymphoblastic leukaemia cells ) evidence was provided for tq to target the level of / tubulin proteins in cancer cells . the degradation found was associated with the upregulation of the tumor suppressor p73 with subsequent induction of apoptosis . no effect on / tubulin protein expression these data indicate that tq exerts a selective effect on / tubulin in cancer cells ( 68 ) . furthermore , tq effects on human topoisomerase ii were investigated and demonstrated that it enhances enzyme - mediated dna cleavage 5-fold , which is similar to the anti - cancer drug etoposide indicating that tq can be considered as human type ii topoisomerase poison ( 69 ) . the majority of patients with glioblastoma , the most aggressive malignant astrocytic brain tumor in adults , experience a recurrence of the tumor because of these cells ` resistance to apoptotic cell death following ionizing radiation and chemotherapy with temozolomide ( tmz ) , and an increased autophagy , tq proved to induce caspase - dependent apoptosis and to inhibit autophagy of glioblastoma cells ( 70 ) . by studying the mechanisms of cytotoxicity on neuroblastoma ( neuro-2a ) cells it was additionally found that tq induces apoptosis by increasing the bax / bcl-2 ratio , which leads to the release of cytochrome c from mitochondria into the cytoplasm . tq treatment also directs the activation of caspase-3 followed by the cleavage of poly ( adp - ribose ) polymerase ( parp ) and down - regulates the caspase inhibitor xiap ( 71 ) . cytotoxicity of tq was also tested in triple - negative breast cancer ( tnbc ) cells that lack functional tumor suppressor p53 . tq treated cells showed g1 phase cell cycle arrest and apoptosis characterized by the loss of mitochondrial membrane integrity as evidenced by release of cytochrome c and caspase 9 activation ( 72 ) . thymoquinone treatment also inhibits the proliferation of multiple myeloma ( mm ) cells and potentiates the apoptotic effect of bortezomib in various mm cell lines via the activation of caspase-3 , resulting in the cleavage of parp . tq treatment also inhibits chemotaxis and invasion induced by c - x - c motif chemokine 12 ( cxcl12 ) in mm cells in vitro and a xenograft mouse model ( 73 ) . it increases levels of ros and mrnas of the oxidative stress - related genes , nqo1 and ho-1 . pretreatment of hepg2 cells with n - acetylcysteine , a scavenger of ros , prevented tq - induced cell death . tq treatment also stimulated mrna expression of pro - apoptotic bcl - xs and trail death receptors , and inhibited expression of the anti - apoptotic gene bcl-2 . conclusively , tq enhanced trail - induced death of hepg2 cells , in part by upregulating trail death receptors , inhibiting nf-b and il-8 and stimulating apoptosis . these manifold molecular mechanisms of tq - dependent suppression of hcc cell growth underscore the potential of this compound as anti - hcc drug ( 74 ) . in conclusion , it is evident that thymoquinone , the predominant constituent of n. sativa volatile oil has a wide spectrum of favorable effects . in our review we concentrated on four properties of tq : hepatoprotective , anti - inflammatory , antioxidant and anti - cancer effects , which are supported by evidence - based research elaborating the molecular mechanisms . these beneficial effects of thymoquinone support the use of this natural compound as a drug with a wide range of medical applications .

nigella sativa has attracted healers in ancient civilizations and researchers in recent times . traditionally , it has been used in different forms to treat many diseases including asthma , hypertension , diabetes , inflammation , cough , bronchitis , headache , eczema , fever , dizziness and influenza . experimentally , it has been demonstrated that n. sativa extracts and the main constituent of their volatile oil , thymoquinone , possess antioxidant , anti - inflammatory and hepatoprotective properties.in this review we aimed at summarizing the most recent investigations related to a few and most important effects of thymoquinone . it is concluded that thymoquinone has evidently proved its activity as hepatoprotective , anti - inflammatory , antioxidant , cytotoxic and anti - cancer chemical , with specific mechanisms of action , which provide support to consider this compound as an emerging drug . further research is required to make thymoquinone a pharmaceutical preparation ready for clinical trials .

Introduction Methods Constituents of Nigella sativa Hepatoprotective effects Anti-inflammatory effects Antioxidant effects Anti-cancer and antitumor activity Conclusion

nigella sativa l. ( ranunculaceae ) ( n. sativa ) is an annual herbaceous plant native to ( and cultivated in ) south west asia , and cultivated and naturalized in europe and north africa . n. sativa seeds are commonly known as black cumin , and have been used as a spice and a condiment . in traditional medicine , n. sativa has been used in different forms to treat many diseases including asthma , hypertension , diabetes , inflammation , cough , bronchitis , headache , eczema , fever , dizziness and influenza ( 1 , 2 ) . recent research reports conducted in muslim countries have shown that n. sativa is very commonly used by cancer patients as dietary supplement ( ds ) in complementary and alternative medicine ( cam ) along with chemotherapy ( 3 , 4 ) . n. sativa seed extract , fixed oil and essential oil showed a wide spectrum of favorable biological activities , the most prominent being antioxidant ( 2 , 5 - 7 ) , anti - inflammatory ( 2 , 8 , 9 ) , antibacterial ( 10 - 12 ) , hepatoprotective ( 13 - 17 ) , antimutagenic ( 18 , 19 ) and antitumor ( 20 - 22 ) activities . searching the database pubmed for the keyword , black cumin , gives 645 results , and searching for the keyword , nigella sativa , gives more than 582 results . in preparing this review article we used the key words , nigella sativa and thymoquinone , and the most recently published articles are cited in this review . n. sativa seeds contain fixed oil , proteins , alkaloids , saponins , and essential oil . the biological effects of n. sativa are attributed to the various characterized constituents ( 1 ) . thymoquinone ( tq ) , the most prominent constituent of n. sativa seeds essential oil has been intensively investigated , 406 research reports have been posted on the pubmed database about tq since 1960 . selected pharmacological effects of thymoquinone to investigate the cytoprotective effects of tq against acetaminophen - induced hepatotoxicity , wistar albino rats were given 500 mg / kg acetaminophen orally , followed by three doses of tq at a total dose of 15 mg / kg within an 18 hr time interval ( three times 5 mg / kg oral thymoquinone for every six hr ) . supplementation of tq ( 2 mg / kg / day ) for 5 days before acetaminophen administration reversed the acetaminophen - induced increase in alt , total nitrate / nitrite and lipid peroxide , and the decrease of reduced gsh and atp . treatment with anti - cancer drugs like the alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide ( cb 1954 ) is associated with significant hepatotoxicity . furthermore , the effects of aqueous extracts of n. sativa seeds ( 50 mg / kg ) or tq ( 5 mg / kg in corn oil ) applied by gavage for 5 days were investigated on detoxifying enzymes and glutathione by comparing healthy and ccl4-challenged ( 1 ml / kg in corn oil , intraperitoneally , a single dose ) rats . both n. sativa and tq reduced the increased levels of serum alt activity , the levels of oxidized glutathione , and the stress ratio caused by ccl4 . tq supplementation also normalized liver reduced glutathione ( gsh ) and decreased the levels of mda and caspase-3 activity in the liver , and reduced serum tumor necrosis factor - alpha ( tnf - alpha ) , serum total bilirubin and the activities of alkaline phosphatase ( alp ) and gamma - glutamyl transferase ( gamma - gt ) enzymes . furthermore , an acute treatment like this does not allow any adaptive response , which will gradually establish . there are many reports on the anti - inflammatory activity of tq ( 30 - 50 ) . kundu et al ( 30 ) , stated that the anti - inflammatory effect of tq is caused by the upregulated expression of heme - oxygenase 1 ( ho-1 ) in human keratinocytes ( hacat ) by activating nuclear factor ( nf)-erythroid2-(e2)-related factor-2 ( nrf2 ) via reactive oxygen species ( ros)-mediated phosphorylation of protein kinase b ( pkb / akt ) and cyclic amp - activated protein kinase - alpha ( ampkalpha ) . according to bai et al ( 37 ) , tq attenuated thioacetamide ( taa)-induced liver fibrosis accompanied by reduced protein and mrna expression of of -smooth muscle actin ( -sma ) , collagen - i and tissue inhibitor of toll - like receptor 4 ( tlr4 ) and decreased pro - inflammatory cytokine levels . tq has also been reported to inhibit the effects of 12-o - tetradecanoylphorbol-13-acetate ( tpa)-induced expression of cyclooxygenase-2 ( cox-2 ) and nuclear factor kappa - light - chain - enhancer of activated b cells ( nf-b ) ( 38 ) . the anti - ulcerative effect of n. sativa and tq was demonstrated by kanter et al ( 40 , 41 ) by investigating ethanol induced mucosal ulceration in rats , which was inhibited by pretreatment with tq and n. sativa . furthermore , oral administration of tq in wistar rats at 5mg / kg body weight for 21 days led to a significant reduction of the levels of different pro - inflammatory mediators ( il-1 , il-6 , tnf , ifn and pge(2 ) ) ( 42 ) . the effects of tq on airway inflammation in a mouse model of allergic asthma were investigated by intraperitoneal injection of tq before airway challenge of ovalbumin ( ova)-sensitized mice , and caused a marked decrease in lung eosinophilia and elevated th2 cytokines - both in vivo and in vitro - following stimulation of lung cells with ova . histological examination of lung tissue demonstrated that the compound significantly inhibited allergen - induced lung eosinophilic inflammation and mucus - producing goblet cells ( 44 ) . using an asthmatic murine model , tq has also been demonstrated to have a high potential in inhibiting the inflammatory changes associated with asthma , especially the aggregation of inflammatory cells in bronchoalveolar lavage ( bal ) fluid and in lung tissues . in experiments on ovalbumin - sensitized guinea pigs and sulfur mustard exposed guinea pigs , an outstanding evidence of the preventive anti - inflammatory effects of tq and n. sativa has been reported ( 46 - 50 ) . pretreatment of male nmri rats with tq and n. sativa oil significantly decreased lipid peroxidation levels measured as mda in hippocampus portion following cerebral ischemia - reperfusion injury ( iri ) ( 53 ) . n. sativa oil and its active component , tq have also been shown to protect brain tissue from radiation - induced nitrosative stress ( 55 ) . furthermore , the glycation of sod by glucose or methylglyoxal ( mg ) and its protection by tq has been investigated . at the same time , it prevented erythrocyte damage in dmh - induced colon post initiation carcinogenesis in rats ( 58 ) . tq and n. sativa oil possess cytoprotective effects against the anti - cancer drugs cyclophosphamide ( ctx ) via maintenance of hemoglobin and blood sugar levels , and the activities of liver enzymes , bilirubin , urea , creatinine , lipids ( triglyceride , cholesterol and low - density lipoprotein ( ldl)-cholesterol ) and lipid peroxidation in the liver . the cytoprotective effects of n. sativa oil and tq were associated with induction of antioxidant mechanisms ( 59 ) . it has also been shown to inhibit mitochondrial membrane potential depolarization and the generation of reactive oxygen species caused by a1 - 42 , and to restore synaptic vesicle recycling inhibition and to partially reverse the loss of spontaneous firing activity , and a1 - 42 aggregation in vitro ( 60 ) . there has been growing interest in natural compounds with anti - cancer properties because they are presumably non - toxic to healthy cells and are available in a readily digestible form . there is a wide consensus in cancer research that tq has promising anti - cancer activity . thus it may be useful as a dietary supplement to enhance the effects of anti - cancer drugs . investigating the anti - cancer effects of tq on a549 non - small cell lung cancer cells exposed to benzo(a)pyrene , ulasli et al ( 62 ) found that tq treatment up - regulated bax and down - regulated bcl2 proteins , and increased the bax / bcl2 ratio . treatment of human breast carcinoma in both in vitro and in vivo models demonstrated antiproliferative and proapoptotic effects of tq , which are mediated by its inductive effect on p38 and ros signaling . tq possesses anti - tumor effects in breast tumor xenograft mice and it potentiates the antitumor effect of doxorubicin ( 64 ) . investigating the putative anti - cancer activities of tq on / tubulin expression in human astrocytoma cells ( cell line u87 , solid tumor model ) and in jurkat cells ( t lymphoblastic leukaemia cells ) evidence was provided for tq to target the level of / tubulin proteins in cancer cells . furthermore , tq effects on human topoisomerase ii were investigated and demonstrated that it enhances enzyme - mediated dna cleavage 5-fold , which is similar to the anti - cancer drug etoposide indicating that tq can be considered as human type ii topoisomerase poison ( 69 ) . the majority of patients with glioblastoma , the most aggressive malignant astrocytic brain tumor in adults , experience a recurrence of the tumor because of these cells ` resistance to apoptotic cell death following ionizing radiation and chemotherapy with temozolomide ( tmz ) , and an increased autophagy , tq proved to induce caspase - dependent apoptosis and to inhibit autophagy of glioblastoma cells ( 70 ) . by studying the mechanisms of cytotoxicity on neuroblastoma ( neuro-2a ) cells it was additionally found that tq induces apoptosis by increasing the bax / bcl-2 ratio , which leads to the release of cytochrome c from mitochondria into the cytoplasm . tq treatment also stimulated mrna expression of pro - apoptotic bcl - xs and trail death receptors , and inhibited expression of the anti - apoptotic gene bcl-2 . these manifold molecular mechanisms of tq - dependent suppression of hcc cell growth underscore the potential of this compound as anti - hcc drug ( 74 ) . n. sativa seeds contain fixed oil , proteins , alkaloids , saponins , and essential oil . the biological effects of n. sativa are attributed to the various characterized constituents ( 1 ) . thymoquinone ( tq ) , the most prominent constituent of n. sativa seeds essential oil has been intensively investigated , 406 research reports have been posted on the pubmed database about tq since 1960 . selected pharmacological effects of thymoquinone to investigate the cytoprotective effects of tq against acetaminophen - induced hepatotoxicity , wistar albino rats were given 500 mg / kg acetaminophen orally , followed by three doses of tq at a total dose of 15 mg / kg within an 18 hr time interval ( three times 5 mg / kg oral thymoquinone for every six hr ) . supplementation of tq ( 2 mg / kg / day ) for 5 days before acetaminophen administration reversed the acetaminophen - induced increase in alt , total nitrate / nitrite and lipid peroxide , and the decrease of reduced gsh and atp . treatment with anti - cancer drugs like the alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide ( cb 1954 ) is associated with significant hepatotoxicity . furthermore , the effects of aqueous extracts of n. sativa seeds ( 50 mg / kg ) or tq ( 5 mg / kg in corn oil ) applied by gavage for 5 days were investigated on detoxifying enzymes and glutathione by comparing healthy and ccl4-challenged ( 1 ml / kg in corn oil , intraperitoneally , a single dose ) rats . both n. sativa and tq reduced the increased levels of serum alt activity , the levels of oxidized glutathione , and the stress ratio caused by ccl4 . serum / glucose deprivation - induced dna damage was significantly decreased in pc12 cells pretreated with n. sativa extract and tq ( 29 ) . furthermore , an acute treatment like this does not allow any adaptive response , which will gradually establish . there are many reports on the anti - inflammatory activity of tq ( 30 - 50 ) . kundu et al ( 30 ) , stated that the anti - inflammatory effect of tq is caused by the upregulated expression of heme - oxygenase 1 ( ho-1 ) in human keratinocytes ( hacat ) by activating nuclear factor ( nf)-erythroid2-(e2)-related factor-2 ( nrf2 ) via reactive oxygen species ( ros)-mediated phosphorylation of protein kinase b ( pkb / akt ) and cyclic amp - activated protein kinase - alpha ( ampkalpha ) . n. sativa and tq treatment also suppressed the expression of the cox-2 enzyme in the pancreatic tissue of streptozotocin ( stz)-induced diabetic rats ( 39 ) . the anti - ulcerative effect of n. sativa and tq was demonstrated by kanter et al ( 40 , 41 ) by investigating ethanol induced mucosal ulceration in rats , which was inhibited by pretreatment with tq and n. sativa . furthermore , oral administration of tq in wistar rats at 5mg / kg body weight for 21 days led to a significant reduction of the levels of different pro - inflammatory mediators ( il-1 , il-6 , tnf , ifn and pge(2 ) ) ( 42 ) . the effects of tq on airway inflammation in a mouse model of allergic asthma were investigated by intraperitoneal injection of tq before airway challenge of ovalbumin ( ova)-sensitized mice , and caused a marked decrease in lung eosinophilia and elevated th2 cytokines - both in vivo and in vitro - following stimulation of lung cells with ova . histological examination of lung tissue demonstrated that the compound significantly inhibited allergen - induced lung eosinophilic inflammation and mucus - producing goblet cells ( 44 ) . using an asthmatic murine model , tq has also been demonstrated to have a high potential in inhibiting the inflammatory changes associated with asthma , especially the aggregation of inflammatory cells in bronchoalveolar lavage ( bal ) fluid and in lung tissues . in experiments on ovalbumin - sensitized guinea pigs and sulfur mustard exposed guinea pigs , an outstanding evidence of the preventive anti - inflammatory effects of tq and n. sativa has been reported ( 46 - 50 ) . different extracts , mainly aqueous extracts , from n. sativa seeds proved to possess relaxant ( bronchodilatory ) effects on tracheal chains of guinea pigs ( 51 ) . furthermore , the glycation of sod by glucose or methylglyoxal ( mg ) and its protection by tq has been investigated . at the same time , it prevented erythrocyte damage in dmh - induced colon post initiation carcinogenesis in rats ( 58 ) . tq and n. sativa oil possess cytoprotective effects against the anti - cancer drugs cyclophosphamide ( ctx ) via maintenance of hemoglobin and blood sugar levels , and the activities of liver enzymes , bilirubin , urea , creatinine , lipids ( triglyceride , cholesterol and low - density lipoprotein ( ldl)-cholesterol ) and lipid peroxidation in the liver . the cytoprotective effects of n. sativa oil and tq were associated with induction of antioxidant mechanisms ( 59 ) . it has also been shown to inhibit mitochondrial membrane potential depolarization and the generation of reactive oxygen species caused by a1 - 42 , and to restore synaptic vesicle recycling inhibition and to partially reverse the loss of spontaneous firing activity , and a1 - 42 aggregation in vitro ( 60 ) . there has been growing interest in natural compounds with anti - cancer properties because they are presumably non - toxic to healthy cells and are available in a readily digestible form . there is a wide consensus in cancer research that tq has promising anti - cancer activity . thus it may be useful as a dietary supplement to enhance the effects of anti - cancer drugs . investigating the anti - cancer effects of tq on a549 non - small cell lung cancer cells exposed to benzo(a)pyrene , ulasli et al ( 62 ) found that tq treatment up - regulated bax and down - regulated bcl2 proteins , and increased the bax / bcl2 ratio . treatment of human breast carcinoma in both in vitro and in vivo models demonstrated antiproliferative and proapoptotic effects of tq , which are mediated by its inductive effect on p38 and ros signaling . tq possesses anti - tumor effects in breast tumor xenograft mice and it potentiates the antitumor effect of doxorubicin ( 64 ) . investigating the putative anti - cancer activities of tq on / tubulin expression in human astrocytoma cells ( cell line u87 , solid tumor model ) and in jurkat cells ( t lymphoblastic leukaemia cells ) evidence was provided for tq to target the level of / tubulin proteins in cancer cells . furthermore , tq effects on human topoisomerase ii were investigated and demonstrated that it enhances enzyme - mediated dna cleavage 5-fold , which is similar to the anti - cancer drug etoposide indicating that tq can be considered as human type ii topoisomerase poison ( 69 ) . by studying the mechanisms of cytotoxicity on neuroblastoma ( neuro-2a ) cells it was additionally found that tq induces apoptosis by increasing the bax / bcl-2 ratio , which leads to the release of cytochrome c from mitochondria into the cytoplasm . these manifold molecular mechanisms of tq - dependent suppression of hcc cell growth underscore the potential of this compound as anti - hcc drug ( 74 ) . in conclusion , it is evident that thymoquinone , the predominant constituent of n. sativa volatile oil has a wide spectrum of favorable effects . in our review we concentrated on four properties of tq : hepatoprotective , anti - inflammatory , antioxidant and anti - cancer effects , which are supported by evidence - based research elaborating the molecular mechanisms . these beneficial effects of thymoquinone support the use of this natural compound as a drug with a wide range of medical applications .

niche partitioning in ecological communities may entail differential responses to a large number of environmental axes ( hutchinson 1957 ) . this partitioning is often considered in spatial terms , but the partitioning of resources through time can also facilitate the coexistence of ecologically similar species ( townsend et al . evidence of temporal partitioning may be found at various scales , from differing activity times within the diel cycle ( kronfeldschor and dayan 2003 ) , to interspecific variation in reproductive effort between years ( silvertown 2004 ) . however , differences in phenology are the most familiar forms of temporal partitioning within ecological communities . here , examples include sympatric mantids whose annual life cycles are staggered to allow coexistence ( hurd and eisenberg 1989 ) and temporal segregation of larval development in odonate communities to reduce competitive and predatory interactions ( crowley and johnson 1982 ) . in communities of amphibians , interspecific variation in reproductive phenology and its implications for coexistence has received some attention , particularly for species whose reproductive activity can be tracked by male advertisement calls ( wells 2007 ) . advertisement calling is exhibited by most species of frogs and toads . within species , calls convey information about the location and fitness of males to prospective partners and competitors ; among species , calls evolve distinct acoustics that act as an important premating isolation mechanism ( wells 2007 ) . however , phenological differences also represent a key source of interspecific variation in advertisement calling in anuran communities , signaling considerable variation in the timing of reproduction . in tropical environments , calling occurs primarily during the rainy season , but there may be considerable heterogeneity in the timing of calling linked to the wide variety of reproductive modes displayed . for example , among 31 species of frogs studied by gottsberger and gruber ( 2004 ) in french guiana , species could be characterized as obligate early season callers ( species that lay foam nests or display direct development ) , mid or late season callers ( species with embryonic development over water ) , sporadic and explosive callers ( species that lay eggs directly into water ) , or generalists that call right through the rainy season ( species with parental care of larvae ) . a comparable diversity of strategies has been reported for other tropical anuran communities , including species that call exclusively during the dry season ( aichinger 1987 ; donnelly and guyer 1994 ; bertoluci 1998 ; bertoluci and rodrigues 2002 ) . temperate anurans may also vary substantially in calling seasonality . among 13 species in eastern texas , ( 2006 ) documented calling seasons ranging from 2 to 12 months . in ontario , de solla et al . ( 2006 ) monitored a community of eight species and found little or no overlap in the calling seasons of several taxa . sympatric anurans can also vary markedly in their meteorological cues for calling . even among aquaticbreeding amphibians from the same community , responses to rainfall can vary considerably , from explosive initiation of calling in response to rain , to dampening or even cessation of calling in response to rain ( oseen and wassersug 2002 ; gottsberger and gruber 2004 ; saenz et al . positive and negative relationships between environmental temperature and calling activity are also known in anuran communities , with some species favoring warm conditions and others cooler conditions ( gottsberger and gruber 2004 ; steelman and dorcas 2010 ) . similarly , while the fundamental effects of relative humidity and wind strength on water balance and thermoregulation in anurans ( tracy 1976 ) suggest that the former should be positively correlated with calling activity and the latter negatively , the relationship between these variables and calling may be idiosyncratic ( oseen and wassersug 2002 ) . while it is clear that the phenology of calling can vary widely within anuran communities , it is also true that the available data on the subject are overwhelmingly derived from anuran communities in the americas ( wells 2007 ) . few , if any , studies are available for large parts of africa and eurasia . the same is true for australia . of the 241 frogs so far described in australia ( anstis 2013 ) , only nine species ( 4% ) have been the focus of dedicated phenological studies on calling and reproduction to our knowledge ( macnally 1984 ; williamson and bull 1992 ; driscoll 1998 ; brooke et al . likewise , we are aware of only seven studies of the phenology of calling across an australian frog community ( humphries 1979 ; gillespie 2001 ; morrison 2001 ; lemckert and mahony 2008 ; lemckert and grigg 2010 ; dostine et al . , we describe the phenology of advertisement calling in an anuran community from the temperate climes of southern australia , focusing on interspecific variation in both the seasonality of calling and the meteorological cues for calling . we combined data collected over 11 years to develop models of the nightly probability of calling by the focal species . as has been documented for tropical and temperate anuran communities elsewhere , our data reveal considerable interspecific variation in the seasonality of calling and the weather conditions that favor calling , which may be indicative of temporal partitioning of reproductive activity in this community . we conducted 1432 nocturnal surveys for frogs between 2001 and 2012 at 253 sites across the volcanic plains north and west of melbourne , victoria , australia . sites were distributed in the catchments of the darebin , merri , moonee ponds , and kororoit creeks . the landscape is undulating , rising to a maximum elevation of ~300 m asl at the study sites . upper catchments are dominated by grazing land , graduating to industrial and urban estates in the lower catchments . the climate is temperate , with cool winters ( july mean daily temperature range = 5.413.1c ) and warm summers ( february mean daily temperature range = 14.126.6c ) ( bom 2014 ) . rainfall averages 538 mm , with the highest monthly rainfall occurring in late winter and spring ( august sites included slow flowing pools along the main channel and tributaries of the streams listed above , as well as lentic wetlands such as farm dams , flooded quarries , swamps , and water treatment ponds . site selection was haphazard , including both randomized protocols ( mostly for selecting pools along streams ) and nonrandomized protocols that aimed to maximize geographic coverage while accounting for both access and logistical constraints ( for further details , see canessa et al . surveys were conducted between september and april ( inclusive ) in each year , encompassing the austral spring , summer , and autumn . survey timing sought to optimize detection rates of litoria raniformis in most cases ; this endangered species having been studied intensively in this region since 2001 ( heard et al . 2012a , b , 2013 , 2015 ) . however , aural surveys during the spring and early summer months optimize detection rates across the frog community in this region ( canessa and parris 2013 ) . surveys commenced at least 30 min after dark and began with a period of 510 min listening quietly for male advertisement calls . surveys extended up to 265 min , with up to 10 repeat surveys at a site in any given year . surveys were completed under permits 10001816 , 10003005 , and 10005649 issued by the victorian department of environment , land , water and planning . to supplement the above survey dataset , we collated data from 263 nocturnal surveys for frogs completed at the same study sites in the same period by private consultants , other researchers , and local naturalists . all surveys were conducted during the austral spring , summer , or autumn , and followed surveys protocols analogous to our own . survey data were gathered from the relevant research report or thesis , or through personal communications . air temperature ( dry bulb , c ) and relative humidity ( % ) were recorded during most surveys with the aid of a compact whirling hygrometer ( brannan p / l , cumbria , uk ) . wind strength was recorded subjectively on an ordinal scale between 0 ( still ) and 3 ( strong , regular gusts ) . rainfall ( mm ) was derived from the australian bureau of meteorology ( bom 2014 ) , taking measurements from the melbourne airport weather station , located roughly in the center of the study area ( 37.67 s , 144.83 e ) . two measures of rainfall were used : rainfall on the day of the survey and cumulative rainfall over the preceding 7 days ( lagged rainfall ) . we used bayesian logistic regression to model the nightly probability of calling for those species with a sufficient number of detections . for each of these species , we began by filtering the nondetection data to exclude records from sites in years when no detections of calling were made . hence , for each species , only detection and nondetection data from sites that were known to be occupied by reproductively active males in a given year were included . we also removed any surveys when the focal wetland was dry ( which precludes calling among the focal species ) and randomly filtered the remaining dataset for each species such that only one site was included for any given survey night . repeat surveys of the same site in a given year were assumed to be independent . hence , for each species , the detection ( 1 ) or nondetection ( 0 ) of calling on survey night i at a site known to be occupied by reproductively active males was treated as a bernoulli variable with probability p i. covariate effects on p i were modeled using a logistic equation and linear link function : ( 1)logpi1pi=+xxi , where is the intercept , and x is the regression coefficient for covariate x. the underlying seasonal variation in p i was modeled using a fourier series approach following burnham and anderson ( 2002 , p. 188 ) . the approach treats phenomena as displaying a regular pattern that may be described using cosine functions of increasing complexity . the change in p i with days since the start of the austral spring ( september 1 , daysi ) was modeled as : ( 2)logpi1pi=+1cos2daysi365+2sin2daysi365 . with the exception of wind strength ( which did not show any obvious seasonality ) , raw meteorological data for each survey were first adjusted to represent the anomaly from the seasonal mean . ( 2012 ) , we fitted up to fourth degree polynomial regressions between each meteorological variable and survey date using maximum likelihood in r version 3.0.3 ( r core team 2014 ) . the bestfitting model for each meteorological variable was selected using akaike 's information criterion ( burnham and anderson 2002 ) , and the residuals from this model used as inputs for the logistic models . fourteen models of p i were fitted to the detection data for each species . each model contained one of the two rainfall variables , plus one or more of temperature , relative humidity , and wind strength . models were fitted twice for each species ; once with temperature represented by air temperature and once with temperature represented by water temperature . correlations between predictors were weak in the model set ( pearson 's r correlations ranged from 0.13 to 0.25 ) , except for a moderate negative correlation between air temperature and humidity ( pearson 's r of 0.46 ) . an effect of survey effort ( survey duration multiplied by the number of observers ) was included in all models to account for the fact that the probability of detecting males that are calling sporadically increases with survey duration , and multiple surveyors have a higher cumulative chance of hearing sporadic or weakly calling individuals . a random year models were fitted to the data using markov chain monte carlo ( mcmc ) sampling in openbugs version 3.2.3 ( thomas et al . 2006 ) , called from r. uninformative priors were used for each parameter . with the exception of survey date , parameter estimates and their 95% credible intervals ( 95% ci ) were drawn from 40,000 mcmc samples after a burnin of 60,000 samples . was used to predict their nightly probability of calling across 366 days , beginning and ending on september 1 . in turn , these predictions were used to estimate the seasonal peak in the probability of calling for each species . we conducted 1432 nocturnal surveys for frogs between 2001 and 2012 at 253 sites across the volcanic plains north and west of melbourne , victoria , australia . sites were distributed in the catchments of the darebin , merri , moonee ponds , and kororoit creeks . the landscape is undulating , rising to a maximum elevation of ~300 m asl at the study sites . upper catchments are dominated by grazing land , graduating to industrial and urban estates in the lower catchments . the climate is temperate , with cool winters ( july mean daily temperature range = 5.413.1c ) and warm summers ( february mean daily temperature range = 14.126.6c ) ( bom 2014 ) . rainfall averages 538 mm , with the highest monthly rainfall occurring in late winter and spring ( august sites included slow flowing pools along the main channel and tributaries of the streams listed above , as well as lentic wetlands such as farm dams , flooded quarries , swamps , and water treatment ponds . site selection was haphazard , including both randomized protocols ( mostly for selecting pools along streams ) and nonrandomized protocols that aimed to maximize geographic coverage while accounting for both access and logistical constraints ( for further details , see canessa et al . surveys were conducted between september and april ( inclusive ) in each year , encompassing the austral spring , summer , and autumn . survey timing sought to optimize detection rates of litoria raniformis in most cases ; this endangered species having been studied intensively in this region since 2001 ( heard et al . 2012a , b , 2013 , 2015 ) . however , aural surveys during the spring and early summer months optimize detection rates across the frog community in this region ( canessa and parris 2013 ) . surveys commenced at least 30 min after dark and began with a period of 510 min listening quietly for male advertisement calls . surveys extended up to 265 min , with up to 10 repeat surveys at a site in any given year . surveys were completed under permits 10001816 , 10003005 , and 10005649 issued by the victorian department of environment , land , water and planning . to supplement the above survey dataset , we collated data from 263 nocturnal surveys for frogs completed at the same study sites in the same period by private consultants , other researchers , and local naturalists . all surveys were conducted during the austral spring , summer , or autumn , and followed surveys protocols analogous to our own . survey data were gathered from the relevant research report or thesis , or through personal communications . six meteorological variables were considered as potential determinants of calling activity . air temperature ( dry bulb , c ) and relative humidity ( % ) were recorded during most surveys with the aid of a compact whirling hygrometer ( brannan p / l , cumbria , uk ) . wind strength was recorded subjectively on an ordinal scale between 0 ( still ) and 3 ( strong , regular gusts ) . rainfall ( mm ) was derived from the australian bureau of meteorology ( bom 2014 ) , taking measurements from the melbourne airport weather station , located roughly in the center of the study area ( 37.67 s , 144.83 e ) . two measures of rainfall were used : rainfall on the day of the survey and cumulative rainfall over the preceding 7 days ( lagged rainfall ) . we used bayesian logistic regression to model the nightly probability of calling for those species with a sufficient number of detections . for each of these species , we began by filtering the nondetection data to exclude records from sites in years when no detections of calling were made . hence , for each species , only detection and nondetection data from sites that were known to be occupied by reproductively active males in a given year were included . we also removed any surveys when the focal wetland was dry ( which precludes calling among the focal species ) and randomly filtered the remaining dataset for each species such that only one site was included for any given survey night . repeat surveys of the same site in a given year were assumed to be independent . hence , for each species , the detection ( 1 ) or nondetection ( 0 ) of calling on survey night i at a site known to be occupied by reproductively active males was treated as a bernoulli variable with probability p i. covariate effects on p i were modeled using a logistic equation and linear link function : ( 1)logpi1pi=+xxi , where is the intercept , and x is the regression coefficient for covariate x. the underlying seasonal variation in p i was modeled using a fourier series approach following burnham and anderson ( 2002 , p. 188 ) . the approach treats phenomena as displaying a regular pattern that may be described using cosine functions of increasing complexity . the change in p i with days since the start of the austral spring ( september 1 , daysi ) was modeled as : ( 2)logpi1pi=+1cos2daysi365+2sin2daysi365 . with the exception of wind strength ( which did not show any obvious seasonality ) , raw meteorological data for each survey were first adjusted to represent the anomaly from the seasonal mean . ( 2012 ) , we fitted up to fourth degree polynomial regressions between each meteorological variable and survey date using maximum likelihood in r version 3.0.3 ( r core team 2014 ) . the bestfitting model for each meteorological variable was selected using akaike 's information criterion ( burnham and anderson 2002 ) , and the residuals from this model used as inputs for the logistic models . fourteen models of p i were fitted to the detection data for each species . each model contained one of the two rainfall variables , plus one or more of temperature , relative humidity , and wind strength . models were fitted twice for each species ; once with temperature represented by air temperature and once with temperature represented by water temperature . correlations between predictors were weak in the model set ( pearson 's r correlations ranged from 0.13 to 0.25 ) , except for a moderate negative correlation between air temperature and humidity ( pearson 's r of 0.46 ) . an effect of survey effort ( survey duration multiplied by the number of observers ) was included in all models to account for the fact that the probability of detecting males that are calling sporadically increases with survey duration , and multiple surveyors have a higher cumulative chance of hearing sporadic or weakly calling individuals . a random year models were fitted to the data using markov chain monte carlo ( mcmc ) sampling in openbugs version 3.2.3 ( thomas et al . 2006 ) , called from r. uninformative priors were used for each parameter . with the exception of survey date , parameter estimates and their 95% credible intervals ( 95% ci ) were drawn from 40,000 mcmc samples after a burnin of 60,000 samples . 2002 ) . the bestfitting model for each species was used to predict their nightly probability of calling across 366 days , beginning and ending on september 1 . in turn , these predictions were used to estimate the seasonal peak in the probability of calling for each species . eight frog species were detected during this study : crinia signifera , crinia parinsignifera , limnodynastes dumerilii , limnodynastes tasmaniensis , litoria ewingii , litoria peronii , litoria raniformis , and litoria verreauxii . six species were commonly detected : c. signifera ( 396 detections total , 177 detections on independent nights ) , lim . although there was considerable temporal overlap in calling activity ( fig . 1 ) , the seasonality and predicted peak of calling varied among species ( figs . 2 , 3 ) . of 15 possible speciestospecies comparisons of the predicted peak in the nightly probability of calling , the 95% credible intervals overlapped in only four cases ( c. signifera & lim . the proportion of surveys in each month during which calling was recorded for the six commonly detected species . only surveys at sites that were known to be occupied in a given year are included for each species ( calling was detected at least once at those sites in those years ) . the estimated nightly probability of calling over 366 days for the six commonly detected species , beginning and ending on the september 1 . estimates are taken from the top model for each species and assume that all other variables influencing the detection of calls ( meteorological variables and survey effort ) are at their mean values . models were fitted to survey data from sites that were known to be occupied in a given year ( calling was detected at least once at those sites in those years ) . the estimated seasonal peak of the nightly probability of calling for the six commonly detected species . estimates are taken from the top model for each species and assume that all other variables influencing the detection of calls ( meteorological variables and survey effort ) are at their mean values . ewingii were recorded calling in all survey months , but most frequently in early spring ( fig . 3 ) , with a steep decline in the nightly probability of calling from november to february , followed by a rapid recovery during autumn ( fig . 2 ) . limnodynastes tasmaniensis displayed a similar ( although weaker ) pattern , with observed calling in all survey months ( fig . 1 ) , but an estimated peak in the nightly probability of calling in september and trough in march ( figs . 2 , 3 ) . , the proportion of surveys in which calling was detected declined in a roughly linear fashion after september ( fig . 1 ) ; however , the estimated seasonal peak in the probability of calling was in october ( fig . 3 ) . thereafter , the nightly probability of calling declined sharply , reaching near zero in midautumn ( fig . 2 ) . the nightly probability of calling was estimated to peak in early december for both species ( fig . 3 ) , before declining to zero or slightly thereabouts during autumn and winter ( fig . 2 ) . interspecific variation was also apparent in relationships between the nightly probability of calling and the meteorological variables considered ( tables 1 , 2 ) . relatively high rainfall in the preceding 7 days increased the probability of calling by c. signifera , lim . however , there was a clear negative effect of lagged rainfall on the probability of calling by lit . raniformis ( mean estimate = 0.60 , 95% ci = 1.10 , 0.11 ; table 2 ) . likewise , while the probability of calling by both lim . raniformis increased as the temperature anomaly increased , the former responded more strongly to air temperature , while the later responded to water temperature ( tables 1 , 2 ) . ewingii : mean estimate = 1.15 , 95% ci = 0.39 , 1.96 ; lit . verreauxii : mean estimate = 1.1 , 95% ci = 0.06 , 2.22 ; table 2 ) . the five topranked models of the nightly probability of calling for the six commonly detected species . all models include cosine effects of days since september 1 and a linear effect of survey effort ( to account for the fact that the probability of detecting calls increases with effort ) . they are as follows : air or water temperature anomaly ( temp ) , humidity anomaly ( hum ) , wind strength ( wind ) , rainfall anomaly ( mm ) either for the day of survey ( rain ) or for the preceding 7 days ( rain lag ) . model selection statistics are the deviance information criterion ( dic ) , distance from the top model ( dic ) , and model weight ( w ) temp is air temperature for these species . variables are as follows : days since september 1 ( days ) , air or water temperature anomaly ( temp ) , humidity anomaly ( hum ) , wind strength ( wind ) , rainfall anomaly ( mm ) either for the day of survey ( rain ) or for the preceding 7 days ( rain lag ) . coefficients for the effect of survey effort are not shown temp is air temperature for these species . there are two obvious mechanisms through which temporal partitioning of reproductive effort may facilitate coexistence in anuran communities . intraspecifically , male anurans compete acoustically for mates in various ways , including extrinsic factors such as caller location ( littlejohn 1977 ; parris 2002 ) and intrinsic factors such as call duration and acoustic frequency ( littlejohn 1977 ; wagner 1992 ; welch et al . 1998 ) . for example , littlejohn and martin ( 1969 ) demonstrated inhibition of calling by pseudophryne semimarmorata in direct response to acoustic interference from sympatric geocrinia victoriana in southern australia . likewise , schwartz and wells ( 1984 ) reported reductions in the attractiveness of calls of hyla ebraccata in panama when the primary notes overlapped with calls of the sympatric h. microcephala . in an extensive study of 39 species across three anuran communities in brazil , duellman and pyles ( 1983 ) demonstrated considerable overlap in the acoustic properties of calls . they concluded that temporal partitioning of calling , both in terms of the seasonal timing and environmental conditions under which calling takes place , was an important mechanism by which acoustic interference is mitigated ( and coexistence facilitated ) in these communities . the second mechanism by which partitioning of reproductive phenology may facilitate coexistence in anuran communities is mitigation of larval competition . competitive interactions between larval anurans are a key determinant of larval survival and metamorphic success ( alford 1999 ) . resource competition among larvae also has important carry over effects on the fitness and survival of postmetamorphic anurans ( chelgren et al . hence , larval competition represents a key determinant of anuran population dynamics and community structure ( wells 2007 ) . the early work of alford and wilbur on the subject of larval competition in anuran communities produced evidence both for and against temporal partitioning as a mechanism for coexistence ( alford and wilbur 1985 ; wilbur and alford 1985 ; alford 1989a , b ) . temporal partitioning was important for the outcomes of some interactions ( alford and wilbur 1985 ; wilbur and alford 1985 ; alford 1989a ) , but not others ( alford 1989b ) . later work confirmed that the strength of larval competition between species varies intrinsically , and mediates the importance of temporal partitioning for larval fitness ( gascon 1992 ) . it also showed that the temporal order in which interactions take place ( socalled priority effects ) may be an important determinant of the strength of competitive interactions among anuran larvae ( lawler and morin 1993 ) . we can not demonstrate that the interspecific variation in calling phenology documented here is adaptive with regard to coexistence , but there are several interesting points to be made in this regard . in the focal community , two species pairs could be expected to benefit most from partitioning of reproductive phenology : lit . these frogs are closely related , have analogous larval ecologies and similar advertisement calls ( loftushills 1973 ; watson et al . 1985 ; anstis 2013 ) . however , there was considerable uncertainty surrounding our estimates of calling seasonality for lit . verreauxii , and we note that an earlier , winter peak in calling activity has been recorded elsewhere for this species ( humphries 1979 ; smith et al . 2003 ; lemckert and grigg 2010 ) . verreauxii suggested by our study could be indicative of temporal partitioning when in sympatry with lit . our data are very limited for c. parinsignifera ; however , they suggest that the peak calling period of c. parinsignifera occurs later than that of c. signifera , and this agrees with observations elsewhere ( humphries 1979 ; macnally 1979 ) . indeed , macnally ( 1979 ) reported that c. signifera truncates its spring calling period to minimize overlap with c. parinsignifera when the two species occur in sympatry . it is also notable that the species pairs with the closest seasonal patterns of calling activity c. signifera & lit . obs . ) . as such , resource competition between larval c. signifera and lit . 2009 ) , the former calls from concealed positions under vegetation or crevices , while lit . elevated perches can significantly increase the effective distance of calls ( parris 2002 ) ; hence , acoustic interference of lit . dumerilli calls from hidden positions under banks and rocks , or in emergent vegetation ( anstis 2013 ; pers . . these two species also favor slightly different meteorological conditions for calling , and there calls differ acoustically ( dominant frequency of 680 hz for lim . our data revealed both expected and unexpected relationships with regard to the meteorological correlates of calling in this community . the probability of calling increased with increasing rainfall over the preceding 7 days for c. signifera , lim . hence , the influence of rainfall on calling activity is most likely mediated by its effect on wetland water levels , as is often the case for aquaticbreeding anurans ( wells 2007 ) . although somewhat counterintuitive , this relationship has been reported for ecologically analogous ranid frogs from north america . ( 2006 ) demonstrated a negative relationship between calling activity and rainfall for both lith . clamitans are all largely aquatic , summer breeding frogs that occupy permanent wetlands ( heard et al . they also all favor nights with warmer water temperatures for calling ( oseen and wassersug 2002 ; saenz et al . 2006 ; this study ) . while it may be supposed that water temperature is lower during rainy periods , and this could account for a negative effect of rainfall on calling activity by lit . raniformis , lagged rainfall and water temperature were unrelated in our dataset ( pearson 's r = 0.04 ) . ( 2006 ) hypothesized that acoustic interference from storms or rainfall itself may be the underlying mechanism . raniformis reduce calling activity following rainfall to reduce acoustic interference with other species in the community , with calling by four species being positively related to lagged rainfall ( as above ) . raniformis may stem from the sensitivity of this species to rising water levels in lotic situations , where increased flows represent a threat to eggs and weakly swimming earlystage tadpoles . of the frogs detected in our study area , lit . raniformis was the most frequently observed in lotic habitats . in line with lemckert and grigg ( 2010 ) , our data suggest that calling by lim . dumerilii is a terrestrial species that can range up to 1 km from wetlands during the nonbreeding season ( humphries 1979 ; anstis 2013 ) . dumerilii to call on nights with comparatively warm air temperatures relates to the suitability of such nights for female migration to breeding sites . finally , the positive relationship between the probability of calling and relative humidity demonstrated here for lit . verreauxii may be readily explained by their propensity to call from elevated perches or on land , rather than in the water ( anstis 2013 ; pers . higher relative humidity facilitates hydroregulation in these circumstances ( tracy 1976 ) , allowing prolonged calling . other ecological mechanisms could account for the variation in reproductive phenology observed during this study . for example , the focal species may vary in their susceptibility to terrestrial or aquatic predators , and time reproduction to minimize exposure to particular predators ( e.g. , lips 2001 ) . invertebrates and fish are important larval predators for some of the frogs studied here ; however , the available evidence suggests that they respond spatially rather than temporally to these predators ( hamer and parris 2010 , 2013 ) . we encourage further research on the reproductive phenology of amphibians and the mechanisms underlying interspecific variation in this trait within communities . as well as insights into niche partitioning and community dynamics , research on reproductive phenology can inform assessments of the effects of anthropogenic disturbances on amphibian communities , including disruptions from climate change ( todd et al . 2011 ) . in particular , we encourage research on the reproductive phenology of amphibians from australia , africa , and eurasia , for which data are scarce .

abstractspatial and temporal partitioning of resources underlies the coexistence of species with similar niches . in communities of frogs and toads , the phenology of advertisement calling provides insights into temporal partitioning of reproductive effort and its implications for community dynamics . this study assessed the phenology of advertisement calling in an anuran community from melbourne , in southern australia . we collated data from 1432 surveys of 253 sites and used logistic regression to quantify seasonality in the nightly probability of calling and the influence of meteorological variables on this probability for six species of frogs . we found limited overlap in the predicted seasonal peaks of calling among these species . those shown to have overlapping calling peaks are unlikely to be in direct competition , due to differences in larval ecology ( crinia signifera and litoria ewingii ) or differences in calling behavior and acoustics ( limnodynastes dumerilii and litoria raniformis ) . in contrast , closely related and ecologically similar species ( crinia signfera and crinia parinsignifera;litoria ewingii and litoria verreauxii ) appear to have staggered seasonal peaks of calling . in combination with interspecific variation in the meteorological correlates of calling , these results may be indicative of temporal partitioning of reproductive activity to facilitate coexistence , as has been reported for tropical and temperate anurans from other parts of the globe .

Introduction Methods Study sites and surveys Meteorological variables Modeling Results Discussion Conflict of Interest

this partitioning is often considered in spatial terms , but the partitioning of resources through time can also facilitate the coexistence of ecologically similar species ( townsend et al . evidence of temporal partitioning may be found at various scales , from differing activity times within the diel cycle ( kronfeldschor and dayan 2003 ) , to interspecific variation in reproductive effort between years ( silvertown 2004 ) . however , differences in phenology are the most familiar forms of temporal partitioning within ecological communities . here , examples include sympatric mantids whose annual life cycles are staggered to allow coexistence ( hurd and eisenberg 1989 ) and temporal segregation of larval development in odonate communities to reduce competitive and predatory interactions ( crowley and johnson 1982 ) . in communities of amphibians , interspecific variation in reproductive phenology and its implications for coexistence has received some attention , particularly for species whose reproductive activity can be tracked by male advertisement calls ( wells 2007 ) . advertisement calling is exhibited by most species of frogs and toads . however , phenological differences also represent a key source of interspecific variation in advertisement calling in anuran communities , signaling considerable variation in the timing of reproduction . in tropical environments , calling occurs primarily during the rainy season , but there may be considerable heterogeneity in the timing of calling linked to the wide variety of reproductive modes displayed . for example , among 31 species of frogs studied by gottsberger and gruber ( 2004 ) in french guiana , species could be characterized as obligate early season callers ( species that lay foam nests or display direct development ) , mid or late season callers ( species with embryonic development over water ) , sporadic and explosive callers ( species that lay eggs directly into water ) , or generalists that call right through the rainy season ( species with parental care of larvae ) . a comparable diversity of strategies has been reported for other tropical anuran communities , including species that call exclusively during the dry season ( aichinger 1987 ; donnelly and guyer 1994 ; bertoluci 1998 ; bertoluci and rodrigues 2002 ) . temperate anurans may also vary substantially in calling seasonality . ( 2006 ) monitored a community of eight species and found little or no overlap in the calling seasons of several taxa . even among aquaticbreeding amphibians from the same community , responses to rainfall can vary considerably , from explosive initiation of calling in response to rain , to dampening or even cessation of calling in response to rain ( oseen and wassersug 2002 ; gottsberger and gruber 2004 ; saenz et al . similarly , while the fundamental effects of relative humidity and wind strength on water balance and thermoregulation in anurans ( tracy 1976 ) suggest that the former should be positively correlated with calling activity and the latter negatively , the relationship between these variables and calling may be idiosyncratic ( oseen and wassersug 2002 ) . while it is clear that the phenology of calling can vary widely within anuran communities , it is also true that the available data on the subject are overwhelmingly derived from anuran communities in the americas ( wells 2007 ) . few , if any , studies are available for large parts of africa and eurasia . of the 241 frogs so far described in australia ( anstis 2013 ) , only nine species ( 4% ) have been the focus of dedicated phenological studies on calling and reproduction to our knowledge ( macnally 1984 ; williamson and bull 1992 ; driscoll 1998 ; brooke et al . likewise , we are aware of only seven studies of the phenology of calling across an australian frog community ( humphries 1979 ; gillespie 2001 ; morrison 2001 ; lemckert and mahony 2008 ; lemckert and grigg 2010 ; dostine et al . , we describe the phenology of advertisement calling in an anuran community from the temperate climes of southern australia , focusing on interspecific variation in both the seasonality of calling and the meteorological cues for calling . we combined data collected over 11 years to develop models of the nightly probability of calling by the focal species . as has been documented for tropical and temperate anuran communities elsewhere , our data reveal considerable interspecific variation in the seasonality of calling and the weather conditions that favor calling , which may be indicative of temporal partitioning of reproductive activity in this community . we conducted 1432 nocturnal surveys for frogs between 2001 and 2012 at 253 sites across the volcanic plains north and west of melbourne , victoria , australia . sites were distributed in the catchments of the darebin , merri , moonee ponds , and kororoit creeks . upper catchments are dominated by grazing land , graduating to industrial and urban estates in the lower catchments . rainfall averages 538 mm , with the highest monthly rainfall occurring in late winter and spring ( august sites included slow flowing pools along the main channel and tributaries of the streams listed above , as well as lentic wetlands such as farm dams , flooded quarries , swamps , and water treatment ponds . survey timing sought to optimize detection rates of litoria raniformis in most cases ; this endangered species having been studied intensively in this region since 2001 ( heard et al . to supplement the above survey dataset , we collated data from 263 nocturnal surveys for frogs completed at the same study sites in the same period by private consultants , other researchers , and local naturalists . rainfall ( mm ) was derived from the australian bureau of meteorology ( bom 2014 ) , taking measurements from the melbourne airport weather station , located roughly in the center of the study area ( 37.67 s , 144.83 e ) . two measures of rainfall were used : rainfall on the day of the survey and cumulative rainfall over the preceding 7 days ( lagged rainfall ) . we used bayesian logistic regression to model the nightly probability of calling for those species with a sufficient number of detections . for each of these species , we began by filtering the nondetection data to exclude records from sites in years when no detections of calling were made . hence , for each species , only detection and nondetection data from sites that were known to be occupied by reproductively active males in a given year were included . we also removed any surveys when the focal wetland was dry ( which precludes calling among the focal species ) and randomly filtered the remaining dataset for each species such that only one site was included for any given survey night . repeat surveys of the same site in a given year were assumed to be independent . hence , for each species , the detection ( 1 ) or nondetection ( 0 ) of calling on survey night i at a site known to be occupied by reproductively active males was treated as a bernoulli variable with probability p i. covariate effects on p i were modeled using a logistic equation and linear link function : ( 1)logpi1pi=+xxi , where is the intercept , and x is the regression coefficient for covariate x. the underlying seasonal variation in p i was modeled using a fourier series approach following burnham and anderson ( 2002 , p. 188 ) . the approach treats phenomena as displaying a regular pattern that may be described using cosine functions of increasing complexity . the change in p i with days since the start of the austral spring ( september 1 , daysi ) was modeled as : ( 2)logpi1pi=+1cos2daysi365+2sin2daysi365 . was used to predict their nightly probability of calling across 366 days , beginning and ending on september 1 . in turn , these predictions were used to estimate the seasonal peak in the probability of calling for each species . we conducted 1432 nocturnal surveys for frogs between 2001 and 2012 at 253 sites across the volcanic plains north and west of melbourne , victoria , australia . sites were distributed in the catchments of the darebin , merri , moonee ponds , and kororoit creeks . upper catchments are dominated by grazing land , graduating to industrial and urban estates in the lower catchments . rainfall averages 538 mm , with the highest monthly rainfall occurring in late winter and spring ( august sites included slow flowing pools along the main channel and tributaries of the streams listed above , as well as lentic wetlands such as farm dams , flooded quarries , swamps , and water treatment ponds . surveys were conducted between september and april ( inclusive ) in each year , encompassing the austral spring , summer , and autumn . survey timing sought to optimize detection rates of litoria raniformis in most cases ; this endangered species having been studied intensively in this region since 2001 ( heard et al . to supplement the above survey dataset , we collated data from 263 nocturnal surveys for frogs completed at the same study sites in the same period by private consultants , other researchers , and local naturalists . six meteorological variables were considered as potential determinants of calling activity . rainfall ( mm ) was derived from the australian bureau of meteorology ( bom 2014 ) , taking measurements from the melbourne airport weather station , located roughly in the center of the study area ( 37.67 s , 144.83 e ) . we used bayesian logistic regression to model the nightly probability of calling for those species with a sufficient number of detections . for each of these species , we began by filtering the nondetection data to exclude records from sites in years when no detections of calling were made . hence , for each species , only detection and nondetection data from sites that were known to be occupied by reproductively active males in a given year were included . repeat surveys of the same site in a given year were assumed to be independent . hence , for each species , the detection ( 1 ) or nondetection ( 0 ) of calling on survey night i at a site known to be occupied by reproductively active males was treated as a bernoulli variable with probability p i. covariate effects on p i were modeled using a logistic equation and linear link function : ( 1)logpi1pi=+xxi , where is the intercept , and x is the regression coefficient for covariate x. the underlying seasonal variation in p i was modeled using a fourier series approach following burnham and anderson ( 2002 , p. 188 ) . the approach treats phenomena as displaying a regular pattern that may be described using cosine functions of increasing complexity . the bestfitting model for each meteorological variable was selected using akaike 's information criterion ( burnham and anderson 2002 ) , and the residuals from this model used as inputs for the logistic models . each model contained one of the two rainfall variables , plus one or more of temperature , relative humidity , and wind strength . correlations between predictors were weak in the model set ( pearson 's r correlations ranged from 0.13 to 0.25 ) , except for a moderate negative correlation between air temperature and humidity ( pearson 's r of 0.46 ) . the bestfitting model for each species was used to predict their nightly probability of calling across 366 days , beginning and ending on september 1 . in turn , these predictions were used to estimate the seasonal peak in the probability of calling for each species . eight frog species were detected during this study : crinia signifera , crinia parinsignifera , limnodynastes dumerilii , limnodynastes tasmaniensis , litoria ewingii , litoria peronii , litoria raniformis , and litoria verreauxii . six species were commonly detected : c. signifera ( 396 detections total , 177 detections on independent nights ) , lim . although there was considerable temporal overlap in calling activity ( fig . 1 ) , the seasonality and predicted peak of calling varied among species ( figs . of 15 possible speciestospecies comparisons of the predicted peak in the nightly probability of calling , the 95% credible intervals overlapped in only four cases ( c. signifera & lim . only surveys at sites that were known to be occupied in a given year are included for each species ( calling was detected at least once at those sites in those years ) . the estimated nightly probability of calling over 366 days for the six commonly detected species , beginning and ending on the september 1 . estimates are taken from the top model for each species and assume that all other variables influencing the detection of calls ( meteorological variables and survey effort ) are at their mean values . models were fitted to survey data from sites that were known to be occupied in a given year ( calling was detected at least once at those sites in those years ) . the estimated seasonal peak of the nightly probability of calling for the six commonly detected species . estimates are taken from the top model for each species and assume that all other variables influencing the detection of calls ( meteorological variables and survey effort ) are at their mean values . ewingii were recorded calling in all survey months , but most frequently in early spring ( fig . 3 ) , with a steep decline in the nightly probability of calling from november to february , followed by a rapid recovery during autumn ( fig . limnodynastes tasmaniensis displayed a similar ( although weaker ) pattern , with observed calling in all survey months ( fig . 1 ) , but an estimated peak in the nightly probability of calling in september and trough in march ( figs . 1 ) ; however , the estimated seasonal peak in the probability of calling was in october ( fig . thereafter , the nightly probability of calling declined sharply , reaching near zero in midautumn ( fig . the nightly probability of calling was estimated to peak in early december for both species ( fig . interspecific variation was also apparent in relationships between the nightly probability of calling and the meteorological variables considered ( tables 1 , 2 ) . relatively high rainfall in the preceding 7 days increased the probability of calling by c. signifera , lim . however , there was a clear negative effect of lagged rainfall on the probability of calling by lit . likewise , while the probability of calling by both lim . raniformis increased as the temperature anomaly increased , the former responded more strongly to air temperature , while the later responded to water temperature ( tables 1 , 2 ) . the five topranked models of the nightly probability of calling for the six commonly detected species . all models include cosine effects of days since september 1 and a linear effect of survey effort ( to account for the fact that the probability of detecting calls increases with effort ) . they are as follows : air or water temperature anomaly ( temp ) , humidity anomaly ( hum ) , wind strength ( wind ) , rainfall anomaly ( mm ) either for the day of survey ( rain ) or for the preceding 7 days ( rain lag ) . model selection statistics are the deviance information criterion ( dic ) , distance from the top model ( dic ) , and model weight ( w ) temp is air temperature for these species . variables are as follows : days since september 1 ( days ) , air or water temperature anomaly ( temp ) , humidity anomaly ( hum ) , wind strength ( wind ) , rainfall anomaly ( mm ) either for the day of survey ( rain ) or for the preceding 7 days ( rain lag ) . coefficients for the effect of survey effort are not shown temp is air temperature for these species . there are two obvious mechanisms through which temporal partitioning of reproductive effort may facilitate coexistence in anuran communities . for example , littlejohn and martin ( 1969 ) demonstrated inhibition of calling by pseudophryne semimarmorata in direct response to acoustic interference from sympatric geocrinia victoriana in southern australia . likewise , schwartz and wells ( 1984 ) reported reductions in the attractiveness of calls of hyla ebraccata in panama when the primary notes overlapped with calls of the sympatric h. microcephala . in an extensive study of 39 species across three anuran communities in brazil , duellman and pyles ( 1983 ) demonstrated considerable overlap in the acoustic properties of calls . they concluded that temporal partitioning of calling , both in terms of the seasonal timing and environmental conditions under which calling takes place , was an important mechanism by which acoustic interference is mitigated ( and coexistence facilitated ) in these communities . the second mechanism by which partitioning of reproductive phenology may facilitate coexistence in anuran communities is mitigation of larval competition . temporal partitioning was important for the outcomes of some interactions ( alford and wilbur 1985 ; wilbur and alford 1985 ; alford 1989a ) , but not others ( alford 1989b ) . later work confirmed that the strength of larval competition between species varies intrinsically , and mediates the importance of temporal partitioning for larval fitness ( gascon 1992 ) . it also showed that the temporal order in which interactions take place ( socalled priority effects ) may be an important determinant of the strength of competitive interactions among anuran larvae ( lawler and morin 1993 ) . we can not demonstrate that the interspecific variation in calling phenology documented here is adaptive with regard to coexistence , but there are several interesting points to be made in this regard . in the focal community , two species pairs could be expected to benefit most from partitioning of reproductive phenology : lit . these frogs are closely related , have analogous larval ecologies and similar advertisement calls ( loftushills 1973 ; watson et al . verreauxii , and we note that an earlier , winter peak in calling activity has been recorded elsewhere for this species ( humphries 1979 ; smith et al . verreauxii suggested by our study could be indicative of temporal partitioning when in sympatry with lit . our data revealed both expected and unexpected relationships with regard to the meteorological correlates of calling in this community . the probability of calling increased with increasing rainfall over the preceding 7 days for c. signifera , lim . hence , the influence of rainfall on calling activity is most likely mediated by its effect on wetland water levels , as is often the case for aquaticbreeding anurans ( wells 2007 ) . although somewhat counterintuitive , this relationship has been reported for ecologically analogous ranid frogs from north america . 2006 ; this study ) . ( 2006 ) hypothesized that acoustic interference from storms or rainfall itself may be the underlying mechanism . of the frogs detected in our study area , lit . finally , the positive relationship between the probability of calling and relative humidity demonstrated here for lit . verreauxii may be readily explained by their propensity to call from elevated perches or on land , rather than in the water ( anstis 2013 ; pers . other ecological mechanisms could account for the variation in reproductive phenology observed during this study . invertebrates and fish are important larval predators for some of the frogs studied here ; however , the available evidence suggests that they respond spatially rather than temporally to these predators ( hamer and parris 2010 , 2013 ) . we encourage further research on the reproductive phenology of amphibians and the mechanisms underlying interspecific variation in this trait within communities . as well as insights into niche partitioning and community dynamics , research on reproductive phenology can inform assessments of the effects of anthropogenic disturbances on amphibian communities , including disruptions from climate change ( todd et al .

conditional gene expression systems such as promoters regulated by tetracycline or doxycycline ( dox ) have several advantages for testing the effects of genes on aging and lifespan . in the ' tet - on ' system , feeding dox to drosophila melanogaster causes high levels of transgene expression in all tissues . by waiting until the young adult stage to administer dox , all of pre - adult development is identical between control and experimental groups , and any difference in lifespan subtle differences in the genetic background of drosophila strains can have significant effects on lifespan . with the tet - on system , control and experimental animals have identical genetic backgrounds , and therefore any differences in lifespan must be due to dox administration and transgene expression . dox itself , and overexpression of control genes such as escherichia coli lacz , have no detectable effects on lifespan . in contrast , several genes have been identified for which dox - regulated overexpression has negative effects on lifespan , and overexpression of the dgmii gene , encoding -mannosidase ii , was associated with slightly increased lifespan . expression of antisense rna has long been known to be able to inhibit gene expression in drosophila and other organisms . double - strand rna ( dsrna ) formed by hybridization of sense and antisense sequences is thought to initiate a pathway in which homologous rna sequences are destroyed . this phenomenon has been referred to as post transcriptional gene silencing ( ptgs ) or rna interference ( rnai ) . expression of inverted - repeat constructs , where the transcript is expected to fold into a dsrna hairpin , has been shown to be an efficient initiator of rnai . in experiments reported here , the tet - on system was used to drive expression of inverted - repeat constructs in drosophila to determine if a conditional system for rnai could be created . in natural populations , lifespan and reproductive period the duration of the reproductive period is subject to natural selection , and lifespan is altered as a consequence . significant experimental support for this model has come from laboratory selection experiments with d. melanogaster . genetically heterogeneous drosophila populations have been selected over hundreds of generations for late - life reproduction . selection is thought to function by altering the frequency of gene alleles present in the starting population . the correlated phenotypes can vary somewhat depending on the starting strains used ; however , increased stress - resistance appears to be common to all or most long - lived strains . in one well studied set of five replicate control ( b ) and long - lived ( o ) strains , lifespan was doubled and was correlated with increased stress - resistance and increased glycogen and lipid stores . starch - gel electrophoresis has been used to assay for changes in the frequency of enzyme electrophoretic alleles in the o and b lines . a change in the frequency of cu / zn - superoxide dismutase ( cu / znsod ) alleles was observed , with the more active allele enriched in the long - lived o strains . consistent with this observation , overexpression of cu / znsod in transgenic drosophila has been shown to be sufficient to cause significant increases in lifespan . the most dramatic change in allele frequency in the o and b lines was observed for the gene for phosphogluconate mutase ( pgm ) . pgm exists in three electrophoretic forms : fast ( pgm ) , medium ( pgm ) and slow ( pgm ) . pgm predominates in the b strains , whereas pgmallele frequency is on average ten - fold higher in the long - lived o strains relative to the b controls . when o strains were taken off selection for several generations ( back - selection ) , lifespan decreased and pgm allele frequency was reduced to levels more like those in b strains . these results suggest that pgm or a closely linked gene is responding to selection , and make pgm a candidate lifespan regulator . pgm encodes the enzyme phosphoglucomutase , which inter - converts glucose 1-phosphate and glucose 6-phosphate . its activity is therefore important for both glycolysis and glycogen synthesis , and the altered pgm allele frequency might therefore be relevant to the increased glycogen and lipid stores of the o strains . if the increased pgm allele frequency is contributing to the unique phenotypes of the long - lived o strains , this might be because pgm has increased or decreased enzyme activity , or enzyme activity has been altered in some way ( perhaps by a change in its regulation ) . one way to begin to test these models experimentally is to engineer transgenic flies with increased or decreased pgm expression and assay for effects on lifespan . multiple strains were generated that were homozygous for each of the f , m and s electrophoretic alleles of pgm , by appropriate crosses to a third chromosome balancer stock . pgm enzyme activity and lifespan varied greatly across the strains with no correlation with pgm allele ( data not shown ) . this result was expected , as differences in genetic background between such purified chromosome strains has profound effects on lifespan and the activities of various enzymes . the pgm coding - region sequences were cloned and sequenced from the pgm , pgm and pgm homozygous strains and from the canton - s wild - type strain . amino - acid substitutions were identified that predicted pi values for the alleles that correlated with their mobility on starch gels ( figure 1 ) . the substitution of t for a in pgm creates the amino - acid sequence ttk ( in the single - letter amino - acid code ) which is a potential phosphorylation site for protein kinase c ( pkc ) that is absent in pgm and pgm . if pgm contributes to the increased lifespan of the o lines , this might be due to increased enzyme activity , decreased enzyme activity , or alteration in enzyme activity in some other way such as in its regulation or subcellular localization . the tet - on system was used to test whether simply increasing or decreasing pgm expression would be sufficient to alter lifespan . a p - element transformation vector called usc1.0 was generated , which had unique ecori and psti sites downstream of the tet - on doxycycline - regulated promoter ( figure 2a ) . the pgm cdna sequences were cloned into usc1.0 to create the pgmsense construct ( figure 2b ) , which should allow overexpression of the pgm enzyme . a fragment of pgm coding region of approximately 1 kilobase ( kb ) ( from + 711 to + 1,794 ) was cloned into usc1.0 in an inverted - repeat orientation to create construct pgminvrpt ( figure 2c ) , which should potentially cause conditional rnai . for each pgm construct five independent transgenic lines were generated using standard methods . as a control , two trangenic lines were generated containing the usc1.0 vector in which no transgene is expressed . expression of the transgenic constructs and the endogenous pgm gene was assayed by northern blot , with or without dox ( figure 3a ) . the pgm inverted - repeat sequences were used as probes and should hybridize to both transgenes and the endogenous pgm transcript . in the sense - construct lines , feeding dox resulted in efficient expression of the transgene at levels five- to ten - fold greater than the endogenous transcript . dox - induced expression of the inverted - repeat construct had two effects detected by northern blot . first , dox produced a smear of hybridization resulting from the inverted - repeat transcript ( figure 3a ) . the smear pattern may have resulted from incomplete denaturation of the hairpin structure and/or instability of the inverted - repeat transcript . second , the level of the endogenous pgm transcript was found to be reduced by dox feeding . the northern blots were also hybridized with a pgm - specific probe corresponding to pgm coding - region sequences ( + 1 to + 700 ) located outside the region used to create the inverted repeat . this probe is therefore specific for the endogenous pgm transcript . using this probe the smear of hybridization derived from the inverted - repeat construct was no longer detected , and the endogenous pgm rna levels were more readily observed and quantitated ( figure 3b ) . dox induced expression of the inverted - repeat construct was found to cause a reduction of endogenous pgm rna levels . the decrease varied across the five transgenic lines , with reductions ranging from 1.3- to 24-fold . the inverted - repeat construct therefore appears to function as expected to induce rnai and cause decreased expression of the endogenous pgm gene . in the control strains where no transgene is expressed , dox administration was found to have no effect on pgm transcript levels ( figure 3c ) . the indirect pgm activity assay was used to assay for changes in pgm gene expression at the protein level . the sense construct yielded conditional overexpression of pgm activity in each transgenic line , with increases ranging from two- to five - fold ( table 1 ) . potential reductions in pgm activity were more difficult to assay because of the inherent limitations of the assay . the pgm activity assay is indirect and is coupled to the conversion of nadp to nadph . the extract will contain other activities capable of converting nadp to nadph , thereby creating a significant background activity . the pgminvrpt construct yielded decreases in pgm activity of as much as 50% ; however , the effect was quite variable from experiment to experiment ( table 1 ) . in the control strains where no transgene is expressed , dox administration was found to have no consistent effect on pgm levels ( table 1 ) . to determine whether altered pgm gene expression could affect lifespan , mean lifespan was assayed in multiple pgmsense and pgminvrpt transgenic lines with or without dox treatment . the percentage change in mean lifespan caused by dox and transgene expression is presented for each line . lifespan was observed to vary across transgenic lines and replicate experiments with changes ranging from -7% to + 6% , with one outlier at + 16% ( table 2 ) . however , no change in lifespan was observed that was consistent across the sense or inverted - repeat lines or in multiple experiments . the lifespan assays were repeated at 29c , and again no consistent changes were observed ( table 3 ) . we conclude that under these conditions , altered pgm expression does not have a detectable effect on adult drosophila lifespan . the phenomenon of rnai ( or ptgs ) is of great interest for two reasons . first , it represents an evolutionarily conserved pathway that probably has important functions in the regulation of gene expression and the control of transposable elements . second , it provides a means for researchers to test gene functions by experimentally downregulating expression of specific genes under well controlled conditions . in drosophila , rnai can be initiated by injection of dsrna into embryos , and this has allowed identification of novel phenotypes for genes during development . however , injection has limited potential application in the adult , as it is unlikely that the rnai could be targeted to all tissues , or to specific tissues , and the trauma of injection is likely to have negative effects on phenotypes such as lifespan . transgenic constructs have been used to cause rnai in drosophila , by using various promoters to drive expression of inverted - repeat fragments of gene coding regions . using the conditional tet - on promoter system to drive expression of an inverted - repeat provided conditional rnai of the pgm gene . this conditional system should have many applications , both in the study of the rnai pathway itself and in the design of experiments where rnai is used a research tool . conditional rnai should be particularly useful to study phenotypes in the adult such as aging and lifespan . conditional gene - expression systems have previously been used to identify positive regulators of lifespan , such as cu / znsod and dgmii . in addition , mutations have been used to identify several genes in drosophila that act as negative regulators of lifespan , such as mth , indy , inr and chico . for mth and inr therefore it is only rare hypomorphic alleles or allele combinations that allow the animals to survive to adult stage where increased lifespan can be observed . conditional rnai should provide a powerful means to study these genes and other negative regulators of aging and lifespan . inactivation or downregulation of the genes specifically in the adult using tet - on rnai may allow increased lifespan without confounding effects on development . lam and thummel have recently reported the use of a heat - shock gene promoter to drive expression of dsrna , and the efficient conditional inhibition of gene expression during larval and pupal stages of drosophila development . genetic selection of drosophila populations for late - life reproduction caused the correlated phenotype of increased lifespan and increased the frequency of the slow electrophoretic allele , pgm , of the pgm gene . if pgm contributes to the increased lifespan phenotype of the selected strains , it might be because pgm has increased activity , decreased activity or activity that is altered in some way such as through its regulation or subcellular localization . the pgm alleles were cloned and sequenced and were found to differ by amino - acid substitutions consistent with the relative electrophoretic mobilities of the encoded enzymes . a recent study reported the sequencing of several fast , medium and slow electrophoretic alleles of pgm from a wild population of d. melanogaster . the sequences reported in that study ( verrelli alleles ) are comparable to those reported here ( ives alleles ) as follows : the medium verrelli allele used for alignment is identical in amino - acid sequence to the ives medium allele . the verrelli fast alleles are identical to the ives fast allele in each of the three amino - acid positions at which the ives fast differs from the ives medium ( wild - type ) allele . there are a number of differences between the verrelli slow alleles and the ives slow allele . the ives slow allele differs from the ives medium ( wild - type ) amino - acid sequence at two positions ( amino - acid positions 6 ( e g ) and 9 ( a t ) ) . the amino - acid change in the ives slow allele at position 6 ( e g ) is not reflected in any of the verrelli slow alleles . the second amino - acid change in the ives slow allele ( at position 9 ) in addition , the verrelli slow alleles have ammo - acid substitutions that are not shared by the ives slow allele . the data are consistent with the conclusion that pgm is highly polymorphic and subject to selection in natural populations . consistent with this idea , pgm haplotypes and glycogen content have been found to vary among flies at different latitudes . the tet - on promoter system allowed a test of the hypothesis that increasing or decreasing pgm gene expression would affect lifespan . pgm enzyme levels were increased two- to five - fold using a sense transgenic construct , and pgm rna levels were decreased 1.3- to 24-fold by driving expression of an inverted - repeat construct . no changes in lifespan were detected that were consistent across multiple transgenic lines or replicate experiments . the results indicate that simply increasing or decreasing pgm gene expression does not significantly affect the lifespan of adult drosophila , at least under the conditions tested . the pgm allele used in these experiments was cloned from the ives strain , which is the progenitor of the long - lived selected strains and their controls . the high degree of polymorphism of pgm makes it possible that the slow electrophoretic allele in the ives strain could consist of multiple dna sequence forms . the failure to observe effects on lifespan could therefore conceivably have resulted from overexpression of the wrong dna sequence form of pgm . however , pgm was cloned and sequenced from four independent lines derived from the ives strain and the clones had the same dna sequence . the data therefore suggest that most , if not all , of the pgm alleles in these strains have the same sequence , making sequence heterogeneity an unlikely explanation of the present results . the results suggest several possibilities for the relationship of pgm allele frequency to the increased lifespan of the selected strains . the first is that pgm might in fact contribute to the increased lifespan of the selected strains as a result of an increased or decreased activity , but that the effect on lifespan is too small to be detected in these assays . the lifespan assay appears to have a variability with control strains of approximately -5% to + 5% , as seen in the data presented here and elsewhere ( g.l . , d. bhole and j.t . the second possibility is that pgm allele frequencies are altered because of selection for an unknown linked gene , and this possibility can not be ruled out at this time . the experiments presented here suggest that tet - on regulated overexpression and rnai of genes near pgm may be a promising approach . third , an interesting model is that pgm contributes to increased lifespan of the selected strains because of an alteration in its regulation and/or subcellular localization . the sequence analysis of the pgm alleles reveals a novel potential site in pgm for phosphorylation by pkc ; however , the potential significance of this change is unknown . the pgm allele was used for the overexpression experiments presented here , and presumably the overexpressed enzyme should be subject to any regulation or localization characteristic of pgm . however , the pgm enzyme was overexpressed in a ' wild - type ' pgm background , and it may be that any phenotypic consequences of pgm allele function are not apparent when pgm is also present . finally , another possible explanation for the absence of an effect on longevity is the method of assay . the selected o stocks were handled and assayed with males and females together , repeatedly mating during adult life . this was done because the segregated - sexes assay is sensitive to even small changes in lifespan , and has been used successfully in the past to detect the effects of other genes on lifespan . it is possible that an effect of pgm on lifespan might be identified with different lifespan assay conditions . however , even if this explanation were to be true , the data still indicate that pgm does not have a simple strong effect on longevity . further experiments will be required to distinguish between these interesting possibilities for the link between pgm allele frequency and lifespan . all transgenic drosophila stocks were generated by p - element germline transformation , using a modified microinjection technique . transgenic lines are named as the construct , followed by the chromosome of insertion in parentheses , followed by a unique line designation . for example , pgminvrpt(3)76 is pgminvrpt construct inserted on chromosome 3 , independent transgenic line 76 . multiple strains homozygous for each of the pgm , pgm and pgm alleles were generated by purifying third chromosomes from the ives stock , which is the precursor to the o and b selection stocks . homozygosity for a particular pgm allele was confirmed by starch - gel electrophoresis assay , as previously described ( and data not shown ) . all stocks were grown on standard cornmeal - agar medium and were cultured at 25c . strains homozygous for the transgenic constructs were crossed to rtta(3)e2 strain , homozygous for the rtta transactivator construct , to generate progeny heterozygous for both constructs . control flies were rtta(3)e2/+ , that is , lacking a target construct . to obtain adult flies of defined age , the crosses were cultured at 25c in urine specimen bottles . before eclosion of the majority of pupae , bottles were cleared of adults and newly eclosed flies were allowed to emerge over the next 48 h. most of the males will have mated during this time . the males only were then removed and were designated one day old , and were maintained at 25c at 40 per vial in culture vials with food , and passaged to new vials every 48 h. for certain experiments the adult males were maintained at 29c , as indicated . for certain experiments , those flies being fed dox were kept on food vials supplemented with 250 g / ml dox . all dna sequencing reactions were done using the dideoxy chain - termination protocol and t7 sequenase v2.0 ( amersham ) . the clones were obtained by polymerase chain reaction ( pcr ) amplification of each allele using genomic dna template isolated from strains homozygous for each allele . the primer locations were assigned on the basis of their relative distance from the a in the atg start codon , which was defined as position + 1 . pgm 5'-end-2 ( -27 to -4 ) : 5'-agccagcagccggaaaactccagt-3 ' ; pgm 3'-end ( + 1706 to + 1727 ) : 5'-ggatgggttggtaatctcagtg-3 ' . each pcr product was gel - purified and cloned into the ecorv site of pbluescript ks+ ( stratagene ) . briefly , ten male flies were anesthetized with co2 and placed in 200 l ice - cold 0.1 m tris buffer solution ( ph 7.6 ) . an additional 200 l tris buffer was added for a total volume of 400 l . approximately 200 - 300 l supernatant was removed , taking care to avoid a top layer of oil and debris . for the assay , 30 - 40 l of this supernatant was used in a 3.1 ml assay mixture that contained : 1.5 mm mgcl2 , 26 mm histidine solution ( ph 7.6 ) , 8 m glucose-1,6-diphosphate , 0.45 u / ml glucose-6-phosphate dehydrogenase , and 0.2 mm nadp . this solution was mixed at room temperature and then 5 mm glucose 1-phosphate was added . the solution was mixed again and placed in a spectrophotometer . to insure that readings were being taken in the linear range of the enzyme activity , readings were taken at t = 6 min at od340 and were expressed as od/g / min . all extracts were assayed in triplicate , and the averages and standard deviations are presented . means were compared using unpaired , two - side t - tests and p - values are presented . the enzyme assay measures the conversion of nadp to nadph rather than a direct measure of phosphoglucomutase itself . in other words , it is measuring a secondary , coupled reaction . all inserts for microinjection were cloned into a derivative of the 7t40 construct called usc1.0 . usc1.0 was generated as follows : first , 7t40 was digested with ecori , thus liberating the hsp70 polyadenylation signal sequence . next , the psti site in polylinker 1 was destroyed by partial psti digestion followed by t4 dna polymerase fill - in and ligation . clones were screened for destruction of the correct psti site to generate usc1.0 ( figure 2a ) . the reverse transcription was carried out on total rna isolated from a strain of flies homozygous for the pgm allele , using the following primer : dtbibo : 5'-taacccgggtctacaaagtgatactgcgtaactgactatattttttttttttttttt-3 ' . a nested pcr strategy was then used to amplify the pgm cdna using the following primers : first primer set : bi : 5'-taacccgggtctacaaagtg-3 ' . the resulting pcr product was cloned into the ecorv site of pbluescript ks+ and clones were screened for the correct orientation . the pgm cdna was then liberated by ecori complete digest followed by psti partial digest and cloned into the ecori to psti sites of usc1.0 to generate construct pgmsense ( figure 2b ) . the pgminvrpt construct was made as follows : because the final insert would be a large inverted repeat , pgm was pcr - amplified in two pieces , in opposite orientations . the two sets of primers used for the amplifications were : first set : pgm - sense ( 3 ' ) ( + 1,773 to + 1,794 ) : 5'-agctgaattccacaaactttaataaatccgaaac-3 ' . second set : pgm - anti not ( + 711 to + 731 ) : 5'-agctgcggccgccctgaaccggttgggtgccac-3 ' . each of these pcr products was cloned individually into the ecorv site of pbluescript ks+ , and then restriction digested with ecori and noti ( first - set fragment ) or noti and psti ( second - set fragment ) . each of the liberated fragments was gel purified and triple - ligated into the psti to ecori sites of usc1.0 to generate construct pgminvrpt ( figure 2c ) . rna was extracted using trizol reagent ( gibco brl ) and the amount of rna was quantitated by uv spectrophotometry . approximately 5 - 10 g rna per lane after blotting , the rna was fixed to genescreen nylon membrane ( dupont ) using a uv crosslinker ( stratagene ) . probes were p - labeled using the prime - it ii kit ( stratagene ) . to determine fold increases / decreases , northern blots were exposed onto a phosphor screen and results were analyzed using a phosphorimager ( imagequant , molecular dynamics ) . ribosomal protein 49 gene ( rp49 ) was used as a loading control , and all pgm northern data is normalized to rp49 rna levels . all transgenic drosophila stocks were generated by p - element germline transformation , using a modified microinjection technique . transgenic lines are named as the construct , followed by the chromosome of insertion in parentheses , followed by a unique line designation . for example , pgminvrpt(3)76 is pgminvrpt construct inserted on chromosome 3 , independent transgenic line 76 . multiple strains homozygous for each of the pgm , pgm and pgm alleles were generated by purifying third chromosomes from the ives stock , which is the precursor to the o and b selection stocks . homozygosity for a particular pgm allele was confirmed by starch - gel electrophoresis assay , as previously described ( and data not shown ) . all stocks were grown on standard cornmeal - agar medium and were cultured at 25c . strains homozygous for the transgenic constructs were crossed to rtta(3)e2 strain , homozygous for the rtta transactivator construct , to generate progeny heterozygous for both constructs . control flies were rtta(3)e2/+ , that is , lacking a target construct . to obtain adult flies of defined age , the crosses were cultured at 25c in urine specimen bottles . before eclosion of the majority of pupae , bottles were cleared of adults and newly eclosed flies were allowed to emerge over the next 48 h. most of the males will have mated during this time . the males only were then removed and were designated one day old , and were maintained at 25c at 40 per vial in culture vials with food , and passaged to new vials every 48 h. for certain experiments the adult males were maintained at 29c , as indicated . for certain experiments , all dna sequencing reactions were done using the dideoxy chain - termination protocol and t7 sequenase v2.0 ( amersham ) . the clones were obtained by polymerase chain reaction ( pcr ) amplification of each allele using genomic dna template isolated from strains homozygous for each allele . the primer locations were assigned on the basis of their relative distance from the a in the atg start codon , which was defined as position + 1 . pgm 5'-end-2 ( -27 to -4 ) : 5'-agccagcagccggaaaactccagt-3 ' ; pgm 3'-end ( + 1706 to + 1727 ) : 5'-ggatgggttggtaatctcagtg-3 ' . each pcr product was gel - purified and cloned into the ecorv site of pbluescript ks+ ( stratagene ) . briefly , ten male flies were anesthetized with co2 and placed in 200 l ice - cold 0.1 m tris buffer solution ( ph 7.6 ) . an additional 200 l tris buffer was added for a total volume of 400 l . approximately 200 - 300 l supernatant was removed , taking care to avoid a top layer of oil and debris . for the assay , 30 - 40 l of this supernatant was used in a 3.1 ml assay mixture that contained : 1.5 mm mgcl2 , 26 mm histidine solution ( ph 7.6 ) , 8 m glucose-1,6-diphosphate , 0.45 u / ml glucose-6-phosphate dehydrogenase , and 0.2 mm nadp . this solution was mixed at room temperature and then 5 mm glucose 1-phosphate was added . the solution was mixed again and placed in a spectrophotometer . to insure that readings were being taken in the linear range of the enzyme activity , readings were taken at t = 6 min at od340 and were expressed as od/g / min . all extracts were assayed in triplicate , and the averages and standard deviations are presented . means were compared using unpaired , two - side t - tests and p - values are presented . the enzyme assay measures the conversion of nadp to nadph rather than a direct measure of phosphoglucomutase itself . in other words , it is measuring a secondary , coupled reaction . all inserts for microinjection were cloned into a derivative of the 7t40 construct called usc1.0 . usc1.0 was generated as follows : first , 7t40 was digested with ecori , thus liberating the hsp70 polyadenylation signal sequence . next , the psti site in polylinker 1 was destroyed by partial psti digestion followed by t4 dna polymerase fill - in and ligation . clones were screened for destruction of the correct psti site to generate usc1.0 ( figure 2a ) . the reverse transcription was carried out on total rna isolated from a strain of flies homozygous for the pgm allele , using the following primer : dtbibo : 5'-taacccgggtctacaaagtgatactgcgtaactgactatattttttttttttttttt-3 ' . a nested pcr strategy was then used to amplify the pgm cdna using the following primers : first primer set : bi : 5'-taacccgggtctacaaagtg-3 ' . the resulting pcr product was cloned into the ecorv site of pbluescript ks+ and clones were screened for the correct orientation . the pgm cdna was then liberated by ecori complete digest followed by psti partial digest and cloned into the ecori to psti sites of usc1.0 to generate construct pgmsense ( figure 2b ) . the pgminvrpt construct was made as follows : because the final insert would be a large inverted repeat , pgm was pcr - amplified in two pieces , in opposite orientations . the two sets of primers used for the amplifications were : first set : pgm - sense ( 3 ' ) ( + 1,773 to + 1,794 ) : 5'-agctgaattccacaaactttaataaatccgaaac-3 ' . second set : pgm - anti not ( + 711 to + 731 ) : 5'-agctgcggccgccctgaaccggttgggtgccac-3 ' . each of these pcr products was cloned individually into the ecorv site of pbluescript ks+ , and then restriction digested with ecori and noti ( first - set fragment ) or noti and psti ( second - set fragment ) . each of the liberated fragments was gel purified and triple - ligated into the psti to ecori sites of usc1.0 to generate construct pgminvrpt ( figure 2c ) . rna was extracted using trizol reagent ( gibco brl ) and the amount of rna was quantitated by uv spectrophotometry . approximately 5 - 10 g rna per lane after blotting , the rna was fixed to genescreen nylon membrane ( dupont ) using a uv crosslinker ( stratagene ) . probes were p - labeled using the prime - it ii kit ( stratagene ) . to determine fold increases / decreases , northern blots were exposed onto a phosphor screen and results were analyzed using a phosphorimager ( imagequant , molecular dynamics ) . ribosomal protein 49 gene ( rp49 ) was used as a loading control , and all pgm northern data is normalized to rp49 rna levels . m.j.a . was supported by a pre - doctoral training grant from the national institute on aging ( ag00093 ) . this research was supported by a grant from the department of health and human services to j.t . the amino acids that differ between the pgm , pgm , pgm and wild - type canton - s alleles are indicated in single - letter code , with the predicted pi values at the right . the genbank accession numbers for the sequences are af416982 , af416981 , af416983 and af416984 , respectively . unique psti and ecori sites are located downstream of the tet - on promoter , enabling cloning of cdnas . ( c ) the pgminvrpt construct , containing an approximately 1 kb inverted repeat of pgm coding region , as indicated by inverted arrows . total rna was isolated from adult males of the indicated pgmsense lines , pgminvrpt lines , and control line , with or without dox treatment . ( a ) northern blot hybridized with a probe that will recognize both endogenous and transgenic pgm transcripts and with a probe specific for rp49 as a loading control . ( b ) northern blot hybridized with a probe that will recognize only the endogenous pgm transcript and with a probe specific for rp49 as a loading control . ( c ) northern blot of control strain hybridized with a probe that will recognize the endogenous pgm transcript and with a probe specific for rp49 as a loading control .

backgrounda tetracycline - regulated ( conditional ) system for rna interference ( rnai ) would have many practical applications . such a strategy was developed using rnai of the gene for phosphogluconate mutase ( pgm ) . pgm is a candidate lifespan regulator : pgms allele frequency is increased by selection for increased lifespan , whereas pgmm and pgmf allele frequencies are decreased.resultsthe pgm alleles were cloned and sequenced and were found to differ by amino - acid substitutions consistent with the relative electrophoretic mobilities of the proteins . the ' tet - on ' doxycycline - regulated promoter system was used to overexpress pgms in a wild - type ( pgmm ) background . enzyme activity increases of two- to five - fold were observed in five independent transgenic lines . tet - on was also used to drive expression of an inverted - repeat fragment of pgm coding region . the inverted - repeat transcript was expected to form a dsrna hairpin , induce rnai , and thereby reduce endogenous pgm gene expression at the rna level . endogenous pgm rna levels in adult flies were found to be reduced or eliminated by doxycycline treatment in five independent inverted - repeat transgenic lines . our results show that doxycycline - regulated expression of inverted - repeat constructs can cause a conditional reduction in specific gene expression . the effect of sense and inverted - repeat construct expression on lifespan was assayed in multiple transgenic lines . under the conditions tested , altered pgm gene expression had no detectable effect on adult drosophila lifespan.conclusionsa system for conditional rnai in drosophila adults shows promise for assay of gene functions during aging . our results indicate that pgm does not have a simple strong effect on longevity .

Background Results Discussion Materials and methods None Lifespan assays DNA sequencing Phosphogluconate mutase enzyme activity assays DNA constructs Northern analyses Acknowledgements Figures and Tables

in the ' tet - on ' system , feeding dox to drosophila melanogaster causes high levels of transgene expression in all tissues . by waiting until the young adult stage to administer dox , all of pre - adult development is identical between control and experimental groups , and any difference in lifespan subtle differences in the genetic background of drosophila strains can have significant effects on lifespan . with the tet - on system , control and experimental animals have identical genetic backgrounds , and therefore any differences in lifespan must be due to dox administration and transgene expression . dox itself , and overexpression of control genes such as escherichia coli lacz , have no detectable effects on lifespan . in contrast , several genes have been identified for which dox - regulated overexpression has negative effects on lifespan , and overexpression of the dgmii gene , encoding -mannosidase ii , was associated with slightly increased lifespan . expression of antisense rna has long been known to be able to inhibit gene expression in drosophila and other organisms . double - strand rna ( dsrna ) formed by hybridization of sense and antisense sequences is thought to initiate a pathway in which homologous rna sequences are destroyed . this phenomenon has been referred to as post transcriptional gene silencing ( ptgs ) or rna interference ( rnai ) . expression of inverted - repeat constructs , where the transcript is expected to fold into a dsrna hairpin , has been shown to be an efficient initiator of rnai . in experiments reported here , the tet - on system was used to drive expression of inverted - repeat constructs in drosophila to determine if a conditional system for rnai could be created . in natural populations , lifespan and reproductive period the duration of the reproductive period is subject to natural selection , and lifespan is altered as a consequence . in one well studied set of five replicate control ( b ) and long - lived ( o ) strains , lifespan was doubled and was correlated with increased stress - resistance and increased glycogen and lipid stores . consistent with this observation , overexpression of cu / znsod in transgenic drosophila has been shown to be sufficient to cause significant increases in lifespan . the most dramatic change in allele frequency in the o and b lines was observed for the gene for phosphogluconate mutase ( pgm ) . pgm exists in three electrophoretic forms : fast ( pgm ) , medium ( pgm ) and slow ( pgm ) . pgm predominates in the b strains , whereas pgmallele frequency is on average ten - fold higher in the long - lived o strains relative to the b controls . when o strains were taken off selection for several generations ( back - selection ) , lifespan decreased and pgm allele frequency was reduced to levels more like those in b strains . these results suggest that pgm or a closely linked gene is responding to selection , and make pgm a candidate lifespan regulator . its activity is therefore important for both glycolysis and glycogen synthesis , and the altered pgm allele frequency might therefore be relevant to the increased glycogen and lipid stores of the o strains . if the increased pgm allele frequency is contributing to the unique phenotypes of the long - lived o strains , this might be because pgm has increased or decreased enzyme activity , or enzyme activity has been altered in some way ( perhaps by a change in its regulation ) . multiple strains were generated that were homozygous for each of the f , m and s electrophoretic alleles of pgm , by appropriate crosses to a third chromosome balancer stock . this result was expected , as differences in genetic background between such purified chromosome strains has profound effects on lifespan and the activities of various enzymes . the pgm coding - region sequences were cloned and sequenced from the pgm , pgm and pgm homozygous strains and from the canton - s wild - type strain . amino - acid substitutions were identified that predicted pi values for the alleles that correlated with their mobility on starch gels ( figure 1 ) . the substitution of t for a in pgm creates the amino - acid sequence ttk ( in the single - letter amino - acid code ) which is a potential phosphorylation site for protein kinase c ( pkc ) that is absent in pgm and pgm . if pgm contributes to the increased lifespan of the o lines , this might be due to increased enzyme activity , decreased enzyme activity , or alteration in enzyme activity in some other way such as in its regulation or subcellular localization . the tet - on system was used to test whether simply increasing or decreasing pgm expression would be sufficient to alter lifespan . a p - element transformation vector called usc1.0 was generated , which had unique ecori and psti sites downstream of the tet - on doxycycline - regulated promoter ( figure 2a ) . the pgm cdna sequences were cloned into usc1.0 to create the pgmsense construct ( figure 2b ) , which should allow overexpression of the pgm enzyme . a fragment of pgm coding region of approximately 1 kilobase ( kb ) ( from + 711 to + 1,794 ) was cloned into usc1.0 in an inverted - repeat orientation to create construct pgminvrpt ( figure 2c ) , which should potentially cause conditional rnai . for each pgm construct five independent transgenic lines were generated using standard methods . expression of the transgenic constructs and the endogenous pgm gene was assayed by northern blot , with or without dox ( figure 3a ) . the pgm inverted - repeat sequences were used as probes and should hybridize to both transgenes and the endogenous pgm transcript . in the sense - construct lines , feeding dox resulted in efficient expression of the transgene at levels five- to ten - fold greater than the endogenous transcript . dox - induced expression of the inverted - repeat construct had two effects detected by northern blot . first , dox produced a smear of hybridization resulting from the inverted - repeat transcript ( figure 3a ) . the smear pattern may have resulted from incomplete denaturation of the hairpin structure and/or instability of the inverted - repeat transcript . second , the level of the endogenous pgm transcript was found to be reduced by dox feeding . the northern blots were also hybridized with a pgm - specific probe corresponding to pgm coding - region sequences ( + 1 to + 700 ) located outside the region used to create the inverted repeat . using this probe the smear of hybridization derived from the inverted - repeat construct was no longer detected , and the endogenous pgm rna levels were more readily observed and quantitated ( figure 3b ) . dox induced expression of the inverted - repeat construct was found to cause a reduction of endogenous pgm rna levels . the inverted - repeat construct therefore appears to function as expected to induce rnai and cause decreased expression of the endogenous pgm gene . in the control strains where no transgene is expressed , dox administration was found to have no effect on pgm transcript levels ( figure 3c ) . the indirect pgm activity assay was used to assay for changes in pgm gene expression at the protein level . the sense construct yielded conditional overexpression of pgm activity in each transgenic line , with increases ranging from two- to five - fold ( table 1 ) . the pgminvrpt construct yielded decreases in pgm activity of as much as 50% ; however , the effect was quite variable from experiment to experiment ( table 1 ) . in the control strains where no transgene is expressed , dox administration was found to have no consistent effect on pgm levels ( table 1 ) . to determine whether altered pgm gene expression could affect lifespan , mean lifespan was assayed in multiple pgmsense and pgminvrpt transgenic lines with or without dox treatment . lifespan was observed to vary across transgenic lines and replicate experiments with changes ranging from -7% to + 6% , with one outlier at + 16% ( table 2 ) . however , no change in lifespan was observed that was consistent across the sense or inverted - repeat lines or in multiple experiments . the lifespan assays were repeated at 29c , and again no consistent changes were observed ( table 3 ) . we conclude that under these conditions , altered pgm expression does not have a detectable effect on adult drosophila lifespan . first , it represents an evolutionarily conserved pathway that probably has important functions in the regulation of gene expression and the control of transposable elements . second , it provides a means for researchers to test gene functions by experimentally downregulating expression of specific genes under well controlled conditions . in drosophila , rnai can be initiated by injection of dsrna into embryos , and this has allowed identification of novel phenotypes for genes during development . transgenic constructs have been used to cause rnai in drosophila , by using various promoters to drive expression of inverted - repeat fragments of gene coding regions . using the conditional tet - on promoter system to drive expression of an inverted - repeat provided conditional rnai of the pgm gene . this conditional system should have many applications , both in the study of the rnai pathway itself and in the design of experiments where rnai is used a research tool . conditional gene - expression systems have previously been used to identify positive regulators of lifespan , such as cu / znsod and dgmii . in addition , mutations have been used to identify several genes in drosophila that act as negative regulators of lifespan , such as mth , indy , inr and chico . inactivation or downregulation of the genes specifically in the adult using tet - on rnai may allow increased lifespan without confounding effects on development . lam and thummel have recently reported the use of a heat - shock gene promoter to drive expression of dsrna , and the efficient conditional inhibition of gene expression during larval and pupal stages of drosophila development . genetic selection of drosophila populations for late - life reproduction caused the correlated phenotype of increased lifespan and increased the frequency of the slow electrophoretic allele , pgm , of the pgm gene . if pgm contributes to the increased lifespan phenotype of the selected strains , it might be because pgm has increased activity , decreased activity or activity that is altered in some way such as through its regulation or subcellular localization . the pgm alleles were cloned and sequenced and were found to differ by amino - acid substitutions consistent with the relative electrophoretic mobilities of the encoded enzymes . a recent study reported the sequencing of several fast , medium and slow electrophoretic alleles of pgm from a wild population of d. melanogaster . the sequences reported in that study ( verrelli alleles ) are comparable to those reported here ( ives alleles ) as follows : the medium verrelli allele used for alignment is identical in amino - acid sequence to the ives medium allele . the verrelli fast alleles are identical to the ives fast allele in each of the three amino - acid positions at which the ives fast differs from the ives medium ( wild - type ) allele . the ives slow allele differs from the ives medium ( wild - type ) amino - acid sequence at two positions ( amino - acid positions 6 ( e g ) and 9 ( a t ) ) . the amino - acid change in the ives slow allele at position 6 ( e g ) is not reflected in any of the verrelli slow alleles . the second amino - acid change in the ives slow allele ( at position 9 ) in addition , the verrelli slow alleles have ammo - acid substitutions that are not shared by the ives slow allele . the data are consistent with the conclusion that pgm is highly polymorphic and subject to selection in natural populations . consistent with this idea , pgm haplotypes and glycogen content have been found to vary among flies at different latitudes . the tet - on promoter system allowed a test of the hypothesis that increasing or decreasing pgm gene expression would affect lifespan . pgm enzyme levels were increased two- to five - fold using a sense transgenic construct , and pgm rna levels were decreased 1.3- to 24-fold by driving expression of an inverted - repeat construct . no changes in lifespan were detected that were consistent across multiple transgenic lines or replicate experiments . the results indicate that simply increasing or decreasing pgm gene expression does not significantly affect the lifespan of adult drosophila , at least under the conditions tested . the high degree of polymorphism of pgm makes it possible that the slow electrophoretic allele in the ives strain could consist of multiple dna sequence forms . the failure to observe effects on lifespan could therefore conceivably have resulted from overexpression of the wrong dna sequence form of pgm . however , pgm was cloned and sequenced from four independent lines derived from the ives strain and the clones had the same dna sequence . the data therefore suggest that most , if not all , of the pgm alleles in these strains have the same sequence , making sequence heterogeneity an unlikely explanation of the present results . the results suggest several possibilities for the relationship of pgm allele frequency to the increased lifespan of the selected strains . the first is that pgm might in fact contribute to the increased lifespan of the selected strains as a result of an increased or decreased activity , but that the effect on lifespan is too small to be detected in these assays . the second possibility is that pgm allele frequencies are altered because of selection for an unknown linked gene , and this possibility can not be ruled out at this time . the experiments presented here suggest that tet - on regulated overexpression and rnai of genes near pgm may be a promising approach . third , an interesting model is that pgm contributes to increased lifespan of the selected strains because of an alteration in its regulation and/or subcellular localization . the sequence analysis of the pgm alleles reveals a novel potential site in pgm for phosphorylation by pkc ; however , the potential significance of this change is unknown . the pgm allele was used for the overexpression experiments presented here , and presumably the overexpressed enzyme should be subject to any regulation or localization characteristic of pgm . however , the pgm enzyme was overexpressed in a ' wild - type ' pgm background , and it may be that any phenotypic consequences of pgm allele function are not apparent when pgm is also present . finally , another possible explanation for the absence of an effect on longevity is the method of assay . this was done because the segregated - sexes assay is sensitive to even small changes in lifespan , and has been used successfully in the past to detect the effects of other genes on lifespan . it is possible that an effect of pgm on lifespan might be identified with different lifespan assay conditions . however , even if this explanation were to be true , the data still indicate that pgm does not have a simple strong effect on longevity . further experiments will be required to distinguish between these interesting possibilities for the link between pgm allele frequency and lifespan . multiple strains homozygous for each of the pgm , pgm and pgm alleles were generated by purifying third chromosomes from the ives stock , which is the precursor to the o and b selection stocks . all stocks were grown on standard cornmeal - agar medium and were cultured at 25c . before eclosion of the majority of pupae , bottles were cleared of adults and newly eclosed flies were allowed to emerge over the next 48 h. most of the males will have mated during this time . the males only were then removed and were designated one day old , and were maintained at 25c at 40 per vial in culture vials with food , and passaged to new vials every 48 h. for certain experiments the adult males were maintained at 29c , as indicated . for the assay , 30 - 40 l of this supernatant was used in a 3.1 ml assay mixture that contained : 1.5 mm mgcl2 , 26 mm histidine solution ( ph 7.6 ) , 8 m glucose-1,6-diphosphate , 0.45 u / ml glucose-6-phosphate dehydrogenase , and 0.2 mm nadp . to insure that readings were being taken in the linear range of the enzyme activity , readings were taken at t = 6 min at od340 and were expressed as od/g / min . all extracts were assayed in triplicate , and the averages and standard deviations are presented . all inserts for microinjection were cloned into a derivative of the 7t40 construct called usc1.0 . clones were screened for destruction of the correct psti site to generate usc1.0 ( figure 2a ) . a nested pcr strategy was then used to amplify the pgm cdna using the following primers : first primer set : bi : 5'-taacccgggtctacaaagtg-3 ' . ribosomal protein 49 gene ( rp49 ) was used as a loading control , and all pgm northern data is normalized to rp49 rna levels . multiple strains homozygous for each of the pgm , pgm and pgm alleles were generated by purifying third chromosomes from the ives stock , which is the precursor to the o and b selection stocks . before eclosion of the majority of pupae , bottles were cleared of adults and newly eclosed flies were allowed to emerge over the next 48 h. most of the males will have mated during this time . the males only were then removed and were designated one day old , and were maintained at 25c at 40 per vial in culture vials with food , and passaged to new vials every 48 h. for certain experiments the adult males were maintained at 29c , as indicated . for the assay , 30 - 40 l of this supernatant was used in a 3.1 ml assay mixture that contained : 1.5 mm mgcl2 , 26 mm histidine solution ( ph 7.6 ) , 8 m glucose-1,6-diphosphate , 0.45 u / ml glucose-6-phosphate dehydrogenase , and 0.2 mm nadp . to insure that readings were being taken in the linear range of the enzyme activity , readings were taken at t = 6 min at od340 and were expressed as od/g / min . all extracts were assayed in triplicate , and the averages and standard deviations are presented . all inserts for microinjection were cloned into a derivative of the 7t40 construct called usc1.0 . a nested pcr strategy was then used to amplify the pgm cdna using the following primers : first primer set : bi : 5'-taacccgggtctacaaagtg-3 ' . ribosomal protein 49 gene ( rp49 ) was used as a loading control , and all pgm northern data is normalized to rp49 rna levels . the amino acids that differ between the pgm , pgm , pgm and wild - type canton - s alleles are indicated in single - letter code , with the predicted pi values at the right . unique psti and ecori sites are located downstream of the tet - on promoter , enabling cloning of cdnas . ( c ) the pgminvrpt construct , containing an approximately 1 kb inverted repeat of pgm coding region , as indicated by inverted arrows . total rna was isolated from adult males of the indicated pgmsense lines , pgminvrpt lines , and control line , with or without dox treatment .

staphylococcus aureus is a highly successful opportunistic pathogen . a surprisingly high percentage of healthy humans ( up to 40% ) can be asymptomatically colonized with s. aureus . at the same time , s. aureus can cause a wide variety of diseases , from relatively benign skin infections , such as folliculitis and furunculosis , to life - threatening conditions , including erysipelas , deep - seated abscesses , osteomyelitis , pneumonia , endocarditis , sepsis , and toxic shock syndrome ( tss ) . recent epidemiological studies indicate that , at least in the us , the annual mortality caused by the methicillin - resistant strains of s. aureus is higher than the mortality caused by hiv / aids [ 3 , 4 ] . therefore , it is important to understand the immunopathogenesis of invasive diseases caused by s. aureus . staphylococcal superantigens ( ssag ) are one such family of exotoxins produced by s. aureus . ssag are the most potent , naturally occurring biological activators of t lymphocytes . unlike conventional antigens , ssag bind directly to cell surface mhc class ii molecules outside of the peptide - binding groove without undergoing any processing . subsequently , they bind to certain t cell receptor chain variable region ( tcr v ) families and robustly activate the t cells expressing them . the specificity of the ssag to certain tcr v regions alone , but not the tcr per se , results in activation of a large pool of cd4 as well as cd8 t cells ( 30% to 70% of the total t cells ) . ssag can also activate the antigen presenting cells ( apc ) , such as b cells , monocyte / macrophages , and dendritic cells ( dc ) , by several mechanisms either directly , by crosslinking their mhc class ii molecules , or indirectly , through cytokines / chemokines . this results in a profound elevation in systemic levels of several cytokines / chemokines which lead to clinical syndromes such as systemic inflammatory response syndrome ( sirs ) and multiple organ dysfunction syndrome ( mods ) , which can culminate in the death of the afflicted patients . as ssag are produced readily in vivo , they play a major role in the pathogenesis of serious infections such as sepsis , pneumonia , and infective endocarditis ( reviewed in ) . while the ssag predominantly activate t cells of the adaptive immune system through tcr , certain other staphylococcal components such as the cell wall peptidoglycans , lipoteichoic acid , lipoproteins , unmethylated bacterial dna containing the cpg motifs and even the intact bacteria , collectively called pathogen - associated molecular patterns ( pamps ) , can activate the immune system through pathogen recognition receptors ( prr ) comprising the toll - like receptors ( tlr ) and the nod - like receptors ( nlr ) , expressed by cells of both innate and adaptive arms of the immune system [ 911 ] . this leads to production of several proinflammatory cytokines , chemokines , and other inflammatory mediators [ 6 , 1216 ] . some studies have shown that staphylococcal pamps can even directly activate t cells to a proinflammatory phenotype in a tlr2-myd88 dependent manner , while others have demonstrated that s. aureus and staphylococcal pamps are able to override the immune regulatory functions of t regulatory cells , thereby indirectly promoting inflammation . overall , staphylococcal pamps can also promote inflammation and immunopathology similar to ssag by several different mechanisms . given that both the ssag and the staphylococcal pamps elicit a predominantly proinflammatory type of immune response [ 16 , 19 , 20 ] , it is widely believed that , during invasive s. aureus infections , the pamps would act additively or synergistically with ssag leading to a more pronounced inflammatory response and inflicting severe immunopathology [ 7 , 21 ] . on the contrary , some recent studies have shown that the staphylococcal cell wall components and certain other staphylococcal pamps downregulate the immune response to ssag as well as dampen antistaphylococcal immunity [ 2224 ] . thus , the staphylococcal pamps appear to have opposing immunological properties according to different studies . given the significance of s. aureus infections [ 2 , 25 ] , it is important to understand the interaction between staphylococcal pamps and ssag in vivo using appropriate animal models . as transgenic mice expressing human mhc class ii molecules closely mimic the human immune response to infections caused by toxigenic staphylococci as well as streptococci producing superantigens [ 2629 ] , we examined the modulatory role of staphylococcal pamps in immune response to ssag and the outcome of s. aureus pneumonia using hla - dr3 and hla - dq8 transgenic mice . hla - dr3 transgenic mice expressing hla - dra10101 and hla - drb10301 transgenes and hla - dq8 transgenic mice expressing dqa10301 and dqb10302 on the endogenous mhc class ii - null background were used in this study [ 30 , 31 ] . hereafter , they are referred to as hla - dr3 or hla - dq8 mice , respectively . mice were bred within the barrier facility of mayo clinic immunogenetics mouse colony ( rochester , mn ) and moved to a conventional facility after weaning . the following antibodies were used for flow cytometry : cd4 ( clone : gk1.5 ) , cd8 ( clone : 536.7 ) , tcr v6 ( clone : rr4 - 7 ) , tcr v8 ( clone : f23.1 ) , cd86 ( clone : gl-1 ) , and class ii ( clone : tu39 ) . highly purified , endotoxin - reduced , staphylococcal enterotoxin b ( seb , toxin laboratories , sarasota , fl , usa ) was dissolved in pbs at 1 mg / ml and stored frozen at 80c in aliquots . peptidoglycans ( pgn ) purified from s. aureus , lipoteichoic acid ( lta ) derived from s. aureus , and heat - killed staphylococcus aureus ( hksa ) were purchased from invivogen ( san diego , ca ) , dissolved or resuspended in endotoxin - free pbs , respectively , and stored frozen at 80c . the above - mentioned tlr agonists from this vendor have been extensively used in biomedical research . for t cell proliferation , single - cell suspensions of splenocytes from hla - dr3 and hla - dq8 transgenic mice were depleted of red blood cells by buffered ammonium chloride lysis . cells were cultured in hepes - buffered rpmi 1640 containing 5% fetal calf serum , serum supplement , streptomycin , and penicillin , at a concentration of 1 10 cells / well in 100 l volumes in 96-well round - bottomed tissue culture plates . seb , pgn , and hksa were added at indicated concentrations at the same time . after 24 hours , the cells were pulsed with tritiated thymidine ( 1 g / well ) . cells were harvested 18 hours later and the extent of cell proliferation was determined by a standard thymidine incorporation assay . to study the effect of staphylococcal pamps on expression of mhc class ii molecules and costimulatory molecules , splenocytes from hla - dr3 transgenic mice were cultured with medium alone or with hksa ( 10 bacteria / ml ) , seb ( 1 g / ml ) , or seb + hksa ( 10 bacteria / ml + 1 g / ml , resp . ) in 24-well plates . twenty - four hours later , the cells were obtained , washed , stained with indicated antibodies , and analyzed by flow cytometry . other analyses were performed on cells within this gate . for serum cytokine analyses , hla - dr3 mice were challenged with seb ( 50 g ) , pgn ( 50 g ) , hksa ( 10 bacteria ) , or seb plus pgn or hksa at these concentrations . the doses of these reagents were determined from previously published studies [ 22 , 3234 ] . given the rapidity at which ssag cause immune activation , these staphylococcal pamps were administered immediately after seb . after 3 hrs , blood was collected in serum separation tubes ( bd biosciences ) , and sera were separated and stored frozen at 80c in aliquots . the cytokine concentrations in the sera were determined in duplicate using a multiplex bead assay , per the manufacturer 's protocol , and using their software and hardware ( bio - plex and bio - rad ) . for flow cytometric studies , hla - dr3 mice were challenged with seb ( 10 g ) , pgn ( 50 g ) , lta ( 50 g ) , or seb plus pgn or lta at the above indicated concentrations . mice were killed 3 days later and distribution of cd4 and cd8 t cells bearing tcr v8 and v6 in the spleens was determined by flow cytometry as per standard protocol . to study the modulatory role of staphylococcal pamps in seb - induced mortality without d - gal sensitization , hla - dr3 mice were challenged with seb ( 50 g ) , pgn ( 50 g ) , hksa ( 10 bacteria ) , or seb plus pgn or hksa . animals were monitored closely and the moribund animals were euthanized as per iacuc recommendations . to study the modulatory role of staphylococcal pamps in seb - induced mortality with d - gal sensitization , hla - dr3 and hla - dq8 transgenic mice were pretreated with d - gal ( 30 mg / mouse ) . one hour later , animals were challenged with seb ( 510 g ) , pgn ( 50 g ) , hksa ( 10 bacteria ) , or seb plus pgn or hksa . we have recently demonstrated that hla - dr3 transgenic mice are susceptible to pneumonia induced by a toxigenic strain of s. aureus , idrl-7971 , producing the ssag , sea , and seb as tested by pcr and elisa [ 29 , 35 ] . therefore , to study the modulatory role of staphylococcal pamps in pneumonia induced by a superantigen - producing strain of s. aureus , hla - dr3 transgenic mice were intratracheally challenged as described previously with the s. aureus strain , idrl-7971 ( 1.32.5 10 cfu / mouse ) . immediately following bacterial inoculation , mice were left untreated or injected with hksa intraperitoneally ( 10 bacteria / mouse ) . statistical analyses , charts , and generation of survival curves were done using graphpad prism , version 4.03 . direct binding to cell surface mhc class ii molecules without undergoing processing is the first step in the series of events by which ssag causes immune activation . therefore , any alterations in the cell surface expression of mhc class ii molecules on apcs could modulate the response elicited by ssag . in this context , some studies have shown that staphylococcal pamps upregulate the expression of mhc class ii as well as costimulatory molecules on apcs , while others have shown that staphylococcal pamps rather downregulate the expression of mhc class ii molecules , particularly on monocytes [ 2224 ] . since many types of apcs are involved in the presentation of ssag in addition to monocytes , we investigated the effect of staphylococcal pamps on the expression of mhc class ii molecules on different apcs . as hksa would encompass all staphylococcal pamps , splenocytes from hla - dr3 transgenic mice were cultured with hksa or seb or both and the abilities of these agents to modulate the cell surface expression of hla - dr3 and cd86 on b220 cells ( predominantly b cells ) , cd11b cells ( predominantly monocyte / macrophages ) , and cd11c cells ( predominantly dc ) were determined . we first studied the effects of hksa on mhc class ii expression on cd11b splenocytes . interestingly , unlike splenocytes cultured with medium alone , two distinct subsets of cd11b cells could be appreciated in splenocytes cultured with hksa based on the expression profile of cd11b , with one expressing high levels of cd11b ( cd11b ) and the other expressing lower levels , cd11b ( figure 1 ) . in cells cultured with medium alone , cd11b was the dominant phenotype ( nearly 8090% of the cd11b cells were of the cd11b phenotype ) . however , in cells cultured with hksa , 5060% of the cd11b cells were of the cd11b phenotype . in a similar manner , while 8090% of the cd11b cells cultured with seb were cd11b , > 50% of the cd11b cells were cd11b when cultured with seb + hksa ( figure 1 ) . therefore , we studied the expression profiles of hla - dr3 and cd86 on cd11b as well as cd11b subsets . hksa had very little suppressive effect on the expression of hla - dr3 within the cd11b subset either when cultured alone or with seb ( figure 1 ) . however , the expression of hla - dr3 was significantly reduced in the cd11b subsets that were generated in the presence of hksa either alone or along with seb . overall , hksa lowered the expression of hla - dr3 and cd86 only on a subset of cd11b cells ( figure 1 ) . we next studied the effect of hksa on the expression of hla - dr3 and cd86 on b220 splenocytes . unlike in cd11b cells , culturing with hksa did not skew the b220 cells to either b220 or b220 phenotype ( figure 2 ) . unlike in cd11b cells , hksa significantly increased the expression of hla - dr3 molecules on b220 cells over the basal level ( median fluorescent intensity or mfi ; 2185 36 versus 2451 151 in medium versus hksa treated cells , respectively ; p < 0.05 ; figure 2 ) . culturing with seb also significantly increased the expression of hla - dr3 molecules on b220 cells ( mfi in seb treated cells , 2581 127 and p < 0.05 , compared to untreated cells ) . however , hksa failed to suppress seb - induced upregulation of hla - dr3 on b220 cells . on the contrary , expression of hla - dr3 further increased when cultured with seb + hksa ( mfi in seb + hksa treated cells , 2695 205 and p < 0.05 , compared to medium ) ( figure 2 ) . similar pattern was noticed with respect to cd86 expression on b220 cells . culturing with hksa caused a significant ( p < 0.05 ) upregulation of cd86 levels on b220 cells compared to untreated cells , while seb caused a much stronger upregulation of cd86 compared to hksa . however , culturing with hksa and seb resulted in the highest upregulation of cd86 compared to either of these agents alone . these results indicated that hksa failed to suppress seb - induced upregulation of hla - dr3 and cd86 on b220 cells . hksa , seb , and their combination increased the expression of hla - dr3 and cd86 expression on cd11c cells ( see supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2014/468285 ) . overall , these results suggested that hksa augmented the expression of mhc class ii and cd86 on b220 cells and dc . while hksa had minimal or no modulatory effect on cd11b cells in the presence of seb , it reduced the expression of mhc class ii and cd86 on the cd11b cells , which were seen in the presence of hksa . we next investigated how modulation in the expression of mhc class ii molecules and cd86 induced by staphylococcal pamps translated into changes in ssag - mediated t cell activation in vitro . for this , splenocytes from hla - dr3 transgenic mice were stimulated with seb in the presence or absence of certain staphylococcal pamps . most importantly , hksa failed to suppress seb - induced splenocyte proliferation in vitro ( figure 3(a ) ) . pgn by itself was significantly mitogenic to splenocytes from hla - dr3 mice ( stimulation index ranging from 2.5 to 12 depending on the dose of pgn ; p < 0.05 ; student 's t - test ; figure 3(b ) ) . however , as would be expected of a superantigen , seb was more mitogenic to splenocytes than pgn . conversely , pgn , especially at 10 and 50 g / ml , consistently augmented seb - induced splenocyte proliferation ( p < 0.05 ; student 's t - test ) . similar results were obtained with hla - dq8 transgenic mice using pgn and hksa ( data not shown ) . overall , pgn and hksa failed to suppress seb - induced splenocyte proliferation in vitro . on the contrary , it should be noted that ssag cause a systemic inflammatory disease characterized by failure of several vital organs that often leads to death , and in vitro studies with isolated splenocytes reflect very little about this systemic process . therefore , to accurately determine the modulatory role of staphylococcal pamps in vivo , we subsequently performed a series of in vivo studies using our robust hla - dr3 and hla - dq8 transgenic mice , which closely mimic the human immune responses to ssag . challenging hla - dr3 transgenic mice with ssag , such as seb , elicits a massive and rapid elevation in systemic levels of various cytokines and chemokines leading to sirs , multiple organ failure , and death , analogous to tss in humans . therefore , we next investigated the modulatory effect of staphylococcal pamps on seb - induced systemic inflammatory response syndrome . serum cytokine analysis showed that nave hla - dr3 mice had very low levels of cytokines / chemokines ( figure 4 ) . as expected , the serum levels of several cytokines ( such as il-6 , il12p40 , il-12p70 , il-10 , ifn- , and tnf- ) and chemokines ( mcp-1 , kc , mip-1 , mip-1 , eotaxin , and g - csf ) were significantly elevated in mice challenged with pgn or hksa compared to nave mice , likely resulting from activation of the innate immune system . given the biological property of seb , it is not surprising to find that sera from hla - dr3 mice challenged with seb had profoundly elevated levels of all the cytokines and chemokines tested ( figure 4 ) , consistent with our prior reports . moreover , the serum levels of all cytokines and chemokines tested were significantly higher in seb treated mice compared to pgn or hksa treated mice . this is also expected because ssag are the most potent biological activators of the immune system . more importantly , when pgn and hksa were injected along with seb , they failed to suppress seb - induced cytokine / chemokine production . there were no significant differences in the levels of various cytokines and chemokines tested between mice challenged with seb alone compared to seb + pgn or seb + hksa . previous studies have shown that staphylococcal cell wall derivatives are more potent inducers of il-10 than ssag . on the contrary , our studies show that seb is more potent than staphylococcal pamps in eliciting il-10 . however , they are less potent than ssag and they failed to attenuate seb - induced cytokine production in vivo . administration of ssag , such as seb , into hla class ii transgenic mice results in the expansion of cd4 as well as cd8 t cells expressing certain tcr v families . it is known that this process is dependent on expression of mhc class ii molecules and is cytokine driven . therefore , by comparing the changes in t cell repertoire in mice challenged with seb alone or seb along with staphylococcal pamps , we were able to study the immunomodulatory functions of staphylococcal pamps . compared to nave mice , there was a 2-fold increase of cd4 and cd8 t cells expressing tcr v8 , which specifically binds to seb , in mice challenged with seb ( figure 5 ) . as expected , pgn and lta did not have this ability to induce expansion of t cells bearing specific tcr v. more importantly , neither pgn nor lta was able to suppress seb - induced expansion of tcr v8-bearing t cells ( figure 5 ; p = ns when comparing seb with seb + pgn or seb with seb + lta ) . we next investigated the ability of staphylococcal pamps to protect from ssag - induced tss and death . whereas all mice challenged with pgn or hksa alone remained healthy , all hla - dr3 mice challenged with seb ( 50 g ) alone either succumbed to tss or became very sick that they needed to be removed from the study ( p < 0.005 ; the log - rank ( mantel - cox ) test ; figure 6(a ) ; 48 mice in each group ) . interestingly , unlike in the previous report , administration of either pgn or hksa failed to protect hla - dr3 transgenic mice from seb - induced tss . even though mortality in seb + hksa treated mice was delayed initially compared to seb or seb + pgn groups , all mice in this group succumbed earlier to tss compared to other groups . overall , there were no statistical differences in the survival between seb , seb + pgn , and seb + hksa groups ( p = ns ; the log - rank ( mantel - cox ) test ) . however , the survival of all seb - challenged mice ( seb alone , seb + pgn , or seb + hksa ) was significantly reduced compared to nave and pgn or hksa challenged mice ( p < 0.005 ; figure 6(a ) ; the log - rank ( mantel - cox ) test ) . it should be noted that the above experiments were done without d - gal sensitization . as conventional laboratory mouse strains are resistant to ssag - induced shock , they are often presensitized with d - gal . d - gal is a hepatotoxic agent and sensitizes hepatocytes to tnf--mediated cell death [ 32 , 39 ] . therefore , in the next set of experiments , we pretreated hla - dr3 mice with d - gal and one hour later mice were challenged with seb ( 510 g ) , pgn ( 50 g ) , hksa ( 10 bacteria ) , seb plus pgn , or seb plus hksa , similar to the study by chau et al . . while all mice treated with d - gal alone , d - gal plus pgn , or d - gal plus hksa remained healthy , all animals treated with d - gal and seb ( either alone or along with pgn or hksa ) rapidly became hypothermic and lethargic and failed to survive beyond 9 hours ( 68 mice in each group ; table 1 ) . there were no significant differences in survival between d - gal plus seb , d - gal plus seb plus pgn , and d - gal plus seb plus hksa groups . to rule out the possibility that the lack of protection from tss by pgn and hksa may be specific for hla - dr3 , we repeated this study with hla - dq8 transgenic mice . hla - dq8 transgenic mice were pretreated with d - gal as described earlier and subsequently challenged with seb ( 10 g ) , pgn ( 10 or 50 g ) , hksa ( 10 bacteria ) , seb plus pgn , or seb plus hksa . as seen with hla - dr3 transgenic mice , pgn and hksa failed to protect from seb - induced tss in hla - dq8 transgenic mice ( 46 mice in each group ; table 1 ) . overall , staphylococcal pamps conferred little protection from ssag - induced tss . in the final in vivo study , we investigated the immunomodulatory effects of staphylococcal pamps on pneumonia induced superantigen - producing s. aureus [ 29 , 35 ] . since hksa would encompass all the staphylococcal pamps , infected animals were treated with hksa . as shown by us earlier , there was significantly higher mortality ( p < 0.01 ; the log - rank ( mantel - cox ) test ) in hla - dr3 mice infected with the toxigenic s. aureus strain idrl-7971 compared to mice treated with hksa alone [ 29 , 35 ] . moreover , the mortality remained significantly high in hla - dr3 transgenic mice that were infected with idrl-7971 and treated with hksa . the survival curves of mice with idrl-7971-induced pneumonia and those with idrl-7971-induced pneumonia treated with hksa were not significantly different ( p = 0.62 ; the log - rank ( mantel - cox ) test ) . overall , these results suggested that staphylococcal pamps do not confer significant protection in certain staphylococcal diseases such as tss and pneumonia . the pattern recognition receptors ( prr ) expressed by the cells of the innate immune system rapidly sense pamps and mount an inflammatory response to contain infection [ 40 , 41 ] . pamps associated with s. aureus are no exceptions [ 9 , 20 , 42 ] . however , the superantigen exotoxins produced by s. aureus ( ssag ) predominantly activate the t cell arm of adaptive immunity by directly binding to mhc class ii molecules and subsequently stimulating the t lymphocytes expressing certain tcr v families . given that both staphylococcal pamps and ssag activate the immune system , exposure to both of these staphylococcal entities , as might occur in many clinical conditions such as staphylococcal sepsis , pneumonia , or toxic shock syndrome ( tss ) , is believed to result in synergistic activation of the innate and adaptive immune system and a worse disease outcome [ 7 , 21 , 43 ] . however , some recent studies have suggested that staphylococcal pamps may actually suppress the immune response to ssag and cause immune tolerance or even immune deviation following s. aureus infections [ 2224 ] . given these conflicting reports , we investigated the immunomodulatory role of staphylococcal pamps in immune responses elicited by ssag during tss and in staphylococcal pneumonia . our studies , which used the robust hla class ii transgenic mouse model , showed that staphylococcal pamps do not suppress ssag - induced t cell proliferation , do not dampen ssag - induced systemic inflammatory response , and fail to protect from ssag - induced tss or staphylococcal pneumonia . one of the major hypotheses put forth by the previous in vitro studies addressing the immunomodulatory properties of staphylococcal pamps is that they cause immunomodulation by downregulating the expression of mhc class ii on macrophages [ 22 , 24 ] . these studies demonstrated that the staphylococcal cell wall components bind to tlr2 , which are constitutively expressed on monocyte / macrophages and downregulate their mhc class ii molecule expression [ 22 , 24 ] . this diminishes the ability of monocyte / macrophages to present ssag , causing reduced t cell activation , immunosuppression / immune deviation , and protection from tss [ 22 , 24 ] . however , these studies did not take into consideration the role of other apcs that are present in vivo . it is well known that , in addition to monocytes , hla class ii molecules are constitutively expressed at very high levels on several apc types , including dc and b cells . particularly , b cells express high levels of mhc class ii molecules and can efficiently present ssag . while we were able to reproduce the findings of frodermann et al . and wang et al . that hksa could downregulate the expression of mhc class ii on a subset of cd11b cells [ 23 , 24 ] , surprisingly hksa had little or no suppressive effect on the expression of mhc class ii molecules on b220 cells ( figures 1 and 2 ) and cd11c ( supplementary figure 1 ) . rather hksa augmented the expression of mhc class ii as well as cd86 , an important costimulatory molecule on these apc subsets . since b cells and dc vastly outnumber the monocyte / macrophages in the spleen , it is not surprising that staphylococcal pamps had no net immunosuppressive effect in vitro and in vivo . the discrepancy between previous studies and our study could be attributed to the use of human cells versus cells from hla class ii transgenic mice , respectively . in addition to professional apc , even human t cells are known to express hla class ii molecules upon activation . in a similar manner , in addition to the conventional apcs , mhc class ii ( hla - dr3 ) molecules are also expressed on activated t cells in our transgenic mice , as shown by rt - pcr and flow cytometry . t cells from hla - dr3 transgenic mice can present antigens and ssag to other t cells . thus , hla class ii molecules are widely expressed by several professional and nonprofessional apcs . given the abundance of hla class ii molecules in vivo , the affinities of ssag for hla class ii molecules , the rapidity ( ssag can elicit an immune response within minutes ) and robustness with which ssag elicit an immune response , and the presentation of ssag by professional and nonprofessional apcs ( including t lymphocytes ) might rapidly activate the immune system and override the regulatory effects of tlr2 . another mechanism by which staphylococcal pamps mediate immune modulation is through induction of il-10 , specifically by the monocytes [ 23 , 24 ] . however , staphylococcal pamp - driven , il-10-mediated immunomodulation has been shown to be abolished by inf- [ 23 , 24 ] . we and others have consistently shown that ssag readily induce ifn- and systemic levels of ifn- are elevated ( both ifn- mrna and protein ) in mice ( even in the current study ) and humans undergoing tss [ 27 , 32 , 38 ] . therefore , while staphylococcal pamps could suppress the immune response through monocyte / macrophage - derived il-10 , elevated levels of proinflammatory cytokines ( such as il-12 , ifn- , and il-17 ) might overcome this suppressive effect . in this context , other studies have shown that even human pbmcs stimulated with ssag and exposed to staphylococcal pamps or hksa produced significantly higher levels of proinflammatory cytokines . even staphylococcal pamps by themselves can induce the production of proinflammatory cytokines by human pbmc and promote immunopathology [ 34 , 4648 ] . finally , the animal model used to investigate the interaction between staphylococcal pamps and ssag also plays a major role in drawing apt conclusions . we have shown that our hla class ii transgenic mice respond robustly to ssag and are highly susceptible to s. aureus - induced pneumonia as well as ssag - induced tss without requiring any sensitizing agents such as lps or d - galactosamine . therefore , the heightened potency of ssag in hla class ii transgenic mice ( and in humans ) may be beyond the realm of immunoregulation by staphylococcal pamps . taken together , our results suggest that staphylococcal pamps offer little protection during staphylococcal tss and pneumonia conditions wherein higher amounts of ssag are present . high mortality rates associated with invasive s. aureus infections underscore the fact that even in humans staphylococcal pamps are unable to effectively mitigate sirs and mods . however , as suggested in a recent study , it is possible that staphylococcal pamps might play an immunosuppressive or immune deviatory role during s. aureus colonization where elevated levels of ssag are not expected to be present .

staphylococcus aureus is capable of causing a spectrum of human illnesses . during serious s. aureus infections , the staphylococcal pathogen - associated molecular patterns ( pamps ) such as peptidoglycan , lipoteichoic acid , and lipoproteins and even intact s. aureus , are believed to act in conjunction with the staphylococcal superantigens ( ssag ) to activate the innate and adaptive immune system , respectively , and cause immunopathology . however , recent studies have shown that staphylococcal pamps could suppress inflammation by several mechanisms and protect from staphylococcal toxic shock syndrome , a life - threatening systemic disease caused by toxigenic s. aureus . given the contradictory pro- and anti - inflammatory roles of staphylococcal pamps , we examined the effects of s. aureus - derived molecular patterns on immune responses driven by ssag in vivo using hla - dr3 and hla - dq8 transgenic mice . our study showed that neither s. aureus - derived peptidoglycans ( pgn ) , lipoteichoic acid ( lta ) , nor heat - killed staphylococcus aureus ( hksa ) inhibited ssag - induced t cell proliferation in vitro . they failed to antagonize the immunostimulatory effects of ssag in vivo as determined by their inability to attenuate systemic cytokine / chemokine response and reduce ssag - induced t cell expansion . these staphylococcal pamps also failed to protect hla - dr3 as well as hla - dq8 transgenic mice from either ssag - induced toxic shock or pneumonia induced by a ssag - producing strain of s. aureus .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

at the same time , s. aureus can cause a wide variety of diseases , from relatively benign skin infections , such as folliculitis and furunculosis , to life - threatening conditions , including erysipelas , deep - seated abscesses , osteomyelitis , pneumonia , endocarditis , sepsis , and toxic shock syndrome ( tss ) . recent epidemiological studies indicate that , at least in the us , the annual mortality caused by the methicillin - resistant strains of s. aureus is higher than the mortality caused by hiv / aids [ 3 , 4 ] . staphylococcal superantigens ( ssag ) are one such family of exotoxins produced by s. aureus . ssag can also activate the antigen presenting cells ( apc ) , such as b cells , monocyte / macrophages , and dendritic cells ( dc ) , by several mechanisms either directly , by crosslinking their mhc class ii molecules , or indirectly , through cytokines / chemokines . as ssag are produced readily in vivo , they play a major role in the pathogenesis of serious infections such as sepsis , pneumonia , and infective endocarditis ( reviewed in ) . while the ssag predominantly activate t cells of the adaptive immune system through tcr , certain other staphylococcal components such as the cell wall peptidoglycans , lipoteichoic acid , lipoproteins , unmethylated bacterial dna containing the cpg motifs and even the intact bacteria , collectively called pathogen - associated molecular patterns ( pamps ) , can activate the immune system through pathogen recognition receptors ( prr ) comprising the toll - like receptors ( tlr ) and the nod - like receptors ( nlr ) , expressed by cells of both innate and adaptive arms of the immune system [ 911 ] . some studies have shown that staphylococcal pamps can even directly activate t cells to a proinflammatory phenotype in a tlr2-myd88 dependent manner , while others have demonstrated that s. aureus and staphylococcal pamps are able to override the immune regulatory functions of t regulatory cells , thereby indirectly promoting inflammation . given that both the ssag and the staphylococcal pamps elicit a predominantly proinflammatory type of immune response [ 16 , 19 , 20 ] , it is widely believed that , during invasive s. aureus infections , the pamps would act additively or synergistically with ssag leading to a more pronounced inflammatory response and inflicting severe immunopathology [ 7 , 21 ] . on the contrary , some recent studies have shown that the staphylococcal cell wall components and certain other staphylococcal pamps downregulate the immune response to ssag as well as dampen antistaphylococcal immunity [ 2224 ] . thus , the staphylococcal pamps appear to have opposing immunological properties according to different studies . given the significance of s. aureus infections [ 2 , 25 ] , it is important to understand the interaction between staphylococcal pamps and ssag in vivo using appropriate animal models . as transgenic mice expressing human mhc class ii molecules closely mimic the human immune response to infections caused by toxigenic staphylococci as well as streptococci producing superantigens [ 2629 ] , we examined the modulatory role of staphylococcal pamps in immune response to ssag and the outcome of s. aureus pneumonia using hla - dr3 and hla - dq8 transgenic mice . hla - dr3 transgenic mice expressing hla - dra10101 and hla - drb10301 transgenes and hla - dq8 transgenic mice expressing dqa10301 and dqb10302 on the endogenous mhc class ii - null background were used in this study [ 30 , 31 ] . hereafter , they are referred to as hla - dr3 or hla - dq8 mice , respectively . peptidoglycans ( pgn ) purified from s. aureus , lipoteichoic acid ( lta ) derived from s. aureus , and heat - killed staphylococcus aureus ( hksa ) were purchased from invivogen ( san diego , ca ) , dissolved or resuspended in endotoxin - free pbs , respectively , and stored frozen at 80c . for t cell proliferation , single - cell suspensions of splenocytes from hla - dr3 and hla - dq8 transgenic mice were depleted of red blood cells by buffered ammonium chloride lysis . to study the effect of staphylococcal pamps on expression of mhc class ii molecules and costimulatory molecules , splenocytes from hla - dr3 transgenic mice were cultured with medium alone or with hksa ( 10 bacteria / ml ) , seb ( 1 g / ml ) , or seb + hksa ( 10 bacteria / ml + 1 g / ml , resp . ) for serum cytokine analyses , hla - dr3 mice were challenged with seb ( 50 g ) , pgn ( 50 g ) , hksa ( 10 bacteria ) , or seb plus pgn or hksa at these concentrations . given the rapidity at which ssag cause immune activation , these staphylococcal pamps were administered immediately after seb . for flow cytometric studies , hla - dr3 mice were challenged with seb ( 10 g ) , pgn ( 50 g ) , lta ( 50 g ) , or seb plus pgn or lta at the above indicated concentrations . to study the modulatory role of staphylococcal pamps in seb - induced mortality without d - gal sensitization , hla - dr3 mice were challenged with seb ( 50 g ) , pgn ( 50 g ) , hksa ( 10 bacteria ) , or seb plus pgn or hksa . to study the modulatory role of staphylococcal pamps in seb - induced mortality with d - gal sensitization , hla - dr3 and hla - dq8 transgenic mice were pretreated with d - gal ( 30 mg / mouse ) . we have recently demonstrated that hla - dr3 transgenic mice are susceptible to pneumonia induced by a toxigenic strain of s. aureus , idrl-7971 , producing the ssag , sea , and seb as tested by pcr and elisa [ 29 , 35 ] . therefore , to study the modulatory role of staphylococcal pamps in pneumonia induced by a superantigen - producing strain of s. aureus , hla - dr3 transgenic mice were intratracheally challenged as described previously with the s. aureus strain , idrl-7971 ( 1.32.5 10 cfu / mouse ) . in this context , some studies have shown that staphylococcal pamps upregulate the expression of mhc class ii as well as costimulatory molecules on apcs , while others have shown that staphylococcal pamps rather downregulate the expression of mhc class ii molecules , particularly on monocytes [ 2224 ] . since many types of apcs are involved in the presentation of ssag in addition to monocytes , we investigated the effect of staphylococcal pamps on the expression of mhc class ii molecules on different apcs . as hksa would encompass all staphylococcal pamps , splenocytes from hla - dr3 transgenic mice were cultured with hksa or seb or both and the abilities of these agents to modulate the cell surface expression of hla - dr3 and cd86 on b220 cells ( predominantly b cells ) , cd11b cells ( predominantly monocyte / macrophages ) , and cd11c cells ( predominantly dc ) were determined . therefore , we studied the expression profiles of hla - dr3 and cd86 on cd11b as well as cd11b subsets . however , the expression of hla - dr3 was significantly reduced in the cd11b subsets that were generated in the presence of hksa either alone or along with seb . overall , hksa lowered the expression of hla - dr3 and cd86 only on a subset of cd11b cells ( figure 1 ) . we next studied the effect of hksa on the expression of hla - dr3 and cd86 on b220 splenocytes . unlike in cd11b cells , hksa significantly increased the expression of hla - dr3 molecules on b220 cells over the basal level ( median fluorescent intensity or mfi ; 2185 36 versus 2451 151 in medium versus hksa treated cells , respectively ; p < 0.05 ; figure 2 ) . however , hksa failed to suppress seb - induced upregulation of hla - dr3 on b220 cells . these results indicated that hksa failed to suppress seb - induced upregulation of hla - dr3 and cd86 on b220 cells . hksa , seb , and their combination increased the expression of hla - dr3 and cd86 expression on cd11c cells ( see supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2014/468285 ) . we next investigated how modulation in the expression of mhc class ii molecules and cd86 induced by staphylococcal pamps translated into changes in ssag - mediated t cell activation in vitro . for this , splenocytes from hla - dr3 transgenic mice were stimulated with seb in the presence or absence of certain staphylococcal pamps . most importantly , hksa failed to suppress seb - induced splenocyte proliferation in vitro ( figure 3(a ) ) . similar results were obtained with hla - dq8 transgenic mice using pgn and hksa ( data not shown ) . overall , pgn and hksa failed to suppress seb - induced splenocyte proliferation in vitro . therefore , to accurately determine the modulatory role of staphylococcal pamps in vivo , we subsequently performed a series of in vivo studies using our robust hla - dr3 and hla - dq8 transgenic mice , which closely mimic the human immune responses to ssag . challenging hla - dr3 transgenic mice with ssag , such as seb , elicits a massive and rapid elevation in systemic levels of various cytokines and chemokines leading to sirs , multiple organ failure , and death , analogous to tss in humans . therefore , we next investigated the modulatory effect of staphylococcal pamps on seb - induced systemic inflammatory response syndrome . serum cytokine analysis showed that nave hla - dr3 mice had very low levels of cytokines / chemokines ( figure 4 ) . as expected , the serum levels of several cytokines ( such as il-6 , il12p40 , il-12p70 , il-10 , ifn- , and tnf- ) and chemokines ( mcp-1 , kc , mip-1 , mip-1 , eotaxin , and g - csf ) were significantly elevated in mice challenged with pgn or hksa compared to nave mice , likely resulting from activation of the innate immune system . given the biological property of seb , it is not surprising to find that sera from hla - dr3 mice challenged with seb had profoundly elevated levels of all the cytokines and chemokines tested ( figure 4 ) , consistent with our prior reports . more importantly , when pgn and hksa were injected along with seb , they failed to suppress seb - induced cytokine / chemokine production . previous studies have shown that staphylococcal cell wall derivatives are more potent inducers of il-10 than ssag . however , they are less potent than ssag and they failed to attenuate seb - induced cytokine production in vivo . administration of ssag , such as seb , into hla class ii transgenic mice results in the expansion of cd4 as well as cd8 t cells expressing certain tcr v families . therefore , by comparing the changes in t cell repertoire in mice challenged with seb alone or seb along with staphylococcal pamps , we were able to study the immunomodulatory functions of staphylococcal pamps . we next investigated the ability of staphylococcal pamps to protect from ssag - induced tss and death . interestingly , unlike in the previous report , administration of either pgn or hksa failed to protect hla - dr3 transgenic mice from seb - induced tss . however , the survival of all seb - challenged mice ( seb alone , seb + pgn , or seb + hksa ) was significantly reduced compared to nave and pgn or hksa challenged mice ( p < 0.005 ; figure 6(a ) ; the log - rank ( mantel - cox ) test ) . therefore , in the next set of experiments , we pretreated hla - dr3 mice with d - gal and one hour later mice were challenged with seb ( 510 g ) , pgn ( 50 g ) , hksa ( 10 bacteria ) , seb plus pgn , or seb plus hksa , similar to the study by chau et al . to rule out the possibility that the lack of protection from tss by pgn and hksa may be specific for hla - dr3 , we repeated this study with hla - dq8 transgenic mice . hla - dq8 transgenic mice were pretreated with d - gal as described earlier and subsequently challenged with seb ( 10 g ) , pgn ( 10 or 50 g ) , hksa ( 10 bacteria ) , seb plus pgn , or seb plus hksa . as seen with hla - dr3 transgenic mice , pgn and hksa failed to protect from seb - induced tss in hla - dq8 transgenic mice ( 46 mice in each group ; table 1 ) . overall , staphylococcal pamps conferred little protection from ssag - induced tss . in the final in vivo study , we investigated the immunomodulatory effects of staphylococcal pamps on pneumonia induced superantigen - producing s. aureus [ 29 , 35 ] . as shown by us earlier , there was significantly higher mortality ( p < 0.01 ; the log - rank ( mantel - cox ) test ) in hla - dr3 mice infected with the toxigenic s. aureus strain idrl-7971 compared to mice treated with hksa alone [ 29 , 35 ] . moreover , the mortality remained significantly high in hla - dr3 transgenic mice that were infected with idrl-7971 and treated with hksa . overall , these results suggested that staphylococcal pamps do not confer significant protection in certain staphylococcal diseases such as tss and pneumonia . however , the superantigen exotoxins produced by s. aureus ( ssag ) predominantly activate the t cell arm of adaptive immunity by directly binding to mhc class ii molecules and subsequently stimulating the t lymphocytes expressing certain tcr v families . given that both staphylococcal pamps and ssag activate the immune system , exposure to both of these staphylococcal entities , as might occur in many clinical conditions such as staphylococcal sepsis , pneumonia , or toxic shock syndrome ( tss ) , is believed to result in synergistic activation of the innate and adaptive immune system and a worse disease outcome [ 7 , 21 , 43 ] . however , some recent studies have suggested that staphylococcal pamps may actually suppress the immune response to ssag and cause immune tolerance or even immune deviation following s. aureus infections [ 2224 ] . given these conflicting reports , we investigated the immunomodulatory role of staphylococcal pamps in immune responses elicited by ssag during tss and in staphylococcal pneumonia . our studies , which used the robust hla class ii transgenic mouse model , showed that staphylococcal pamps do not suppress ssag - induced t cell proliferation , do not dampen ssag - induced systemic inflammatory response , and fail to protect from ssag - induced tss or staphylococcal pneumonia . since b cells and dc vastly outnumber the monocyte / macrophages in the spleen , it is not surprising that staphylococcal pamps had no net immunosuppressive effect in vitro and in vivo . the discrepancy between previous studies and our study could be attributed to the use of human cells versus cells from hla class ii transgenic mice , respectively . in a similar manner , in addition to the conventional apcs , mhc class ii ( hla - dr3 ) molecules are also expressed on activated t cells in our transgenic mice , as shown by rt - pcr and flow cytometry . t cells from hla - dr3 transgenic mice can present antigens and ssag to other t cells . given the abundance of hla class ii molecules in vivo , the affinities of ssag for hla class ii molecules , the rapidity ( ssag can elicit an immune response within minutes ) and robustness with which ssag elicit an immune response , and the presentation of ssag by professional and nonprofessional apcs ( including t lymphocytes ) might rapidly activate the immune system and override the regulatory effects of tlr2 . therefore , while staphylococcal pamps could suppress the immune response through monocyte / macrophage - derived il-10 , elevated levels of proinflammatory cytokines ( such as il-12 , ifn- , and il-17 ) might overcome this suppressive effect . in this context , other studies have shown that even human pbmcs stimulated with ssag and exposed to staphylococcal pamps or hksa produced significantly higher levels of proinflammatory cytokines . we have shown that our hla class ii transgenic mice respond robustly to ssag and are highly susceptible to s. aureus - induced pneumonia as well as ssag - induced tss without requiring any sensitizing agents such as lps or d - galactosamine . therefore , the heightened potency of ssag in hla class ii transgenic mice ( and in humans ) may be beyond the realm of immunoregulation by staphylococcal pamps . taken together , our results suggest that staphylococcal pamps offer little protection during staphylococcal tss and pneumonia conditions wherein higher amounts of ssag are present . high mortality rates associated with invasive s. aureus infections underscore the fact that even in humans staphylococcal pamps are unable to effectively mitigate sirs and mods . however , as suggested in a recent study , it is possible that staphylococcal pamps might play an immunosuppressive or immune deviatory role during s. aureus colonization where elevated levels of ssag are not expected to be present .

the neurogliaform family of cells is one of the major groups of gabaergic neurons expressing nitric oxide synthase ( nnos ) . neurogliaform cells , originally described by cajal as arachniform cells ( ramn y cajal , 1922 ; ramn y cajal , 1999 ) , together with the closely related ivy cells , which were only recently characterized ( fuentealba et al . , 2008 ) , are estimated to be the most abundant gabaergic cell type in area ca1 of the hippocampus ( fuentealba et al . , 2008 ) . however , the neurogliaform family of cells is found in large numbers not only throughout ca1 , but also across a range of brain regions , including the entire hippocampal formation ( vida et al . , 1998 ; price et al . , 2005 ; elfant et al . , 2008 ; price et al . , 2008 ; karayannis et al . , 2010 ; szabadics et al . , 2010 ; armstrong et al . , 2011 ; krook - magnuson et al . , 2011 ; markwardt et al . , 2011 ) , the neocortex ( tamas et al . , 2003 ; simon et al . , 2005 ; olah et al . , 2007 ; szabadics et al . , 2007 ; olah et al . , 2009 ) , the piriform cortex ( suzuki and bekkers , 2012 ) , striatum ( ibanez - sandoval et al . , 2011 ) , and the habenula of the thalamus ( weiss and veh , 2011 ) . their function in the context of local circuit dynamics is only beginning to be unraveled . here , in addition to reviewing the known anatomical , synaptic , neuromodulatory , and connective properties of neurogliaform family cells , we discuss their potential network functions , particularly in the hippocampal formation . despite their wide distribution , cells within the neurogliaform family have very similar characteristics across brain regions , and these unique characteristics clearly set them apart from other gabaergic cell types . neurogliaform cells have a dense local axonal plexus and generally small , round , somata ( figure 1 ) . the axons of neurogliaform cells have frequent , small en passant boutons , which despite their small size can form synaptic contacts ( vida et al . , 1998 ; the synapses formed by neurogliaform family cell boutons , however , do not necessarily form classical synapses like those of other gabaergic cell classes , in that the synaptic cleft at identified synapses is unusually wide and some boutons , complete with synaptic vesicles , do not have an easily identifiable postsynaptic target in the classical sense ( vida et al . their unique synaptic and axonal morphologies underlie some key features of neurogliaform synaptic transmission ( figure 2 ) , namely ( 1 ) the ability of neurogliaform cells to mediate gabaergic volume transmission , affecting virtually any processes within their dense axonal plexus , ( 2 ) the production of a slow gabaa current in the postsynaptic cell ( gabaa , slow ) , and ( 3 ) the postsynaptic gabab response to even a single neurogliaform action potential ( tamas et al . , 2003 ; price et al . , 2005 ; szabadics et al . , 2007 ; price et al . , 2008 ; olah et al . , 2009 ; karayannis et al . , 2010 ; capogna and pearce , 2011 ) neurogliaform and ivy cells in the hippocampus : ( a ) dentate gyrus neurogliaform cell , ( b ) ca3 ivy cell , ( c ) ca1 ivy cell , ( d ) ca1 neurogliaform cell ; these cells are characterized by their dense local axonal plexus ( black : axon ; blue : somata and dendrites ) . these cells also show a characteristic late - spiking firing pattern in response to depolarizing current steps [ insets , ( a , c , d ) ; scale bars 20 mv , 200 ms ] . ml , molecular layer ; gcl , granule cell layer ; so , stratum oriens ; sp , stratum pyramidale ; sl , stratum lucidum ; sr , stratum radiatum ; slm , stratum lacunosum - moleculare . cells represented in ( a , b ) are from rat ( e ) schematic diagram of the hippocampus , illustrating the approximate relative locations of cells in ( a d ) ( scale bar = 100 m ) . note that the ivy cell reconstructed in ( c ) targets proximal dendrites , while the neurogliaform cell in ( d ) targets distal dendrites of ca1 pyramidal cells . ( f ) recorded , biocytin - filled cells confirmed as neurogliaform cells in the dentate gyrus ( three separate cells , numbered 13 ) express a variety of markers , including coup tfii , nnos , reelin , and npy ( scale bar = 20 m ) among others ( see text ) . ( g ) light microscopic image , showing the characteristic neurogliaform axonal arborization , with multiple axonal branches passing through a single plane of focus and frequent , small en passant boutons [ same cell as ( a ) , scale bar 20 m ] . ( h ) in addition to the classical late - spiking pattern characteristic of these cells , a high percentage of neurogliaform and ivy cells display the recently described phenomenon referred to as persistent firing . after hundreds of action potentials induced by repeated depolarizing current steps ( note that only the last depolarizing step is shown here ) , the cell continues to fire action potentials after the cessation of depolarizing input ( bottom trace : schematic of current injection ; top trace : current clamp recording , dashed line indicates 60 mv ) . note the apparent low action potential threshold ( arrow ) . due to the long duration of the persistent firing state , the trace has segments omitted ( indicated by hash marks ) for illustration purposes . reproduced and modified with permission from armstrong et al . ( a ) action potentials in a neurogliaform cell ( top trace , example from neocortex ) produce a slow ipsc ( gabaa , slow bottom trace ) in the postsynaptic cell . ( b ) in contrast , action potentials in fast - spiking basket cell ( top trace ) produce an ipsc with fast kinetics in the postsynaptic cell ( bottom trace ) . ( c ) neurogliaform cells ( averaged examples from dentate gyrus ) produce a biphasic postsynaptic response , consisting of a slow gabaa and a gabab component , which can be distinguished by application of the gabab antagonist cgp55845 ( red trace ) . this biphasic response can be seen following even a single presynaptic action potential ( inset ) . ( d ) neurogliaform cells ( ngfc , black traces ) can form both electrical and chemical synaptic connections with other gabaergic cell types . in this example , an electrical and unidirectional synaptic connection between a ngfc ( black traces ) and a non - ngfc ( green traces ) left traces : a hyperpolarizing current step ( pre , current clamp responses shown in the upper traces of each example ) , evokes an outward current in the electrically connected cell ( post , voltage clamp responses shown in the lower traces of each example ) . right : when an action potential is evoked in the presynaptic non - ngfc , only a short inward current , due to electrical coupling , is observed in the connected ngfc , while both electrical and chemical synaptic responses can be appreciated in the postsynaptic non - ngfc in response to ngfc stimulation ( upper traces , both pre and postsynaptic traces are averaged ) . note the electrical responses in the non - ngfc riding on top of the slow gaba - mediated ipsc . reproduced and modified with permission from szabadics et al . neurogliaform cells ability to induce a biphasic current in the postsynaptic cell , including both a gabaa - mediated and a gabab - mediated component , is a property which has been consistently observed across brain regions ( tamas et al . , 2003 ; price et al . , 2005 ; the gabaa and gabab - mediated components can be separated based on reversal potential , since the k - mediated gabab component and the largely cl - mediated gabaa component reverse at different membrane potentials . additionally , the gabaa and gabab - mediated components can be pharmacologically separated using specific antagonists for gabaa and gabab receptors ( figure 2 ) . importantly , this biphasic gaba - mediated current results in a large inhibitory charge transfer in the postsynaptic target , and involves even traditionally extrasynaptic receptors , such as subunit - containing gabaa receptors and gabab receptors , as well as classically synaptic receptors , e.g. , benzodiazepine - sensitive subunit - containing gabaa receptors ( szabadics et al . , 2007 ; olah et al . , 2009 ; karayannis et al . , 2010 ) . the kinetics of the neurogliaform cell - evoked gabaa response are also considerably slower than the kinetics of responses to other gabaergic cell types , and these events , referred to as gabaa , slow ( pearce , 1993 ; reviewed in capogna and pearce , 2011 ) , were shown to arise from neurogliaform cells ( tamas et al . , 2003 ; price et al . , 2005 ; szabadics et al . , 2007 ) . in theory , . however , the kinetics of the gabaa , slow response are not affected by altering release probability through variation of external ca concentration , such that vesicular release properties can not explain the kinetics ( szabadics et al . , 2007 ) . dendritic filtering of neurogliaform cell input to distal dendrites also can not explain the slow kinetics , since other dendritically targeting cells ( in this case , martinotti cells ) do not induce gabaa , slow ( szabadics et al . , 2007 ) . further , while spillover of gaba does occur , affecting both gabaa , slow and gabab - mediated events , the extreme kinetics of the responses are not fully explained by gaba spillover . importantly , the postsynaptic gabaa , slow response is highly sensitive to low - affinity competitive gabaa receptor antagonists , indicating that low concentrations of gaba at the postsynaptic membrane contribute to the slow unitary kinetics of neurogliaform cell connections ( szabadics et al . , 2007 ) . these data suggest that the nature of the gaba transient ( the spatiotemporal concentration profile of gaba at the synapse ) is largely responsible for the kinetics of neurogliaform connections ( krook - magnuson and huntsman , 2007 ; szabadics et al . , this unique gaba transient can be explained by the morphology of the synapse ( i.e. , the relatively small area combined with the relatively large distances between pre- and post - synaptic processes ; szabadics et al . interestingly , although in vivo neurogliaform cells do fire in a phase - locked manner with theta rhythms ( fuentealba et al . , 2010 ) , paired neurogliaform cell connections in acute slices demonstrate profound depression with repeated stimulation ( tamas et al . , 2003 ) , an effect which persists in the presence of gabab antagonists ( karayannis et al . , 2010 ) . the axonal arbors of neurogliaform cells described above are one of the most distinctive features of these cells . while the axons of neurogliaform cells form a unique dense , local plexus , they can also cross boundaries of brain regions . for example , neurogliaform cells were recently identified in the molecular layer of the dentate gyrus ( armstrong et al . , 2011 ) , where importantly , it was found that their axons can cross the hippocampal fissure and extend into the adjacent ca1 or subiculum ( armstrong et al . , similarly , axons of neurogliaform cells in the ca1 can cross into dentate molecular layer ( ceranik et al . this extension of axons across boundaries is a notable feature among gabaergic cells , suggesting that neurogliaform cells may serve to share information between , and coordinate the activities of , distinct brain regions . beyond their remarkable chemical synapses , in contrast , neurogliaform cells frequently form gap junctions with a wide variety of other gabaergic cell types ( figure 2 ; simon et al . , 2005 ; , 2007 ; zsiros and maccaferri , 2008 ; armstrong et al . , 2011 ) . this is important when considering the role of neurogliaform cells in hippocampal networks , which will be discussed below . despite the similarities in axonal and synaptic properties of individual cells of the neurogliaform family , there are some notable differences between them as well . members of the neurogliaform family may differ in where they reside within a given brain region , the input they receive , and the domain of the postsynaptic cells their axons target , as well as in the neuronal markers that they express . as mentioned above neurogliaform cells classically target the distal dendrites of principal cells while ivy cells , which have to date been definitively identified in the ca1 and the ca3 of the hippocampus ( fuentealba et al . , 2008 ; szabadics and soltesz , 2009 ; szabadics et al . , 2010 ) , have a unique position in and around the pyramidal cell layer , where they may have different incoming and outgoing connectivity when compared to neurogliaform cells . in the ca1 , where ivy cells were first described ( fuentealba et al . , 2008 ) , neurogliaform cells in and near the stratum lacunosum - moleculare receive excitatory inputs from both the temporo - ammonic pathway and ca3 schaffer collaterals , and in turn target the distal dendrites of ca1 pyramidal cells ( figures 1 and 3 ; price et al . , 2005 ; fuentealba et al . , in contrast , ivy cells reside in or near the stratum pyramidale , receive excitatory input from local pyramidal cells as well as presumably from ca3 schaffer collaterals , and target the proximal dendrites of ca1 pyramidal cells ( fuentealba et al . , 2008 ) . this differential input means that , while neurogliaform cells near the ca1 lacunosum - moleculare serve a primarily feedforward inhibitory role , ivy cells near the pyramidale are poised to mediate both feedforward and feedback inhibition to ca1 pyramidal cells . ivy and neurogliaform cells can also differ in their in vivo firing properties and dendritic morphologies ( fuentealba et al . , 2008 ; fuentealba et al . , 2010 ) , and differences in network functions of neurogliaform and ivy cells in vivo will be discussed further below . entorhinal cortical input ( green ) directly excites ngfcs ( red cells ) both in the dentate gyrus and in the ca1 . these cells provide feedforward inhibition to principal cells ( blue ) of the dentate gyrus or ca1 , respectively . in the dentate , neurogliaform family cells targeting proximal dendrites ( ivy , brown ) provide inhibition to both adult granule cells ( gc ) and newly born granule cells ( ngc ) . granule cells of the dentate , as well as granule cells of the ca3 provide mossy fiber input to ca3 ivy cells , which provide both feedforward inhibition to pyramidal cells ( pyr ) and feedback inhibition to ca3 , but not dentate , granule cells . schaffer collateral input to the ca1 contacts ca1 ngfcs and presumably , ivy cells which both provide feedforward inhibition to ca1 pyramidal cells . both ngfcs and ivy cells in the ca1 express the -opioid receptor ( or , yellow triangle ) . in this simplified schematic , excitatory input to excitatory cells has been omitted . ( a ) indicates the observed connectivity of ngfcs ( red ) in the dentate gyrus with other interneurons ( light blue ) , consisting of five different connectivity motifs ( 1 : bidirectional chemical synaptic ; 2 : unidirectional chemical synaptic ; 3 : electrical only ; 4 electrical and bidirectional chemical synaptic ; and 5 : electrical and unidirectional chemical synaptic ) . ( b ) shows the response in a dentate gyrus ngfc to perforant path stimulation . ( c ) illustrates that individual mossy fiber boutons ( mf , blue trace ) form strong unitary connections ( left box ) with postsynaptic ivy cells in the ca3 ( brown traces ) while unitary connections between mossy fiber boutons and fast - spiking basket cells ( fsbc , orange traces ) are smaller in amplitude . however ( right box ) , while ivy cells receive relatively fewer individual connections from mossy fibers , fsbcs receive more frequent mossy fiber bouton input . ( d ) demonstrates , in a paired recording , that ivy cells ( action potential , brown trace ) provide inhibition to ca3 gcs ( ipscs , black traces ) . ( e ) shows the effect of the -opioid receptor agonist , damgo on an ivy cell ( brown trace ) to ca1 pyramidal cell ( lower traces ) pair . the paired connection ( control acsf , black ) is nearly abolished by application of damgo ( light blue ) , and can be restored by addition of the -opioid receptor antagonist , ctap ( damgo + ctap , purple ) . ( f ) indicates the relative timing of ngfc and ivy cell firing during theta rhythms recorded from the pyramidal cell layer in vivo . ngfcs tend to fire right after the peak of theta , shortly following entorhinal input , while ivy cells tend to fire right after the trough , shortly following ca3 input and just after the firing of ca1 cells . ( 2011 ) ( a , b ) ; szabadics and soltesz ( 2009 ) ( c ) ; szabadics et al . ( 2011 ) ( e ) . even within the different types of neurogliaform family cells thus , while neuropeptide y ( npy ) , nnos , coup tfii , -actinin , gabaa1 , gabaa , and reelin are notably found in neurogliaform cells ( figure 1 ) , there is significant heterogeneity in marker expression from cell to cell such that no one marker or combination of markers captures , uniquely , all neurogliaform cells ( deller and leranth , 1990 ; ratzliff and soltesz , 2001 ; price et al . , 2005 ; , 2009 ; olah et al . , 2009 ; fuentealba et al . , 2010 ; tricoire et al . , ivy cells of the ca1 seem to share many of the same markers as neurogliaform cells , but have not been observed to express reelin ( fuentealba et al . , 2010 ) . interestingly , the presence or absence of nnos may serve as a marker of developmental origin for both neurogliaform and ivy cells , that is whether the cells arise from the medial or caudal ganglionic eminences , both of which can generate neurogliaform family cells ( tricoire et al . , 2010 ) . as might be expected due to their differential inputs and placement within the ca1 , ivy and neurogliaform cells of the ca1 differ also in their in vivo firing patterns . in anesthetized animals , when both theta and gamma oscillations are recorded from the stratum pyramidale , individual ivy cells fire at a low frequency shortly after the trough of theta that is , after ca3 input and just after ca1 pyramidal cells fire and during the trough of gamma oscillations , while staying primarily silent during ripples ( fuentealba et al . , 2008 ; klausberger and somogyi , 2008 ; mizuseki et al . , 2009 ; fuentealba et al . , on the other hand , neurogliaform cells fire just after the peak of theta , following input to ca1 from the entorhinal cortex ; are phase - coupled with the locally recorded gamma ; and are either unmodulated by , or show a decrease in firing during ripples ( buzsaki , 2002 ; fuentealba et al . , 2010 ) . in awake animals , putative ivy cells recorded using tetrodes have similar firing properties during theta oscillations and ripples as ivy cells in anaesthetized animals ( fuentealba et al . , 2008 ) . despite these differences in firing patterns in vivo , when recordings are made from slices in whole - cell patch configuration , both ivy and neurogliaform cells exhibit a characteristic late - spiking firing pattern , often with a depolarizing ramp and subthreshold oscillations in the gamma frequency range leading up to the first spikes ( szabadics et al . , 2007 ; armstrong et al . , 2011 ; krook - magnuson et al . , 2011 ; weiss and veh , 2011 ; while neurogliaform cells of rodents typically do not exhibit a sag upon hyperpolarization , neurogliaform cells of humans and monkeys do ( olah et al . both ivy and neurogliaform cells exhibit the recently described phenomenon of persistent firing ( krook - magnuson et al . , 2011 ; persistent firing is a state of continued firing in the absence of continued input ( sheffield et al . , 2011 ) . experimentally , it is induced by repeated somatic current injections , causing the cell to fire hundreds of action potentials over the span of minutes . during this time period , there appears to be an integration of spiking information , eventually resulting in the induction of persistent firing . once this state of persistent firing is achieved , spiking occurs without further input for tens of seconds ( though in rare instances it can persist for over 10 min ; sheffield et al . , persistent firing is not affected by blocking gabaa , gabab , ampa , and nmda receptors ( sheffield et al . , 2011 ) , but induction is inhibited by activation of -opioid receptors ( krook - magnuson et al . , 2011 ) . the apparent threshold for firing during persistent firing is very low when recording from the soma , and it has been shown that this is due to the action potentials starting in the axons themselves and back - propagating to the soma ( sheffield et al . , 2011 ) . somatically recorded spikes have an initial component , representing spiking in the axon , and a subsequent component , indicative of a somato - dendritic spike ( sheffield et al . , 2011 ) . the mechanism permitting the integration of spiking information over the span of minutes , required for the induction of persistent firing , is a matter of open debate and investigation , as are the physiological consequences of persistent firing . importantly , however , not all interneurons display persistent firing ( krook - magnuson et al . indeed , less than 20% of fast - spiking parvalbumin expressing basket cells display persistent firing , in contrast to over 80% of neurogliaform family cells ( krook - magnuson et al . , 2011 ) . neurogliaform family cells have recently been described in the dentate gyrus ( armstrong et al . , 2011 ; markwardt et al . , 2011 ) . neurogliaform cells located in the middle and outer molecular layers receive entorhinal input and synapse on distal dendrites of granule cells , meaning that they are capable of providing feedforward inhibition ( armstrong et al . , 2011 ; figures 1 and 3 ) . compared to granule cells , neurogliaform cells in the dentate molecular layer receive fewer spontaneous inputs ( consisting of excitatory and inhibitory events at roughly equal frequencies ) to their small dendritic arbors than granule cells . however , like granule cells , they do receive direct perforant path input from the entorhinal cortex . this perforant path input is facilitating at high frequencies , and suggests that neurogliaform cells may act to inhibit granule cells primarily during high frequency incoming activity ( armstrong et al . , 2011 ) . in regions like the dentate gyrus , where granule cells exhibit sparse firing , feedforward gabaergic input has been predicted to play a major role in normal function ( ferrante et al . , 2009 ) , including for spatial navigation , as well as in pathological states , such as in epilepsy . because neurogliaform cells target the distal dendrites of principal cells , they may have stronger effects on the processing of incoming input in dendritic compartments than on spike timing control per se . however , inhibitory dendritic input , in addition to its role in dendritic processing , has also recently been shown to have major effects on the firing of postsynaptic cells ( lovett - barron et al . , 2012 ) . thus , new studies will be necessary to determine what the major effect of neurogliaform cell activation may be during concurrent dendritic input to granule cells from other sources . the fact that neurogliaform cells form both chemical and electrical synapses with other classes of gabaergic neurons means that they can display a variety of connectivity motifs . in the dentate gyrus molecular layer , for example , every possible two - cell connectivity motif between neurogliaform cells and other types of interneurons was observed ( electrical only , unidirectional synaptic , bidirectional synaptic , and combinations of electrical and synaptic connections ; armstrong et al . , 2011 ; this arrangement could serve a number of important roles in the network , possibly synchronizing neuronal subgroups , inhibiting nearby neuronal subgroups , or acting as low - pass filters on incoming input to distinct cell types ( mitchell and silver , 2003 ; zsiros et al . , 2007 ) . computational studies will help to determine the significance of this connectivity for overall network function ( santhakumar et al . morgan and soltesz , 2008 ; ferrante et al . , 2009 ; cutsuridis and hasselmo , 2012 ) . in the dentate , where neurogenesis of granule cells occurs throughout life , cells of the neurogliaform family play another important role , being among the first sources of input to newly born granule cells ( markwardt et al . , dentate neurogliaform family cells not only inhibit adult granule cells directly , but also inhibit other interneurons ( indeed , burst firing in neurogliaform family cells during 4ap application coincided with robust suppression of spontaneous firing of other interneuronal types ) and may therefore also disinhibit mature cells ( markwardt et al . , 2011 ) . in this way , neurogliaform family cells might coordinate activity of both newly born and adult granule cells , via direct depolarization of newborn granule cells ( through depolarizing gaba effects ) and coincident disinhibition of mature cells ( markwardt et al . , 2011 ) . the advent of novel ways to probe the roles of specific neuronal subgroups in vivo , such as optogenetic methods , provide ways of investigating how neurogliaform family cells may enhance or direct the integration of newly born cells into the network , and to better understand under what conditions neurogliaform cells have a predominately inhibitory action on adult granule cells ( e.g. , feedforward inhibition ) , and under what conditions neurogliaform cells produce a net disinhibition of granule cells ( by inhibiting other interneurons ) . just as neurogliaform cells can mediate feedforward inhibition to dentate granule cells , ivy cells in the ca3 region are similarly positioned to mediate feedforward inhibition to ca3 pyramidal cells ( szabadics and soltesz , 2009 ) . in paired recordings between mossy fiber boutons of granule cells and postsynaptic ca3 interneurons , ivy cells received fewer , but stronger unitary connections from mossy fibers than fast - spiking basket cells ( szabadics and soltesz , 2009 ) . this arrangement suggests that individual large amplitude inputs from individual mossy fibers to ivy cells may be capable of inducing feedforward inhibition to ca3 pyramidal cells , while other interneurons , such as fast - spiking basket cells , require greater convergent input from a number of mossy fiber terminals in order to produce feedforward inhibition . interestingly , granule cells have recently been identified within the ca3 , where their excitatory inputs from entorhinal cortex and mossy fiber outputs to both glutamatergic and gabaergic cells of the ca3 , as well as their electrophysiological properties , are similar to those of granule cells located in the dentate ( szabadics et al . , 2010 ) . unlike dentate granule cells , however , ca3 granule cells have reciprocal connections with ca3 ivy cells , as well as with other gabaergic cells of the ca3 ( szabadics et al . , 2010 ; figure 3 ) . therefore , ca3 granule cells represent a unique , local glutamatergic neuronal subtype which in addition to both exciting ca3 pyramidal cells and gabaergic cells to drive feedforward inhibition , also receive feedback input from the ca3 gabaergic network . ivy cells providing simultaneous feedforward inhibition to ca3 pyramidal cells and feedback inhibition of ca3 granule cells , may have important implications for oscillatory activity and spike timing within this brain region . finally , neurogliaform cells in ca1 are also positioned to mediate feedforward inhibition , as they receive input from both ca3 and entorhinal cortex , and in turn inhibit ca1 pyramidal cells ( in addition to interneurons ; vida et al . , 1998 ; price et al . , 2005 ; price et al . , 2008 ) . feedforward inhibition in ca1 can be very strong ; following the stimulation of the temporo - ammonic pathway , a predominately inhibitory response is generated in ca1 pyramidal cells ( soltesz and jones , 1995 ) . relevant to our discussion , this inhibitory response consists of both postsynaptic gabaa and gabab components ( empson and heinemann , 1995 ) , which may implicate involvement of neurogliaform cells . neuromodulatory influences onto and arising from neurogliaform family cells are only beginning to be uncovered . one recent interesting finding involves the effects of opioids on these cells in the ca1 . in studies of input to ca1 from ca3 and entorhinal cortex , it was noted that stimulating the temporo - ammonic pathway one theta cycle before stimulating schaffer collateral input leads to inhibition of the second excitatory input to ca1 pyramidal cells in a gabab - dependent , opioid - sensitive manner ( dvorak - carbone and schuman , 1999 ; mcquiston , 2011 ) . in the context of the known properties of neurogliaform family cells , this observation suggests that neurogliaform cells could be modulated by opioids . in fact , both ivy and neurogliaform cells were recently found to be highly modulated by -opioid receptors ( krook - magnuson et al . , 2011 ) . activation of -opioid receptors produced both somatic hyperpolarization and inhibition of neurotransmitter release from terminals . indeed , in paired recordings between ivy and pyramidal cells , activation of -opioid receptors nearly abolished the ivy cell - mediated inhibitory postsynaptic current ( ipsc ; figure 3 ) . together these findings and others suggest not only that neurogliaform cells can mediate feedforward inhibition , and that feedforward inhibition interacts with the timing of impinging excitatory inputs to ca1 pyramidal cells , but also that this inhibition is highly modulated by opioids . this has implications both for understanding the mechanisms of action of drugs of abuse that act at the -opioid receptor ( such as heroin ) , as well as for the physiological function of ca1 during exploration , learning , and memory . indeed , -opioid receptor knock - out mice show reduced radial - arm maze and morris water maze performance ( jamot et al . , 2003 ) . further , alterations in the hippocampal opioid system are also seen , for example , in epilepsy and alzheimer s , indicating a potential role in these disorders ( laorden et al . , 1985 ; gall , 1988 ; gall et al . , 1988 ; dintino et al . , 2006 ; rocha et al . , 2007 ; cuellar - herrera et al . , 2012 ) moreover , in a model of alzheimer s it was found that the reported increase in enkephalin ( an endogenous ligand for -opioid receptors ) contributed to the cognitive difficulties associated with the disease ( meilandt et al . , 2008 ) , indicating that changes in the hippocampal opioid system can have significant functional consequences . in addition to new insights into the neuromodulation of neurogliaform family output , the expression of such neuroactive markers as npy and nnos suggests that neurogliaform family cells may be important sources of both npy and no in the brain . npy is a neuropeptide with a range of reported functions , including stimulating dentate neurogenesis ( howell et al . , 2007 ) . additionally , npy is implicated in anxiolysis , in the mechanisms of antidepressants ( reviewed in heilig , 2004 ) , and in response to stress veterans exposed to traumatic experiences but who had higher plasma levels of npy experienced fewer stress - related sequelae such as post - traumatic stress disorder ( ptsd ) and depression than those with lower npy levels ( morgan et al . , 2002 ; yehuda et al . , 2006 ) . interestingly , npy may also be important in epilepsy ; both the level of npy protein and its receptor subtypes are robustly changed after seizures , and npy overexpression is protective against acute and chronic seizures . this suggests that neurogliaform family cells may be important in suppressing seizure activity in epilepsy ( vezzani et al . , 1999 ; the expression of nnos by cells of the neurogliaform family is especially interesting since it is not yet known how the interneuronal production of no may contribute to overall network activity . while the known functions of no in neuronal and non - neuronal tissues are numerous , the roles of nnos and no in specific gabaergic cell types , such as neurogliaform family cells , has yet to be determined . however , no is a well - known retrograde modulator , and nnos in neurogliaform cells may therefore involve synapse- and cell type - specific regulation of transmission from both excitatory and inhibitory inputs . this may serve to modulate levels of neurogliaform activity ( and thus levels of gabaergic volume transmission ) during specific input patterns ( szabadits et al . , 2007 ; feil and kleppisch , 2008 ; maggesissi et al . , 2009 ; 2011 ) , or to affect long term synaptic plasticity ( shin and linden , 2005 ; anwyl , 2006 ; lange et al . , 2012 ) . in addition , the known vasodilatory role of no in the peripheral circulatory system and in the cns ( hall and behbehani , 1998 ; cauli et al . , 2004 ; corsani et al . , 2008 ; melikian et al . , 2009 ) suggests the possibility that neurogliaform family cells might play a major role in increasing perfusion to regions that are particularly active ( tamas et al . , 2003 ; simon et al . , 2005 ) . more studies are clearly needed to definitively address the role of nnos in the function of neurogliaform family cells . neurogliaform family cells have unique intrinsic and synaptic properties and are found in a range of brain regions , including throughout the hippocampal formation . in addition to functions related to their expression of npy and nnos , roles in influencing network synchrony and oscillatory activity have been proposed ( tamas et al . , 2003 ; simon et al . , 2005 ; zsiros et al . , 2007 ; fuentealba et al . , 2008 ; price et al . , 2008 ; their modulation by -opioid receptors suggests that volume transmission by neurogliaform family cells may play a major role in the effects of opiates , and may be of importance for future pharmacological insights . interestingly , some gabaergic populations have been observed to select postsynaptic partners based on long - range projection patterns . for example , in the entorhinal cortex , cck - positive basket cells selectively target only those principal cells which project to contralateral extrahippocampal structures , avoiding principal cells which project to the ipsilateral hippocampus and form the perforant path ( varga et al . , 2010 ) . in contrast , due to their unique volume neurotransmission , neurogliaform cells are likely to indiscriminately inhibit all cells with processes within their axonal arbors . thus , neurogliaform cells may represent a gabaergic cell class which coordinates the activity , or level of activity , between neurons processing information destined for distinct brain regions ( krook - magnuson et al . , 2012 ) . this idea is particularly interesting when considering that cells with different long - range projection patterns may intermingle within a single cell layer ( varga et al . , 2010 ) . moreover , as neurogliaform cells have been found to cross boundaries ( e.g. , the hippocampal fissure ) , they may also directly regulate concurrent activity in distinct brain regions ( ceranik et al . , 1997 ; price et al . , 2005 ; fuentealba et al . , 2010 ; armstrong et al . , 2011 ) . the role of neurogliaform family cells as important feedforward inhibitors is supported by in vitro and existing in vivo data , but the wide connectivity and unique properties of these cells with other interneurons complicates our understanding of their true network functions . ultimately , more studies , including studies in vivo in awake , behaving animals are needed to determine how neurogliaform and ivy cells behave in information processing and network activities during different behavioral states . a comprehensive understanding of the roles of these unique neurons and their peculiar connective properties promises to shed light on new mechanisms by which microcircuits interact within and across brain areas . 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 .

neurogliaform and ivy cells are members of an abundant family of neuronal nitric oxide synthase ( nnos ) expressing gabaergic interneurons found in diverse brain regions . these cells have a defining dense local axonal plexus , and display unique synaptic properties including a biphasic postsynaptic response with both a slow gabaa component and a gabab component following even a single action potential . the type of transmission displayed by these cells has been termed volume transmission , distinct from both tonic and classical synaptic transmission . electrical connections are also notable in that , unlike other gabaergic cell types , neurogliaform family cells will form gap junctions not only with other neurogliaform cells , but also with non - neurogliaform family gabaergic cells . in this review , we focus on neurogliaform and ivy cells throughout the hippocampal formation , where recent studies highlight their role in feedforward inhibition , uncover their ability to display a phenomenon called persistent firing , and reveal their modulation by opioids . the unique properties of this family of cells , their abundance , rich connectivity , and modulation by clinically relevant drugs make them an attractive target for future studies in vivo during different behavioral and pharmacological conditions .

Introduction Unique Axons, Unique Connections Diversity and Divisions Electrophysiological Properties of Neurogliaform Family Cells Network and Functional Roles of Neurogliaform and Ivy Cells Neuromodulators and Neurogliaform Family Cells Summary and Outlook Conflict of Interest Statement

the neurogliaform family of cells is one of the major groups of gabaergic neurons expressing nitric oxide synthase ( nnos ) . neurogliaform cells , originally described by cajal as arachniform cells ( ramn y cajal , 1922 ; ramn y cajal , 1999 ) , together with the closely related ivy cells , which were only recently characterized ( fuentealba et al . however , the neurogliaform family of cells is found in large numbers not only throughout ca1 , but also across a range of brain regions , including the entire hippocampal formation ( vida et al . here , in addition to reviewing the known anatomical , synaptic , neuromodulatory , and connective properties of neurogliaform family cells , we discuss their potential network functions , particularly in the hippocampal formation . despite their wide distribution , cells within the neurogliaform family have very similar characteristics across brain regions , and these unique characteristics clearly set them apart from other gabaergic cell types . neurogliaform cells have a dense local axonal plexus and generally small , round , somata ( figure 1 ) . , 1998 ; the synapses formed by neurogliaform family cell boutons , however , do not necessarily form classical synapses like those of other gabaergic cell classes , in that the synaptic cleft at identified synapses is unusually wide and some boutons , complete with synaptic vesicles , do not have an easily identifiable postsynaptic target in the classical sense ( vida et al . their unique synaptic and axonal morphologies underlie some key features of neurogliaform synaptic transmission ( figure 2 ) , namely ( 1 ) the ability of neurogliaform cells to mediate gabaergic volume transmission , affecting virtually any processes within their dense axonal plexus , ( 2 ) the production of a slow gabaa current in the postsynaptic cell ( gabaa , slow ) , and ( 3 ) the postsynaptic gabab response to even a single neurogliaform action potential ( tamas et al . , 2010 ; capogna and pearce , 2011 ) neurogliaform and ivy cells in the hippocampus : ( a ) dentate gyrus neurogliaform cell , ( b ) ca3 ivy cell , ( c ) ca1 ivy cell , ( d ) ca1 neurogliaform cell ; these cells are characterized by their dense local axonal plexus ( black : axon ; blue : somata and dendrites ) . cells represented in ( a , b ) are from rat ( e ) schematic diagram of the hippocampus , illustrating the approximate relative locations of cells in ( a d ) ( scale bar = 100 m ) . ( f ) recorded , biocytin - filled cells confirmed as neurogliaform cells in the dentate gyrus ( three separate cells , numbered 13 ) express a variety of markers , including coup tfii , nnos , reelin , and npy ( scale bar = 20 m ) among others ( see text ) . ( h ) in addition to the classical late - spiking pattern characteristic of these cells , a high percentage of neurogliaform and ivy cells display the recently described phenomenon referred to as persistent firing . ( c ) neurogliaform cells ( averaged examples from dentate gyrus ) produce a biphasic postsynaptic response , consisting of a slow gabaa and a gabab component , which can be distinguished by application of the gabab antagonist cgp55845 ( red trace ) . this biphasic response can be seen following even a single presynaptic action potential ( inset ) . ( d ) neurogliaform cells ( ngfc , black traces ) can form both electrical and chemical synaptic connections with other gabaergic cell types . in this example , an electrical and unidirectional synaptic connection between a ngfc ( black traces ) and a non - ngfc ( green traces ) left traces : a hyperpolarizing current step ( pre , current clamp responses shown in the upper traces of each example ) , evokes an outward current in the electrically connected cell ( post , voltage clamp responses shown in the lower traces of each example ) . neurogliaform cells ability to induce a biphasic current in the postsynaptic cell , including both a gabaa - mediated and a gabab - mediated component , is a property which has been consistently observed across brain regions ( tamas et al . , 2005 ; the gabaa and gabab - mediated components can be separated based on reversal potential , since the k - mediated gabab component and the largely cl - mediated gabaa component reverse at different membrane potentials . the kinetics of the neurogliaform cell - evoked gabaa response are also considerably slower than the kinetics of responses to other gabaergic cell types , and these events , referred to as gabaa , slow ( pearce , 1993 ; reviewed in capogna and pearce , 2011 ) , were shown to arise from neurogliaform cells ( tamas et al . the axonal arbors of neurogliaform cells described above are one of the most distinctive features of these cells . while the axons of neurogliaform cells form a unique dense , local plexus , they can also cross boundaries of brain regions . for example , neurogliaform cells were recently identified in the molecular layer of the dentate gyrus ( armstrong et al . , 2011 ) , where importantly , it was found that their axons can cross the hippocampal fissure and extend into the adjacent ca1 or subiculum ( armstrong et al . this extension of axons across boundaries is a notable feature among gabaergic cells , suggesting that neurogliaform cells may serve to share information between , and coordinate the activities of , distinct brain regions . beyond their remarkable chemical synapses , in contrast , neurogliaform cells frequently form gap junctions with a wide variety of other gabaergic cell types ( figure 2 ; simon et al . despite the similarities in axonal and synaptic properties of individual cells of the neurogliaform family , there are some notable differences between them as well . members of the neurogliaform family may differ in where they reside within a given brain region , the input they receive , and the domain of the postsynaptic cells their axons target , as well as in the neuronal markers that they express . as mentioned above neurogliaform cells classically target the distal dendrites of principal cells while ivy cells , which have to date been definitively identified in the ca1 and the ca3 of the hippocampus ( fuentealba et al . , 2010 ) , have a unique position in and around the pyramidal cell layer , where they may have different incoming and outgoing connectivity when compared to neurogliaform cells . in the ca1 , where ivy cells were first described ( fuentealba et al . , 2008 ) , neurogliaform cells in and near the stratum lacunosum - moleculare receive excitatory inputs from both the temporo - ammonic pathway and ca3 schaffer collaterals , and in turn target the distal dendrites of ca1 pyramidal cells ( figures 1 and 3 ; price et al . , in contrast , ivy cells reside in or near the stratum pyramidale , receive excitatory input from local pyramidal cells as well as presumably from ca3 schaffer collaterals , and target the proximal dendrites of ca1 pyramidal cells ( fuentealba et al . this differential input means that , while neurogliaform cells near the ca1 lacunosum - moleculare serve a primarily feedforward inhibitory role , ivy cells near the pyramidale are poised to mediate both feedforward and feedback inhibition to ca1 pyramidal cells . , 2010 ) , and differences in network functions of neurogliaform and ivy cells in vivo will be discussed further below . in the dentate , neurogliaform family cells targeting proximal dendrites ( ivy , brown ) provide inhibition to both adult granule cells ( gc ) and newly born granule cells ( ngc ) . granule cells of the dentate , as well as granule cells of the ca3 provide mossy fiber input to ca3 ivy cells , which provide both feedforward inhibition to pyramidal cells ( pyr ) and feedback inhibition to ca3 , but not dentate , granule cells . both ngfcs and ivy cells in the ca1 express the -opioid receptor ( or , yellow triangle ) . in this simplified schematic , excitatory input to excitatory cells has been omitted . ( c ) illustrates that individual mossy fiber boutons ( mf , blue trace ) form strong unitary connections ( left box ) with postsynaptic ivy cells in the ca3 ( brown traces ) while unitary connections between mossy fiber boutons and fast - spiking basket cells ( fsbc , orange traces ) are smaller in amplitude . even within the different types of neurogliaform family cells thus , while neuropeptide y ( npy ) , nnos , coup tfii , -actinin , gabaa1 , gabaa , and reelin are notably found in neurogliaform cells ( figure 1 ) , there is significant heterogeneity in marker expression from cell to cell such that no one marker or combination of markers captures , uniquely , all neurogliaform cells ( deller and leranth , 1990 ; ratzliff and soltesz , 2001 ; price et al . , ivy cells of the ca1 seem to share many of the same markers as neurogliaform cells , but have not been observed to express reelin ( fuentealba et al . interestingly , the presence or absence of nnos may serve as a marker of developmental origin for both neurogliaform and ivy cells , that is whether the cells arise from the medial or caudal ganglionic eminences , both of which can generate neurogliaform family cells ( tricoire et al . as might be expected due to their differential inputs and placement within the ca1 , ivy and neurogliaform cells of the ca1 differ also in their in vivo firing patterns . in awake animals , putative ivy cells recorded using tetrodes have similar firing properties during theta oscillations and ripples as ivy cells in anaesthetized animals ( fuentealba et al . despite these differences in firing patterns in vivo , when recordings are made from slices in whole - cell patch configuration , both ivy and neurogliaform cells exhibit a characteristic late - spiking firing pattern , often with a depolarizing ramp and subthreshold oscillations in the gamma frequency range leading up to the first spikes ( szabadics et al . , persistent firing is not affected by blocking gabaa , gabab , ampa , and nmda receptors ( sheffield et al . the apparent threshold for firing during persistent firing is very low when recording from the soma , and it has been shown that this is due to the action potentials starting in the axons themselves and back - propagating to the soma ( sheffield et al . the mechanism permitting the integration of spiking information over the span of minutes , required for the induction of persistent firing , is a matter of open debate and investigation , as are the physiological consequences of persistent firing . indeed , less than 20% of fast - spiking parvalbumin expressing basket cells display persistent firing , in contrast to over 80% of neurogliaform family cells ( krook - magnuson et al . neurogliaform family cells have recently been described in the dentate gyrus ( armstrong et al . neurogliaform cells located in the middle and outer molecular layers receive entorhinal input and synapse on distal dendrites of granule cells , meaning that they are capable of providing feedforward inhibition ( armstrong et al . compared to granule cells , neurogliaform cells in the dentate molecular layer receive fewer spontaneous inputs ( consisting of excitatory and inhibitory events at roughly equal frequencies ) to their small dendritic arbors than granule cells . this perforant path input is facilitating at high frequencies , and suggests that neurogliaform cells may act to inhibit granule cells primarily during high frequency incoming activity ( armstrong et al . in regions like the dentate gyrus , where granule cells exhibit sparse firing , feedforward gabaergic input has been predicted to play a major role in normal function ( ferrante et al . because neurogliaform cells target the distal dendrites of principal cells , they may have stronger effects on the processing of incoming input in dendritic compartments than on spike timing control per se . the fact that neurogliaform cells form both chemical and electrical synapses with other classes of gabaergic neurons means that they can display a variety of connectivity motifs . in the dentate , where neurogenesis of granule cells occurs throughout life , cells of the neurogliaform family play another important role , being among the first sources of input to newly born granule cells ( markwardt et al . , dentate neurogliaform family cells not only inhibit adult granule cells directly , but also inhibit other interneurons ( indeed , burst firing in neurogliaform family cells during 4ap application coincided with robust suppression of spontaneous firing of other interneuronal types ) and may therefore also disinhibit mature cells ( markwardt et al . in this way , neurogliaform family cells might coordinate activity of both newly born and adult granule cells , via direct depolarization of newborn granule cells ( through depolarizing gaba effects ) and coincident disinhibition of mature cells ( markwardt et al . the advent of novel ways to probe the roles of specific neuronal subgroups in vivo , such as optogenetic methods , provide ways of investigating how neurogliaform family cells may enhance or direct the integration of newly born cells into the network , and to better understand under what conditions neurogliaform cells have a predominately inhibitory action on adult granule cells ( e.g. , feedforward inhibition ) , and under what conditions neurogliaform cells produce a net disinhibition of granule cells ( by inhibiting other interneurons ) . just as neurogliaform cells can mediate feedforward inhibition to dentate granule cells , ivy cells in the ca3 region are similarly positioned to mediate feedforward inhibition to ca3 pyramidal cells ( szabadics and soltesz , 2009 ) . this arrangement suggests that individual large amplitude inputs from individual mossy fibers to ivy cells may be capable of inducing feedforward inhibition to ca3 pyramidal cells , while other interneurons , such as fast - spiking basket cells , require greater convergent input from a number of mossy fiber terminals in order to produce feedforward inhibition . interestingly , granule cells have recently been identified within the ca3 , where their excitatory inputs from entorhinal cortex and mossy fiber outputs to both glutamatergic and gabaergic cells of the ca3 , as well as their electrophysiological properties , are similar to those of granule cells located in the dentate ( szabadics et al . unlike dentate granule cells , however , ca3 granule cells have reciprocal connections with ca3 ivy cells , as well as with other gabaergic cells of the ca3 ( szabadics et al . therefore , ca3 granule cells represent a unique , local glutamatergic neuronal subtype which in addition to both exciting ca3 pyramidal cells and gabaergic cells to drive feedforward inhibition , also receive feedback input from the ca3 gabaergic network . ivy cells providing simultaneous feedforward inhibition to ca3 pyramidal cells and feedback inhibition of ca3 granule cells , may have important implications for oscillatory activity and spike timing within this brain region . finally , neurogliaform cells in ca1 are also positioned to mediate feedforward inhibition , as they receive input from both ca3 and entorhinal cortex , and in turn inhibit ca1 pyramidal cells ( in addition to interneurons ; vida et al . neuromodulatory influences onto and arising from neurogliaform family cells are only beginning to be uncovered . one recent interesting finding involves the effects of opioids on these cells in the ca1 . in studies of input to ca1 from ca3 and entorhinal cortex , it was noted that stimulating the temporo - ammonic pathway one theta cycle before stimulating schaffer collateral input leads to inhibition of the second excitatory input to ca1 pyramidal cells in a gabab - dependent , opioid - sensitive manner ( dvorak - carbone and schuman , 1999 ; mcquiston , 2011 ) . in the context of the known properties of neurogliaform family cells , this observation suggests that neurogliaform cells could be modulated by opioids . together these findings and others suggest not only that neurogliaform cells can mediate feedforward inhibition , and that feedforward inhibition interacts with the timing of impinging excitatory inputs to ca1 pyramidal cells , but also that this inhibition is highly modulated by opioids . further , alterations in the hippocampal opioid system are also seen , for example , in epilepsy and alzheimer s , indicating a potential role in these disorders ( laorden et al . this suggests that neurogliaform family cells may be important in suppressing seizure activity in epilepsy ( vezzani et al . while the known functions of no in neuronal and non - neuronal tissues are numerous , the roles of nnos and no in specific gabaergic cell types , such as neurogliaform family cells , has yet to be determined . however , no is a well - known retrograde modulator , and nnos in neurogliaform cells may therefore involve synapse- and cell type - specific regulation of transmission from both excitatory and inhibitory inputs . , 2009 ) suggests the possibility that neurogliaform family cells might play a major role in increasing perfusion to regions that are particularly active ( tamas et al . neurogliaform family cells have unique intrinsic and synaptic properties and are found in a range of brain regions , including throughout the hippocampal formation . , 2008 ; their modulation by -opioid receptors suggests that volume transmission by neurogliaform family cells may play a major role in the effects of opiates , and may be of importance for future pharmacological insights . in contrast , due to their unique volume neurotransmission , neurogliaform cells are likely to indiscriminately inhibit all cells with processes within their axonal arbors . thus , neurogliaform cells may represent a gabaergic cell class which coordinates the activity , or level of activity , between neurons processing information destined for distinct brain regions ( krook - magnuson et al . the role of neurogliaform family cells as important feedforward inhibitors is supported by in vitro and existing in vivo data , but the wide connectivity and unique properties of these cells with other interneurons complicates our understanding of their true network functions . ultimately , more studies , including studies in vivo in awake , behaving animals are needed to determine how neurogliaform and ivy cells behave in information processing and network activities during different behavioral states .

nitric oxide ( no ) is an important biological signaling molecule present in the cardiovascular , immune , and nervous systems . no is released by macrophages during the immune response , controls smooth muscle relaxation , and regulates neurotransmission . dysregulation of no homeostasis is associated with numerous diseases , including diabetes , stroke , alzheimer s disease , and cancer . hno , the one - electron reduced and protonated derivative of no , displays distinctive chemistry and biochemistry from that of no . exogenously applied hno confers vasoprotective effects , increases heart muscle contractility , and inhibits platelet aggregation . hno also exacerbates ischemia - related injury and induces neurotoxicity . under biological conditions , hno is both an electrophile that oxidizes thiols and a nucleophile that can coordinate and reduce metal ions . nitrosation of thiolates induces the release of mobile zinc , which may trigger a variety of signaling pathways . understanding these and other downstream effects of exogenously applied hno is essential if nitroxyl donors are to be used as therapeutic agents . hno can be produced by nitric oxide synthase ( nos ) . under normal physiological conditions , nos catalyzes the conversion of arginine to citrulline and no in the presence of the cofactor tetrahydrobiopterin ( thb ) . in the absence of thb , isolated neuronal nos produces hno instead of no . additionally , hno can be generated by oxidation of hydroxylamine ( nh2oh ) with heme - containing proteins . no and hno interconvert in the presence of superoxide dismutase ( sod ) , and cytochrome c can reduce no to hno . recently , hno production was detected in the reaction between hsno and h2s and in the heme - iron - catalyzed reduction of nitrite with h2s . these results suggest that hno can be produced under biological conditions and might be associated with particular physiological or pathological states . improved methods for detecting hno selectively , with well - defined spatiotemporal resolution in live cells , tissues , and animals , are needed to fully understand the biology of nitroxyl . l - nha = n - hydroxy - l - arginine ; nnos = neuronal nitric oxide synthase ; sod = superoxide dismutase ; thb = tetrahydrobiopterin . a number of fluorescent sensors that detect hno in live cells are available ( chart 1 ) . one approach to hno sensing relies on the use of fluorophores functionalized with amines that bind cu(ii ) . the fluorescence of these sensors is quenched upon binding of the paramagnetic cupric ion . upon exposure to hno , cu(ii ) reduces to cu(i ) , which renders the complex diamagnetic and restores the fluorescence of the probe . sensors based on this principle include bodipy - based complex cubot1 ( chart 1 ) , coumarin - based derivative cucot1 ( chart 1 ) , and the cubrno series of benzoresorufin - based sensors ( chart 1 ) that detect both hno and no . recently , the metal - free probe p - rhod ( chart 1 ) was reported . this sensor exploits the reactivity of hno with triphenylphosphine to produce an azaylide , which undergoes an intramolecular nucleophilic attack that releases rhodol , a bright green fluorophore . despite their value , these probes have shortcomings associated with their lack of selectivity or short emission wavelengths . it would be valuable to have a sensor that not only detects hno with high selectivity but would also emit in the near - infrared ( nir ) region , desired for in vivo imaging because of deeper tissue penetration . moreover , sensors with narrow emission profiles at long wavelengths can be used together with visible - light probes in multicolor microscopy experiments . in the present article we describe the design , synthesis , and implementation of cudhx1 ( chart 1 ) , a novel sensor that satisfies all of these requirements . the structure of cudhx1 comprises a cyclam binding site for cu(ii ) and a dihydroxanthene ( dhx ) nir fluorophore . we chose cyclam because cu(ii ) complexes of this ligand react very slowly with the interfering species h2s and glutathione ( gsh ) under physiological conditions . the dhx fluorophore was selected because it is a bright nir emitter with excellent biocompatibility . here we report the results of our studies using cudhx1 to image hno in live cells as well as multicolor imaging experiments to investigate the ability of angeli s salt , an hno donor , to affect the levels of mobile zinc in live cells . nitric oxide was passed through an ascarite column and a 6 ft coil containing silica gel at 78 c to remove impurities and then collected and stored under nitrogen in a gas storage bulb . h nmr chemical shifts are reported in ppm relative to sime4 ( = 0 ) and were referenced internally with respect to residual protons in the solvent ( = 3.31 for cd3od or = 2.50 for dmso - d6 ) . c nmr chemical shifts are reported in ppm relative to sime4 ( = 0 ) and were referenced internally with respect to solvent signal ( = 39.51 for dmso - d6 ) . f nmr chemical shifts are reported in ppm relative to cfcl3 ( = 0 ) and were referenced internally with respect to 2,2,2-trifluoroethanol ( = 77.03 ) . low - resolution mass spectra ( lrms ) were acquired on an agilent 1100 series lc / msd trap spectrometer ( lcms ) , using electrospray ionization ( esi ) . high - resolution mass spectrometry ( hr - esi - ms ) was conducted by staff at the mit department of chemistry instrumentation facility on a bruker daltonics apexiv 4.7 t ft - icr - ms instrument . silica gel 60 321 ( 0.0150.040 mm ) was used for flash column chromatography . semipreparative hplc separations were carried out on an agilent 1200 hplc instrument with a multiwavelength detector and automated fraction collector using a c18 reverse stationary phase ( zorbax - sb c18 , 5 m , 9.5 250 mm ) and a mobile phase composed of two solvents ( a : 0.1% ( v / v ) trifluoroacetic acid ( tfa ) in h2o ; b : 0.1% ( v / v ) tfa in ch3cn ) . iupac names of all compounds are provided and were determined using cs chembiodrawultra 12.0 . a solution of ir780 ( 300 mg , 0.45 mmol ) in dry dmf ( 5 ml ) was added to 5-(hydroxymethyl)benzene-1,3-diol ( 63 mg , 0.45 mmol ) . triethylamine ( 0.6 ml , 4.49 mmol ) was added , and the mixture was stirred at 110 c for 30 min . the solvent was evaporated under reduced pressure and the solid was purified by column chromatography ( sio2 ; ch2cl2/ch3oh 9:1 ) to give the product as a dark blue solid ( 195 mg , yield 76% ) . h nmr ( 300 mhz , cd3od ) : 1.04 ( t , j = 7.4 hz , 3h ) , 1.80 ( s , 6h ) , 1.891.96 ( m , 4h ) , 2.692.82 ( m , 4h ) , 4.26 ( t , j = 7.2 hz ) , 4.79 ( s , 2h ) , 6.40 ( d , j = 14.7 hz , 1h ) , 6.74 ( d , j = 2 hz , 1h ) , 6.91 ( d , j = 2 hz , 1h ) , 7.367.41 ( m , 1h ) , 7.457.49 ( m , 2h ) , 7.607.63 ( m , 1h ) , 7.70 ( s , 1h ) , 8.69 ( d , j = 14.5 hz , 1h ) . lrms ( esi ) . thionyl chloride ( 75 l , 1.03 mmol ) and dry pyridine ( 83 l , 1.03 mmol ) were dissolved in dry ch2cl2 ( 1 ml ) and cooled to 0 c . compound 1 ( 195 mg , 0.34 mmol ) was dissolved in dry ch2cl2 ( 1 ml ) and dry dmf ( 0.1 ml ) and added slowly to the mixture of pyridine and thionyl chloride . after 30 min , h2o ( 0.1 ml ) was added , and the mixture was dried with na2so4 . the dark blue solid was dissolved in ch2cl2/ch3oh ( 9:1 ) and filtered through a plug of sio2 with ch2cl2/ch3oh ( 9:1 , 200 ml ) . a solution of crude compound 2 in dry ch3cn ( 3 ml ) was added to 1,4,8,11-tetraazacyclotetradecane ( cyclam , 136 mg , 0.68 mmol ) . diisopropylethylamine ( 0.12 ml , 0.68 mmol ) was added , and the mixture was heated to reflux . after 30 min , the solvent was evaporated , and the residue was purified by rp - hplc according to the following protocol : constant flow rate 3 ml min ; isocratic flow 2% b , 05 min ; gradient , 3595% b , 1025 min . all equivalent fractions recovered from independent runs were combined and lyophilized to dryness to yield the tfa salt of compound 3 ( 46 mg , 18% over two steps ) . h nmr ( 500 mhz , dmso - d6 ) : 0.96 ( t , j = 7.4 hz , 3h ) , 1.72 ( s , 7h ) , 1.81 ( m , 7h ) , 2.67 ( m , 10h ) , 3.10 ( m , 10h ) , 3.80 ( s , 2h ) , 4.35 ( t , j = 7.1 hz , 2h ) , 6.53 ( d , j = 15 hz , 1h ) , 6.92 ( m , 1h ) , 7.42 , ( t , j = 7.7 hz , 1h ) , 7.51 ( t , j = 8.3 hz , 1h ) , 7.66 ( d , j = 8.0 hz , 1h ) , 7.72 ( d , j = 7.7 hz , 2h ) , 8.55 ( d , j = 15 hz , 1h ) . c nmr ( 125 mhz , dmso - d6 ) : 11.08 , 20.10 , 20.92 , 23.60 , 27.59 , 28.55 , 40.44 , 45.95 , 49.56 , 50.24 , 101.59 , 103.73 , 113.12 , 113.38 , 113.71 , 115.75 , 115.96 , 118.11 , 120.48 , 122.76 , 125.85 , 126.85 , 127.96 , 128.97 , 136.75 , 141.65 , 141.88 , 144.50 , 154.73 , 158.13 , 158.42 , 158.65 , 158.90 , 160.62 , 161.12 , 177.01 . a solution of compound 3 ( 46 mg , 0.06 mmol ) in ch3cn ( 3 ml ) and ( bromomethyl)benzene ( 4 l , 0.03 mmol ) were combined . diisopropylethylamine ( 22 l , 0.12 mmol ) was added , and the reaction mixture stirred at room temperature for 2 h. the solvent was evaporated , and the product was purified by rp - hplc according to the following protocol : constant flow rate 3 ml min ; isocratic flow 2% b , 05 min ; gradient , 3595% b , 1025 min . all equivalent fractions recovered from independent runs were combined and lyophilized to dryness to yield the tfa salt of compound dhx1 ( 14.2 mg , 28% ) . h nmr ( 500 mhz , dmso - d6 ) : 0.97 ( t , j = 7 hz , 3h ) , 1.72 ( s , 6h ) , 1.80 ( m , 6h ) , 2.06 ( m , 2h ) , 2.66 ( m , 3h ) , 2.72 ( m , 5h ) , 3.14 ( m , 8h ) , 3.83 ( s , 3h ) , 4.35 ( t , j = 7 hz , 2h ) , 6.52 ( d , j = 15 hz , 1h ) , 6.91 ( m , 1h ) , 7.00 ( m , 1h ) , 7.40 ( m , 6h ) , 7.50 ( t , j = 7 , 1h ) , 7.66 ( d , j = 5 hz , 1h ) , 7.72 ( m , 2h ) , 8.54 ( d , j = 15 hz , 1h ) . c nmr ( 125 mhz , dmso - d6 ) : 11.09 , 20.09 , 20.89 , 23.60 , 27.57 , 27.62 , 28.50 , 40.43 , 45.91 , 50.20 , 50.24 , 53.71 , 57.10 , 101.27 , 103.55 , 113.07 , 113.32 , 113.52 , 113.69 , 113.85 , 115.69 , 116.11 , 118.06 , 120.42 , 122.76 , 125.63 , 126.78 , 128.29 , 128.44 , 128.93 , 130.06 , 130.37 , 131.40 , 141.64 , 141.85 , 144.41 , 154.66 , 158.02 , 158.04 , 158.51 , 158.81 , 160.80 , 161.29 , 176.87 . all aqueous solutions were prepared using deionized water with resistivity 18.2 m cm , obtained using a milli - q water purification system . piperazine - n , n-bis(2-ethanesulfonic acid ) ( pipes ) and 99.999% kcl were purchased from calbiochem . stock solutions of compound 3 and ligand dhx1 in dmso were prepared in the 18 mm range and stored at 20 c in 1 ml aliquots and thawed immediately before each experiment . the copper complexes were prepared by dissolving the ligand in 1 ml of ch3oh , adding 1 equiv of cucl2 to each solution , and stirring overnight . the solvent was evaporated , and the resulting sensor was redissolved in 1 ml dmso and stored at 20 c . all spectroscopic measurements were conducted in aqueous buffer containing 50 mm pipes ( ph 7.0 ) and 100 mm kcl , with the exception of those performed in ch3oh and at varying ph values . vis spectra were acquired using a cary 50 spectrometer using quartz cuvettes from starna ( 1 cm path length ) . all measurements were conducted on solutions maintained at 25 c by circulating water baths . extinction coefficients of the ligands and sensors were determined by using 13 m solutions in aqueous buffer . fluorescence quantum yields were determined using the same solutions , ex = 650 nm . quantum yields were referenced to ir780 , which has a reported quantum yield of 0.076 in ch3oh , ex = 725 nm . degassed aliquots of the stock solutions of each sensor were brought into an anaerobic chamber under a nitrogen atmosphere ( o2 < 1 ppm ) dedicated to work with aqueous solutions ( hereafter called the wet box ) . solutions containing 5 m cu-3 and 2 m cudhx1 were prepared using 2 ml of either degassed aqueous buffer or ch3oh in gastight cuvettes . angeli s salt ( na2n2o3 , cayman chemical ) was used as the source of hno , because it decomposes rapidly ( t1/2 = 3 min ) at ph = 7 to produce hno and nano2 . solutions of angeli s salt ( 4 mm ) were prepared in the wet box in degassed 10 mm naoh ( 2 ml ) and brought out of the wet box in a gastight syringe . no gas was removed from the wet box in a gastight syringe and injected into the headspace of each gastight cuvette before measuring fluorescence . cyclic voltammograms were measured in a three - electrode cell with a 2.0 mm diameter glassy carbon working electrode , a platinum auxiliary electrode , and ag / ag pseudoreference electrode in acetonitrile . the measurements were performed at room temperature with a versastat3 potentiostat from princeton applied research operated with v3 studio software . measurements were carried out at a scan rate of 200 mv s on quiescent solutions that had been sparged with n2 for 5 min . low - temperature x - band epr spectra ( 77 k , 9 ghz ) were collected with a bruker ems spectrometer equipped with an er 4199hs cavity and a gunn diode microwave source . 0.8 equiv of cu(mecn)4bf4 were added to 400 m dhx1 or 3 in 350 l ch3oh , stirred overnight , and brought out of the glovebox in sealed epr tubes . angeli s salt ( 100 equiv ) was prepared in 10 mm naoh anaerobically and brought out of the wet box in a gastight syringe . when cucl2 was used to prepare cudhx1 for the hno reactivity epr studies , different results were observed ( figure s21 ) . for the no reactivity test , degassed aliquots from the stock solution of cudhx1 were brought into a wet box . solutions containing 400 m cudhx1 were prepared in 350 l of degassed ch3oh and brought out of the wet box in sealed epr tubes . no gas was taken out of the wet box in a gastight syringe and injected into the headspace of the epr tube . spectra were simulated in matlab using the solid - state / frozen - solution functionality ( pepper ) implemented in easyspin . selectivity of the fluorescence turn - on toward biologically relevant analytes was determined by comparing the fluorescence emission spectra of a 2 m solution of cudhx1 in aqueous buffer at ph = 7 , before and after treatment with 100 equiv of cacl2 , mgcl2 , nacl , zncl2 , kno3 , nano2 , ko2 , h2o2 , naclo , sodium ascorbate , naonoo , l-(+)-cysteine hydrochloride , gluthathione , methionine , na2s , or angeli s salt . no gas , 5000 equiv , and 100 l of 10 mm naoh ( solvent of angeli s salt solutions ) were also tested . in each case , the response was quantified by integrating the emission intensity from 660 to 900 nm and normalized to that of 2 m cudhx1 in aqueous buffer . for the no and hno selectivity studies , the samples were prepared anaerobically . for the no , hno , and na2s studies , fluorescence spectra were acquired every 1 min for 10 min . for the remaining analytes , in addition , the fluorescence turn - on of 2 m cudhx1 in the presence of 100 equiv of ko2 and angeli s salt was measured in ch3cn . to determine the effect of ph on the fluorescence emission of cudhx1 , 2 m solutions of cudhx1 were prepared anaerobically in aqueous buffer ( either 50 mm mes , 100 mm kcl ; ph 4 and 5 or 50 mm pipes , 100 mm kcl ; ph 6 , 7 , and 8) , and fluorescence spectra were recorded before and after addition of 100 equiv of angeli s salt . hela cells were cultured in dulbecco s modified eagle medium ( dmem ; cellgro , mediatec , inc . ) , supplemented with 10% fetal bovine serum ( fbs ; hyclone ) , 1% penicillin - streptomycin , 1% sodium pyruvate , and 1% l - glutamine . the cells were grown to 90% confluence at 37 c with 5% co2 before being passed and plated onto poly - d - lysine - coated plates 48 h before imaging . the growth medium was replaced with phosphate - buffered saline ( pbs ) containing 5 m cudhx1 and 3 m hoechst 33528 dye , and the cells were incubated for 15 min . cells were rinsed with pbs ( 2 2 ml ) followed by addition of fresh pbs ( 2 ml ) and mounted on the microscope . all cell imaging experiments involving addition of hno were carried out in pbs because addition of angeli s salt to plates containing dmem did not lead to any turn - on response ( figure s23 ) , presumably because of reaction of angeli s salt with cysteine or other components of dmem . for cell imaging experiments with zp1 , the growth medium was replaced with dye - free dmem containing 5 m zp1 , 3 m hoechst 33528 dye , and the cells were incubated for 1 h. cells were rinsed with pbs ( 2 2 ml ) before addition of fresh pbs containing 5 m cudhx1 and incubated for 15 min . cells were rinsed with pbs ( 2 2 ml ) followed by addition of fresh pbs ( 2 ml ) and mounted on the microscope . imaging experiments were performed using a zeiss axiovert 200 m inverted epifluorescence microscope equipped with an em - ccd digital camera ( hamamatsu ) and a ms200 xy piezo z stage ( applied scientific instruments ) . the light source was an x - cite 120 metal - halide lamp ( exfo ) , and the fluorescence images were obtained using an oil - immersion objective at 63 or 100 magnification . the filters sets used are defined as blue : excitation g 365 nm , beamsplitter ft 395 nm , emission bp 445/50 nm ; green : excitation bp 470/40 nm , beamsplitter ft 495 nm , emission 525/50 nm ; nir : excitation hq 650/45 nm , beamsplitter q 680 nm , emission hq 710/50 nm . the exposure times for acquisition of fluorescence images were kept constant for each series of images at each channel . to measure analyte - induced fluorescence changes , a solution of angeli s salt , na2s , or s - nitrosoglutathione was added to the plate on the microscope stage to reach a concentration of 1.5 mm , and images were taken immediately after addition and after 5 min . mobilization of intracellular zinc was induced by treating the cells with 3 mm angeli s salt and the released zn was chelated by bathing the cells in a solution of fresh pbs containing 50 m n , quantification of fluorescence intensity was performed using imagej ( version 1.45 , nih ) . the whole cell was selected as the region of interest , and the integrated fluorescence from the background region was subtracted from the cell body region . 5-(hydroxymethyl)benzene-1,2-diol was allowed to react with ir780 , a commercially available tricarbocyanine dye , to produce compound 1 . compound 2 was used immediately in a nucleophilic substitution reaction with 1,4,8,11-tetraazacyclotetradecane ( cyclam ) to give compound 3 . this product was obtained after purification by rp - hplc using a mobile phase containing 0.1% tfa and isolated as the trifluoroacetate salt , as ascertained by c and f nmr spectroscopy . compound 3 was alkylated with benzyl bromide to produce dhx1 , which was purified by rp - hplc and isolated as the trifluoroacetate salt of the trans ( 1,8-substituted cyclam ) isomer . the purity of both 3 and dhx1 was determined to be > 95% by analytical hplc analysis ( figure s6 ) . reagents and conditions : ( a ) et3n , dmf , 110 c , 20 min , 78% ; ( b ) socl2 , pyridine , ch2cl2 , dmf , 0 c , 30 min ; ( c ) cyclam , dipea , ch3cn , reflux , 30 min , 18% ( over steps b and c ) ; ( d ) benzyl bromide , dipea , ch3cn , 25 c , 2 h , 28% . compounds 3 and dhx1 were treated with cucl2 to form their cu(ii ) complexes . esi - ms analysis revealed that under these conditions the complexes cu-3 and cudhx1 were formed with a coordinated trifluoroacetate ligand with m / z = 799.33 and 889.38 , respectively . the photophysical properties of 3 ( figure s12 ) and dhx1 , as well as their cu(ii ) complexes , as measured in buffered aqueous solution ( 50 mm pipes , 100 mm kcl , ph = 7 ) are summarized in table 1 . the absorption spectrum of dhx1 is broad with a maximum at 693 nm ( figure 1 ) . the absorbance of the cu(ii ) complex is also broad and , in addition to a maximum at 693 nm , contains a peak at 650 nm . excitation at 650 nm gives a fluorescence spectrum with a maximum at 715 nm that extends across the nir region ( figure 1 ) . this value is comparable to that of indocyanine green ( 1.1 10 m cm ) , a benchmark nir fluorophore for in vivo imaging , indicating that the photophysical properties of dhx1 are suitable for in vivo applications . the quantum yield of this compound is decreased by more than 1 order of magnitude upon binding of cu(ii ) , a consequence of paramagnetic quenching induced by the transition metal . fluorescence ( dotted lines ) and absorbance ( solid lines ) spectra of dhx1 ( black lines ) and cudhx1 ( red lines ) in aqueous buffer ( 50 mm pipes , 100 mm kcl , ph = 7 ) . the ability of a molecule to sense a particular analyte in a complex biological sample depends on the specificity of its chemical reactions . we tested the reactivity of cu-3 and cudhx1 toward hno , the analyte of interest , and other species that are present in live cells and can potentially interfere with nitroxyl sensing . all reactivity tests were performed in aqueous buffer ( 50 mm pipes , 100 mm kcl , ph = 7 ) . samples of cu-3 and cudhx1 were prepared anaerobically to ensure that the sensors react directly with hno or no and not with an oxidation product . cu-3 displayed no change in fluorescence intensity upon addition of no gas and showed only a very small fluorescence increase upon addition of angeli s salt , an hno donor ( figure s11 ) . upon addition of 100 equiv of angeli s salt , cudhx1 underwent a 5-fold increase in fluorescence intensity ( figure 2 ) after only 2 min ( figure s13 ) . we determined 50 equiv to be the minimum amount of angeli s salt needed to induce turn - on of 2 m cudhx1 in aqueous buffer at ph = 7 . it is difficult to estimate the effective concentration of hno in a solution of angeli s salt because of the high reactivity of nitroxyl . we therefore can only conclude that the detection limit of hno by cudhx1 is 50 equiv . addition of 5000 equiv of no resulted in no detectable change in fluorescence intensity , demonstrating that cudhx1 detects hno selectively over no ( figure 3 ) . the reaction of cudhx1 with hno did not restore the fluorescence to that of the free ligand ( figure 2 ) . moreover , after 2 min , the fluorescence intensity decreased slowly ( figure s13 ) . upon reaction of ligand dhx1 with angeli s salt , there was a decrease in fluorescence intensity ( figure s14 ) , indicating that one of the products of the decomposition of the salt reacts with dhx1 to diminish its emission in buffered solution . fluorescence spectra of 2 m cudhx1 ( black dashed line ) in aqueous buffer ( 50 mm pipes , 100 mm kcl , ph = 7 ) and 2 min after the addition of 100 equiv of angeli s salt ( red solid line ) . angeli s salt , which decomposes readily ( t1/2 = 3 min ) at ph = 7 to give hno and nano2 , is usually handled as an aqueous solution at ph = 12 . to verify that hno is responsible for the observed fluorescence increase , the reactivity of cudhx1 toward nano2 ( 100 equiv ) and 10 mm naoh ( 100 l ) was evaluated . no increase in fluorescence intensity was observed upon reaction of cudhx1 with either 100 equiv of nano2 or 10 mm naoh in aqueous buffer ( figure 3 ) . additionally , no turn - on was induced when 100 equiv of cacl2 , mgcl2 , nacl , zncl2 , kno3 , h2o2 , naclo , sodium ascorbate , naonoo , l-(+)-cysteine , gsh , or methionine were added to a solution of cudhx1 ( figure 3 ) . cudhx1 is selective for hno over superoxide ( ko2 ) both in buffer ( figure 3 ) and in ch3cn ( figure s16 ) . the only analyte that promoted some detectable fluorescence enhancement after 10 min was h2s ( figure 3 ) , which elicited a much slower turn - on compared to that induced by hno . this selectivity is remarkable in view of the susceptibility of some cu - based sensors toward reducing thiols and makes cudhx1 a much more valuable sensor for use in biological samples where thiols are abundant . in addition , the fluorescence of cudhx1 was measured at different ph values . a blue shift and decrease in intensity was observed at ph < 6 , consistent with protonation of the hydroxyl group on the fluorophore ( pka 5.6 ) . at low ph values , hno - induced turn - on was considerably smaller than at neutral ph ( figure s17 ) . normalized integrated fluorescence intensity ( 660900 nm ) of 2 m cudhx1 in aqueous buffer ( 50 mm pipes , 100 mm kcl , ph = 7 ) and 10 min after addition of 100 equiv of the analyte or 2 min after addition of 100 equiv of hno . cyclic voltammetric studies of cudhx1 and cu-3 revealed quasi - reversible cu(ii)/cu(i ) reductions at 370 and 325 mv ( vs fc / fc ) , respectively ( figures s18 and s19 ) . the reduction of no to give hno at ph = 7 occurs at 0.11 v ( vs nhe , ) and the reduction potential of the cu(ii)/cu(i ) couple in cudhx1 is 1.01 v ( vs nhe ) . reduction of the nitrosonium cation ( no ) to no occurs at 1.52 v ( vs nhe ) , and consequently no can not reduce cu(ii ) in cudhx1 . esi - ms studies in buffer ( 50 mm pipes , 100 mm kcl , ph = 7 ) revealed that both cudhx1 and cu-3 exist as mixtures of complexes with cu(ii ) bound to trifluoroacetate or chloride as fifth ligand . addition of 100 equiv of angeli s salt to cudhx1 or cu-3 resulted in loss of the copper ion to give primarily the m / z of ligands dhx1 and 3 , respectively ( figures s9 and s10 ) . in the case of cu-3 , the m / z of the copper complex without the fifth ligand was also detected ( figure s10 ) . the x - band epr spectrum of cudhx1 , measured at 77 k in ch3oh , revealed a rhombic signal ( figure 4 ) , which disappeared upon treatment with 100 equiv of angeli s salt , as expected for reduction of cu(ii ) to cu(i ) . in contrast , addition of 5000 equiv of no gas to a solution of cudhx1 in ch3oh did not evoke a noticeable change in the epr spectrum ( figure 4 ) . the reduced form of cudhx1 could be reoxidized by allowing air into the epr tube , giving a more axially symmetric signal ; simulated traces are provided in figure s20 . notably , we could not observe an epr - silent species after reaction of cu-3 with angeli s salt ( figure s22 ) . this observation suggests that , if cu(i)-3 is formed , it converts immediately to cu(ii ) , even under anaerobic conditions . the nature of the species responsible for this reoxidized copper material was not further investigated . top : cudhx1 before ( black line ) and after addition of 5000 equiv of no ( red line ) . middle : cudhx1 before ( black line ) and after ( red line ) addition of 100 equiv of angeli s salt . bottom : cudhx1 after reduction by hno ( black line ) and after reoxidation by air ( red line ) . collection parameters : temperature , 77 k ; modulation amplitude , 20 g ; microwave power , 0.2 mw at 9.23 ghz . human cervical cancer ( hela ) cells were incubated with 5 m cudhx1 and 3 m hoechst 33528 nuclear stain in pbs for 15 min at 37 c . fluorescence microscopy images revealed only faint fluorescence in the nir channel ( figure 5c ) , consistent with the low brightness of cudhx1 . treatment with 1.5 mm angeli s salt resulted in a 3-fold increase in fluorescence intensity after only 5 min to restore a bright signal in the nir channel ( figure 5d ) . fluorescence microscopy images of cells incubated with cudhx1 in pbs before and after addition of angeli s salt : ( a ) differential interference contrast ( dic ) image ; ( b ) blue channel showing nuclei ; ( c ) nir channel before addition of angeli s salt ; and ( d ) nir channel 5 min after treatment with 1.5 mm angeli s salt . time - lapsed microscopy experiments indicated that , in the absence of hno , cudhx1 displays only a slight turn - on over time in live cells ( figure s24 ) , significantly less than that induced by hno . treatment of hela cells with gsno , an no donor , did not elicit any detectable fluorescence turn - on ( figure s25 ) , demonstrating that the sensor is selective for hno over no and s - nitrosothiols in live cells . in addition , incubation of live cells with na2s resulted in only a very small increase in fluorescence , and subsequent treatment of the same cells with angeli s salt still induced a strong fluorescence turn - on ( figure s26 ) . this experiment proves that the presence of h2s does not interfere with intracellular hno sensing by cudhx1 . an advantage of nir probes is that they can be used in combination with visible - color sensors to image two or more analytes simultaneously . in this kind of experiment , it is possible to investigate in real time the interplay between signaling molecules in live organisms . an example of such a relationship is the release of mobile zinc upon nitrosation of metallothionein . mobilization of endogenous zinc induced by no donors can be detected in live cells by fluorescent sensors , but no is unable to nitrosate thiolates directly . hno , however , reacts directly with thiols and may be capable of releasing chelatable zinc . to investigate this possibility , we incubated hela cells with cudhx1 and the green - fluorescent , zinc - selective , sensor zp1 . before any treatment , the cells showed only very faint fluorescence in both the nir and green channels ( figure 6d , g ) . treatment of these cells with 3 mm angeli s salt led to an increase in fluorescence in both the nir channel and the green channel ( figure 6c , e , h ) . addition of 50 m tpen reduced the fluorescence only in the green channel ( figure 6c , f ) , demonstrating that the observed turn - on corresponded to an increase in the intracellular levels of mobile zinc . addition of tpen did not significantly change the fluorescence of cudhx1 in the nir channel ( figure 6c , i ) . ( a ) dic image ; ( b ) blue channel showing nuclei ; ( c ) quantification of the fluorescence intensity in the green and nir channels ; ( d ) green channel before addition of angeli s salt ; ( e ) green channel after addition of angeli s salt ; ( f ) green channel after addition of tpen ; ( g ) nir channel before addition of angeli s salt ; ( h ) nir channel after addition of angeli s salt ; and ( i ) nir channel after addition of tpen . angeli s salt was handled as a solution in aqueous 10 mm naoh , which produces hno and nano2 . to prove that the mobilization of zn was induced by hno , hela cells were incubated with 3 mm nano2 in aqueous 10 mm naoh . no significant change in fluorescence intensity was detected in the green channel under these conditions ( figure s27 ) . these experiments reveal the value of cudhx1 as a versatile sensor for multicolor imaging , allowing for simultaneous observation of hno and mobile zn . they also prove that release of mobile zn is a downstream effect of addition of hno . to the best of our knowledge , our results provide the first application of a multicolor microscopy experiment to study the cellular chemistry of hno . cudhx1 provides a fast , selective , nir fluorescent sensor for the detection of hno . cudhx1 is selective against a variety of analytes present in live cells , including thiols and h2s , which is an advantage over previous cu(ii)-based hno sensors . epr spectroscopic and esi - ms studies showed that the sensing mechanism relies on reduction of cu(ii ) by hno , with concomitant dissociation of cu(i ) from the cyclam - based ligand . the sensor detects hno in live cells , even in the presence of h2s . the narrow emission of cudhx1 in the nir region makes it useful for multicolor imaging experiments , exemplified here by its use in combination with zp1 to track the increase in intracellular levels of mobile zinc elicited by exogenously applied angeli s salt . these results represent the first direct observation of a relationship between hno and mobile zinc in a biological environment . we anticipate that cudhx1 will be useful for investigating the biology of hno and its interplay with other signaling molecules by multicolor fluorescence microscopy in the nir .

the first near - infrared fluorescent turn - on sensor for the detection of nitroxyl ( hno ) , the one - electron reduced form of nitric oxide ( no ) , is reported . the new copper - based probe , cudhx1 , contains a dihydroxanthene ( dhx ) fluorophore and a cyclam derivative as a cu(ii ) binding site . upon reaction with hno , cudhx1 displays a five - fold fluorescence turn - on in cuvettes and is selective for hno over thiols and reactive nitrogen and oxygen species . cudhx1 can detect exogenously applied hno in live mammalian cells and in conjunction with the zinc - specific , green - fluorescent sensor zp1 can perform multicolor / multianalyte microscopic imaging . these studies reveal that hno treatment elicits an increase in the concentration of intracellular mobile zinc .

Introduction Experimental Section Results and Discussion Summary and Conclusions

nitric oxide ( no ) is an important biological signaling molecule present in the cardiovascular , immune , and nervous systems . hno , the one - electron reduced and protonated derivative of no , displays distinctive chemistry and biochemistry from that of no . exogenously applied hno confers vasoprotective effects , increases heart muscle contractility , and inhibits platelet aggregation . under biological conditions , hno is both an electrophile that oxidizes thiols and a nucleophile that can coordinate and reduce metal ions . nitrosation of thiolates induces the release of mobile zinc , which may trigger a variety of signaling pathways . understanding these and other downstream effects of exogenously applied hno is essential if nitroxyl donors are to be used as therapeutic agents . hno can be produced by nitric oxide synthase ( nos ) . under normal physiological conditions , nos catalyzes the conversion of arginine to citrulline and no in the presence of the cofactor tetrahydrobiopterin ( thb ) . in the absence of thb , isolated neuronal nos produces hno instead of no . no and hno interconvert in the presence of superoxide dismutase ( sod ) , and cytochrome c can reduce no to hno . recently , hno production was detected in the reaction between hsno and h2s and in the heme - iron - catalyzed reduction of nitrite with h2s . these results suggest that hno can be produced under biological conditions and might be associated with particular physiological or pathological states . improved methods for detecting hno selectively , with well - defined spatiotemporal resolution in live cells , tissues , and animals , are needed to fully understand the biology of nitroxyl . l - nha = n - hydroxy - l - arginine ; nnos = neuronal nitric oxide synthase ; sod = superoxide dismutase ; thb = tetrahydrobiopterin . a number of fluorescent sensors that detect hno in live cells are available ( chart 1 ) . one approach to hno sensing relies on the use of fluorophores functionalized with amines that bind cu(ii ) . upon exposure to hno , cu(ii ) reduces to cu(i ) , which renders the complex diamagnetic and restores the fluorescence of the probe . sensors based on this principle include bodipy - based complex cubot1 ( chart 1 ) , coumarin - based derivative cucot1 ( chart 1 ) , and the cubrno series of benzoresorufin - based sensors ( chart 1 ) that detect both hno and no . recently , the metal - free probe p - rhod ( chart 1 ) was reported . it would be valuable to have a sensor that not only detects hno with high selectivity but would also emit in the near - infrared ( nir ) region , desired for in vivo imaging because of deeper tissue penetration . in the present article we describe the design , synthesis , and implementation of cudhx1 ( chart 1 ) , a novel sensor that satisfies all of these requirements . the structure of cudhx1 comprises a cyclam binding site for cu(ii ) and a dihydroxanthene ( dhx ) nir fluorophore . we chose cyclam because cu(ii ) complexes of this ligand react very slowly with the interfering species h2s and glutathione ( gsh ) under physiological conditions . here we report the results of our studies using cudhx1 to image hno in live cells as well as multicolor imaging experiments to investigate the ability of angeli s salt , an hno donor , to affect the levels of mobile zinc in live cells . nitric oxide was passed through an ascarite column and a 6 ft coil containing silica gel at 78 c to remove impurities and then collected and stored under nitrogen in a gas storage bulb . low - resolution mass spectra ( lrms ) were acquired on an agilent 1100 series lc / msd trap spectrometer ( lcms ) , using electrospray ionization ( esi ) . the solvent was evaporated under reduced pressure and the solid was purified by column chromatography ( sio2 ; ch2cl2/ch3oh 9:1 ) to give the product as a dark blue solid ( 195 mg , yield 76% ) . after 30 min , the solvent was evaporated , and the residue was purified by rp - hplc according to the following protocol : constant flow rate 3 ml min ; isocratic flow 2% b , 05 min ; gradient , 3595% b , 1025 min . h nmr ( 500 mhz , dmso - d6 ) : 0.97 ( t , j = 7 hz , 3h ) , 1.72 ( s , 6h ) , 1.80 ( m , 6h ) , 2.06 ( m , 2h ) , 2.66 ( m , 3h ) , 2.72 ( m , 5h ) , 3.14 ( m , 8h ) , 3.83 ( s , 3h ) , 4.35 ( t , j = 7 hz , 2h ) , 6.52 ( d , j = 15 hz , 1h ) , 6.91 ( m , 1h ) , 7.00 ( m , 1h ) , 7.40 ( m , 6h ) , 7.50 ( t , j = 7 , 1h ) , 7.66 ( d , j = 5 hz , 1h ) , 7.72 ( m , 2h ) , 8.54 ( d , j = 15 hz , 1h ) . stock solutions of compound 3 and ligand dhx1 in dmso were prepared in the 18 mm range and stored at 20 c in 1 ml aliquots and thawed immediately before each experiment . solutions of angeli s salt ( 4 mm ) were prepared in the wet box in degassed 10 mm naoh ( 2 ml ) and brought out of the wet box in a gastight syringe . low - temperature x - band epr spectra ( 77 k , 9 ghz ) were collected with a bruker ems spectrometer equipped with an er 4199hs cavity and a gunn diode microwave source . for the no reactivity test , degassed aliquots from the stock solution of cudhx1 were brought into a wet box . selectivity of the fluorescence turn - on toward biologically relevant analytes was determined by comparing the fluorescence emission spectra of a 2 m solution of cudhx1 in aqueous buffer at ph = 7 , before and after treatment with 100 equiv of cacl2 , mgcl2 , nacl , zncl2 , kno3 , nano2 , ko2 , h2o2 , naclo , sodium ascorbate , naonoo , l-(+)-cysteine hydrochloride , gluthathione , methionine , na2s , or angeli s salt . for the no and hno selectivity studies , the samples were prepared anaerobically . for the no , hno , and na2s studies , fluorescence spectra were acquired every 1 min for 10 min . for the remaining analytes , in addition , the fluorescence turn - on of 2 m cudhx1 in the presence of 100 equiv of ko2 and angeli s salt was measured in ch3cn . to determine the effect of ph on the fluorescence emission of cudhx1 , 2 m solutions of cudhx1 were prepared anaerobically in aqueous buffer ( either 50 mm mes , 100 mm kcl ; ph 4 and 5 or 50 mm pipes , 100 mm kcl ; ph 6 , 7 , and 8) , and fluorescence spectra were recorded before and after addition of 100 equiv of angeli s salt . , supplemented with 10% fetal bovine serum ( fbs ; hyclone ) , 1% penicillin - streptomycin , 1% sodium pyruvate , and 1% l - glutamine . all cell imaging experiments involving addition of hno were carried out in pbs because addition of angeli s salt to plates containing dmem did not lead to any turn - on response ( figure s23 ) , presumably because of reaction of angeli s salt with cysteine or other components of dmem . for cell imaging experiments with zp1 , the growth medium was replaced with dye - free dmem containing 5 m zp1 , 3 m hoechst 33528 dye , and the cells were incubated for 1 h. cells were rinsed with pbs ( 2 2 ml ) before addition of fresh pbs containing 5 m cudhx1 and incubated for 15 min . the light source was an x - cite 120 metal - halide lamp ( exfo ) , and the fluorescence images were obtained using an oil - immersion objective at 63 or 100 magnification . to measure analyte - induced fluorescence changes , a solution of angeli s salt , na2s , or s - nitrosoglutathione was added to the plate on the microscope stage to reach a concentration of 1.5 mm , and images were taken immediately after addition and after 5 min . compounds 3 and dhx1 were treated with cucl2 to form their cu(ii ) complexes . the absorbance of the cu(ii ) complex is also broad and , in addition to a maximum at 693 nm , contains a peak at 650 nm . this value is comparable to that of indocyanine green ( 1.1 10 m cm ) , a benchmark nir fluorophore for in vivo imaging , indicating that the photophysical properties of dhx1 are suitable for in vivo applications . the quantum yield of this compound is decreased by more than 1 order of magnitude upon binding of cu(ii ) , a consequence of paramagnetic quenching induced by the transition metal . we tested the reactivity of cu-3 and cudhx1 toward hno , the analyte of interest , and other species that are present in live cells and can potentially interfere with nitroxyl sensing . samples of cu-3 and cudhx1 were prepared anaerobically to ensure that the sensors react directly with hno or no and not with an oxidation product . upon addition of 100 equiv of angeli s salt , cudhx1 underwent a 5-fold increase in fluorescence intensity ( figure 2 ) after only 2 min ( figure s13 ) . we determined 50 equiv to be the minimum amount of angeli s salt needed to induce turn - on of 2 m cudhx1 in aqueous buffer at ph = 7 . it is difficult to estimate the effective concentration of hno in a solution of angeli s salt because of the high reactivity of nitroxyl . the reaction of cudhx1 with hno did not restore the fluorescence to that of the free ligand ( figure 2 ) . upon reaction of ligand dhx1 with angeli s salt , there was a decrease in fluorescence intensity ( figure s14 ) , indicating that one of the products of the decomposition of the salt reacts with dhx1 to diminish its emission in buffered solution . angeli s salt , which decomposes readily ( t1/2 = 3 min ) at ph = 7 to give hno and nano2 , is usually handled as an aqueous solution at ph = 12 . to verify that hno is responsible for the observed fluorescence increase , the reactivity of cudhx1 toward nano2 ( 100 equiv ) and 10 mm naoh ( 100 l ) was evaluated . no increase in fluorescence intensity was observed upon reaction of cudhx1 with either 100 equiv of nano2 or 10 mm naoh in aqueous buffer ( figure 3 ) . additionally , no turn - on was induced when 100 equiv of cacl2 , mgcl2 , nacl , zncl2 , kno3 , h2o2 , naclo , sodium ascorbate , naonoo , l-(+)-cysteine , gsh , or methionine were added to a solution of cudhx1 ( figure 3 ) . cudhx1 is selective for hno over superoxide ( ko2 ) both in buffer ( figure 3 ) and in ch3cn ( figure s16 ) . the only analyte that promoted some detectable fluorescence enhancement after 10 min was h2s ( figure 3 ) , which elicited a much slower turn - on compared to that induced by hno . this selectivity is remarkable in view of the susceptibility of some cu - based sensors toward reducing thiols and makes cudhx1 a much more valuable sensor for use in biological samples where thiols are abundant . in addition , the fluorescence of cudhx1 was measured at different ph values . at low ph values , hno - induced turn - on was considerably smaller than at neutral ph ( figure s17 ) . reduction of the nitrosonium cation ( no ) to no occurs at 1.52 v ( vs nhe ) , and consequently no can not reduce cu(ii ) in cudhx1 . esi - ms studies in buffer ( 50 mm pipes , 100 mm kcl , ph = 7 ) revealed that both cudhx1 and cu-3 exist as mixtures of complexes with cu(ii ) bound to trifluoroacetate or chloride as fifth ligand . in the case of cu-3 , the m / z of the copper complex without the fifth ligand was also detected ( figure s10 ) . the x - band epr spectrum of cudhx1 , measured at 77 k in ch3oh , revealed a rhombic signal ( figure 4 ) , which disappeared upon treatment with 100 equiv of angeli s salt , as expected for reduction of cu(ii ) to cu(i ) . the reduced form of cudhx1 could be reoxidized by allowing air into the epr tube , giving a more axially symmetric signal ; simulated traces are provided in figure s20 . this observation suggests that , if cu(i)-3 is formed , it converts immediately to cu(ii ) , even under anaerobic conditions . fluorescence microscopy images revealed only faint fluorescence in the nir channel ( figure 5c ) , consistent with the low brightness of cudhx1 . treatment with 1.5 mm angeli s salt resulted in a 3-fold increase in fluorescence intensity after only 5 min to restore a bright signal in the nir channel ( figure 5d ) . time - lapsed microscopy experiments indicated that , in the absence of hno , cudhx1 displays only a slight turn - on over time in live cells ( figure s24 ) , significantly less than that induced by hno . treatment of hela cells with gsno , an no donor , did not elicit any detectable fluorescence turn - on ( figure s25 ) , demonstrating that the sensor is selective for hno over no and s - nitrosothiols in live cells . in addition , incubation of live cells with na2s resulted in only a very small increase in fluorescence , and subsequent treatment of the same cells with angeli s salt still induced a strong fluorescence turn - on ( figure s26 ) . in this kind of experiment , it is possible to investigate in real time the interplay between signaling molecules in live organisms . an example of such a relationship is the release of mobile zinc upon nitrosation of metallothionein . mobilization of endogenous zinc induced by no donors can be detected in live cells by fluorescent sensors , but no is unable to nitrosate thiolates directly . hno , however , reacts directly with thiols and may be capable of releasing chelatable zinc . to investigate this possibility , we incubated hela cells with cudhx1 and the green - fluorescent , zinc - selective , sensor zp1 . treatment of these cells with 3 mm angeli s salt led to an increase in fluorescence in both the nir channel and the green channel ( figure 6c , e , h ) . addition of 50 m tpen reduced the fluorescence only in the green channel ( figure 6c , f ) , demonstrating that the observed turn - on corresponded to an increase in the intracellular levels of mobile zinc . addition of tpen did not significantly change the fluorescence of cudhx1 in the nir channel ( figure 6c , i ) . these experiments reveal the value of cudhx1 as a versatile sensor for multicolor imaging , allowing for simultaneous observation of hno and mobile zn . cudhx1 provides a fast , selective , nir fluorescent sensor for the detection of hno . cudhx1 is selective against a variety of analytes present in live cells , including thiols and h2s , which is an advantage over previous cu(ii)-based hno sensors . epr spectroscopic and esi - ms studies showed that the sensing mechanism relies on reduction of cu(ii ) by hno , with concomitant dissociation of cu(i ) from the cyclam - based ligand . the sensor detects hno in live cells , even in the presence of h2s . the narrow emission of cudhx1 in the nir region makes it useful for multicolor imaging experiments , exemplified here by its use in combination with zp1 to track the increase in intracellular levels of mobile zinc elicited by exogenously applied angeli s salt . these results represent the first direct observation of a relationship between hno and mobile zinc in a biological environment .

a total of 354 crc formalin - fixed paraffin - embedded tissues were collected retrospectively from the pathologic archives of yeungnam university medical center , daegu , korea . all samples were derived from patients who underwent radical resection and subsequent adjuvant chemotherapy from 1996 to 2000 in our institution . among these , 49 patients ( 32 patients who had preoperative neoadjuvant chemoradiation therapy , 16 patients who were diagnosed with stage iv disease , and 1 patient who was diagnosed with stage iv and had preoperative neoadjuvant chemoradiation therapy at the same time ) were excluded . patients electronic medical records and pathologic reports were reviewed to obtain information including age , sex , location of tumor , pt / pn categories , presence of lymphatic / venous / perineural invasion , and tumor differentiation . the follow - up period for patients ( from the date of surgery to the date of recurrence or the last follow - up ) ranged from 4 to 243 months and the average disease - free survival ( dfs ) was 85 months . the restaging of tumors and histological re - grading were carried out according to the american joint committee on cancer staging manual , seventh edition . the study was approved by the yeungnam university medical center institutional review board ( no . tissue microarray ( tma ) construction was conducted by reviewing hematoxylin and eosin stained slides and selecting an area that had sufficient tumor with no hemorrhage or necrosis . one representative tissue core ( 2 mm in diameter ) was obtained from a donor block and placed in a recipient block using a trephine apparatus . 1a ) , renal , hepatic parenchymal tissues and three cases of non - neoplastic gastric parenchymal tissues were included in each tma . immunohistochemical ( ihc ) staining with rabbit monoclonal abs against cd9 ( 1:1,200 , epr2949 ; abcam , cambridge , uk ) was performed on each tma block using a benchmark xt immunostainer ( ventana medical system , tucson , az , usa ) following the manufacturer s protocol . immunoreactivity of cd9 expression was assessed in tumor cells ( t - cd9 ) and immune cells ( i - cd9 ) in stroma semiquantitatively . in cases of t - cd9 the intensity was initially scored on a scale of 0 to 3 : negative ( 0 ) , weak positive ( 1 + ) , moderately positive ( 2 + ) , and strongly positive ( 3 + ) ( fig . the extent of staining was categorized into five groups according to the percentages of positively stained cells : 0% ( 0 ) , 25% ( 1 + ) , > 25 and 50% ( 2 + ) , > 50 and 75% ( 3 + ) , and > 75% ( 4 + ) . the final t - cd9 score was determined by multiplying the extent and intensity scores and recording the final score from 0 to 12 . the t - cd9 immunoreactivity score was then dichotomized as low ( score , 0 to 3 ) or high ( score , 4 to 12 ) . in cases of i - cd9 , the percentage of stained immune cells was estimated on a 0 , 1 + , 2 + , and 3 + scale : 0% ( 0 ) , 25% ( 1 + ) , > 25 and 50% ( 2 + ) , and > 50% ( 3 + ) ( fig . the cases of i - cd9 score 0 were classified as low , and when the score was 1 to 3 , they were classified as high . all ihc staining assessments were performed independently by two pathologists ( k .- j.k . and y.k.b . ) , who were blinded to the clinicopathological information . correlation between the categorical variables was analyzed using pearson s chi - square test or fisher exact test . dfs was defined as the period from the date of primary radical resection to the date of tumor recurrence , metastasis , death , or the last follow - up . dfs was assessed by the kaplan - meier method with the log - rank test , and multivariate analysis with the cox regression model was used to adjust variables that had been statistically significant for dfs in univariate analysis . all statistical analyses were two - sided , and statistical significance was considered to be p<.05 . a total of 354 crc formalin - fixed paraffin - embedded tissues were collected retrospectively from the pathologic archives of yeungnam university medical center , daegu , korea . all samples were derived from patients who underwent radical resection and subsequent adjuvant chemotherapy from 1996 to 2000 in our institution . among these , 49 patients ( 32 patients who had preoperative neoadjuvant chemoradiation therapy , 16 patients who were diagnosed with stage iv disease , and 1 patient who was diagnosed with stage iv and had preoperative neoadjuvant chemoradiation therapy at the same time ) were excluded . patients electronic medical records and pathologic reports were reviewed to obtain information including age , sex , location of tumor , pt / pn categories , presence of lymphatic / venous / perineural invasion , and tumor differentiation . the follow - up period for patients ( from the date of surgery to the date of recurrence or the last follow - up ) ranged from 4 to 243 months and the average disease - free survival ( dfs ) was 85 months . the restaging of tumors and histological re - grading were carried out according to the american joint committee on cancer staging manual , seventh edition . the study was approved by the yeungnam university medical center institutional review board ( no . tissue microarray ( tma ) construction was conducted by reviewing hematoxylin and eosin stained slides and selecting an area that had sufficient tumor with no hemorrhage or necrosis . one representative tissue core ( 2 mm in diameter ) was obtained from a donor block and placed in a recipient block using a trephine apparatus . 1a ) , renal , hepatic parenchymal tissues and three cases of non - neoplastic gastric parenchymal tissues were included in each tma . immunohistochemical ( ihc ) staining with rabbit monoclonal abs against cd9 ( 1:1,200 , epr2949 ; abcam , cambridge , uk ) was performed on each tma block using a benchmark xt immunostainer ( ventana medical system , tucson , az , usa ) following the manufacturer s protocol . immunoreactivity of cd9 expression was assessed in tumor cells ( t - cd9 ) and immune cells ( i - cd9 ) in stroma semiquantitatively . in cases of t - cd9 the intensity was initially scored on a scale of 0 to 3 : negative ( 0 ) , weak positive ( 1 + ) , moderately positive ( 2 + ) , and strongly positive ( 3 + ) ( fig . 1b the extent of staining was categorized into five groups according to the percentages of positively stained cells : 0% ( 0 ) , 25% ( 1 + ) , > 25 and 50% ( 2 + ) , > 50 and 75% ( 3 + ) , and > 75% ( 4 + ) . the final t - cd9 score was determined by multiplying the extent and intensity scores and recording the final score from 0 to 12 . the t - cd9 immunoreactivity score was then dichotomized as low ( score , 0 to 3 ) or high ( score , 4 to 12 ) . in cases of i - cd9 , the percentage of stained immune cells was estimated on a 0 , 1 + , 2 + , and 3 + scale : 0% ( 0 ) , 25% ( 1 + ) , > 25 and 50% ( 2 + ) , and > the cases of i - cd9 score 0 were classified as low , and when the score was 1 to 3 , they were classified as high . all ihc staining assessments were performed independently by two pathologists ( k .- j.k . and y.k.b . ) , who were blinded to the clinicopathological information . correlation between the categorical variables was analyzed using pearson s chi - square test or fisher exact test . dfs was defined as the period from the date of primary radical resection to the date of tumor recurrence , metastasis , death , or the last follow - up . dfs was assessed by the kaplan - meier method with the log - rank test , and multivariate analysis with the cox regression model was used to adjust variables that had been statistically significant for dfs in univariate analysis . all statistical analyses were two - sided , and statistical significance was considered to be p<.05 . five normal sample of colorectal tissue and 305 crc samples were examined to identify cd9 expression . of the 305 crc samples , one case t - cd9 sample was excluded from evaluation because repeated sectioning of the tma block resulted in very few tumor cells remaining for evaluation . as a result , 304 cases of t - cd9 and 305 cases of i - cd9 were analyzed . among the five samples of non - neoplastic colorectal mucosae , the expression of cd-9 was negative or weakly positive in almost all epithelial cells ( fig . these weakly positive cells showed similar intensity and were classified into the low expression group . on the other hand , more than 50% of stained stromal immune cells in non - neoplastic colorectal tissues exhibited strong expression of cd9 throughout the entire samples and were classified as the high expression group . t - cd9 ( + ) was detected in 175 of 304 crcs ( 57.6% ) , and i - cd9 ( + ) was detected in 265 of 305 cases ( 86.9% ) . one hundred fifty - four cases ( 50.7% ) showed both t - cd9 ( + ) and i - cd9 ( + ) and 19 cases ( 6.3% ) exhibited both t - cd9 ( ) and i - cd9 ( ) . no statistically significant differences were noted in clinicopathological parameters including age , sex , stage , gross type , location , pt / pn categories , or frequency of angiolymphatic and perineural invasion between the t - cd9 ( + ) and t - cd9 ( ) groups ( table 1 ) . in kaplan - meier survival analysis , the t - cd9 ( + ) group showed a tendency for better dfs than the t - cd9 ( ) group , but statistical significance was not reached ( p=.057 ) ( fig . we additionally subdivided cases into two groups according to tumor location : right - sided tumors ( n=89 ) and left - sided tumors ( n=215 ) . based on the previous literature , crcs were classified into right- and left - sided tumors relative to the splenic flexure . in other words , tumors arising proximal to the splenic flexure ( cecum , ascending colon , and transverse colon ) are considered right - sided , and tumors distal to the splenic flexure ( descending colon , sigmoid colon , and rectum ) are considered left - sided . in left - sided tumors , dfs was significantly better in the t - cd9 ( + ) group compared to the t - cd9 ( ) group ( p=.021 ) ( fig . 3b ) . however , in right - sided tumors , no significant difference was observed between the two groups ( p=.453 ) ( fig . 3c ) . multivariate analysis including stage , lymphatic invasion , vascular invasion , perineural invasion and t - cd9which were significant prognostic factors in univariate analysis revealed that the stage ( p=.002 ) was still an independent prognostic predictor for dfs , whereas t - cd9 expression failed to correlate with prognosis , with no difference between t - cd9 ( + ) and t - cd9 ( ) groups in patients with left - sided crc ( p=.167 ) ( table 2 ) . i - cd9 ( + ) crcs significantly correlated with well / moderately differentiation ( p=.014 ) compared to i - cd9 ( ) crcs . however , no significant associations were found for other parameters including age , sex , stage , gross type , location , pt / pn categories , or frequency of angiolymphatic and perineural invasion ( table 1 ) . in kaplan - meier survival analysis , the i - cd9 ( + ) group had a tendency towards worse dfs compared to the i - cd9 ( ) group , but no significant difference was found ( p=.156 ) ( fig . when the cases were subdivided into left- and right - sided crc groups , i - cd9 ( ) suggested a potential protective role in left - sided tumors ( p=.233 ) , but statistical significance was not reached ( fig . kaplan - meier survival analysis found the longest dfs for patients in the t - cd9 ( + ) /i - cd9 ( ) group , whereas the t - cd9 ( )/i - cd9 ( + ) group had the shortest dfs , but statistical significance was not reached ( p=.054 ) ( fig . when the patients were divided into left- and right - sided crc groups , increased survival differences between subgroups was observed in left - sided tumors ( p=.030 ) ( fig . using a cox proportional hazards model , combined t - cd9/i - cd9 expression was not an independent prognostic factor of dfs in left - sided crcs ( p=.199 ) ( table 2 ) . five normal sample of colorectal tissue and 305 crc samples were examined to identify cd9 expression . of the 305 crc samples , one case t - cd9 sample was excluded from evaluation because repeated sectioning of the tma block resulted in very few tumor cells remaining for evaluation . as a result , 304 cases of t - cd9 and 305 cases of i - cd9 were analyzed . among the five samples of non - neoplastic colorectal mucosae , the expression of cd-9 was negative or weakly positive in almost all epithelial cells ( fig . these weakly positive cells showed similar intensity and were classified into the low expression group . on the other hand , more than 50% of stained stromal immune cells in non - neoplastic colorectal tissues exhibited strong expression of cd9 throughout the entire samples and were classified as the high expression group . t - cd9 ( + ) was detected in 175 of 304 crcs ( 57.6% ) , and i - cd9 ( + ) was detected in 265 of 305 cases ( 86.9% ) . one hundred fifty - four cases ( 50.7% ) showed both t - cd9 ( + ) and i - cd9 ( + ) and 19 cases ( 6.3% ) exhibited both t - cd9 ( ) and i - cd9 ( ) . no statistically significant differences were noted in clinicopathological parameters including age , sex , stage , gross type , location , pt / pn categories , or frequency of angiolymphatic and perineural invasion between the t - cd9 ( + ) and t - cd9 ( ) groups ( table 1 ) . in kaplan - meier survival analysis , the t - cd9 ( + ) group showed a tendency for better dfs than the t - cd9 ( ) group , but statistical significance was not reached ( p=.057 ) ( fig . we additionally subdivided cases into two groups according to tumor location : right - sided tumors ( n=89 ) and left - sided tumors ( n=215 ) . based on the previous literature , crcs were classified into right- and left - sided tumors relative to the splenic flexure . in other words , tumors arising proximal to the splenic flexure ( cecum , ascending colon , and transverse colon ) are considered right - sided , and tumors distal to the splenic flexure ( descending colon , sigmoid colon , and rectum ) are considered left - sided . in left - sided tumors , dfs was significantly better in the t - cd9 ( + ) group compared to the t - cd9 ( ) group ( p=.021 ) ( fig . 3b ) . however , in right - sided tumors , no significant difference was observed between the two groups ( p=.453 ) ( fig . 3c ) . multivariate analysis including stage , lymphatic invasion , vascular invasion , perineural invasion and t - cd9which were significant prognostic factors in univariate analysis revealed that the stage ( p=.002 ) was still an independent prognostic predictor for dfs , whereas t - cd9 expression failed to correlate with prognosis , with no difference between t - cd9 ( + ) and t - cd9 ( ) groups in patients with left - sided crc ( p=.167 ) ( table 2 ) . i - cd9 ( + ) crcs significantly correlated with well / moderately differentiation ( p=.014 ) compared to i - cd9 ( ) crcs . however , no significant associations were found for other parameters including age , sex , stage , gross type , location , pt / pn categories , or frequency of angiolymphatic and perineural invasion ( table 1 ) . in kaplan - meier survival analysis , the i - cd9 ( + ) group had a tendency towards worse dfs compared to the i - cd9 ( ) group , but no significant difference was found ( p=.156 ) ( fig . when the cases were subdivided into left- and right - sided crc groups , i - cd9 ( ) suggested a potential protective role in left - sided tumors ( p=.233 ) , but statistical significance was not reached ( fig . , no statistical significance was observed between the two groups ( p=.643 ) ( fig . kaplan - meier survival analysis found the longest dfs for patients in the t - cd9 ( + ) /i - cd9 ( ) group , whereas the t - cd9 ( )/i - cd9 ( + ) group had the shortest dfs , but statistical significance was not reached ( p=.054 ) ( fig . when the patients were divided into left- and right - sided crc groups , increased survival differences between subgroups was observed in left - sided tumors ( p=.030 ) ( fig . using a cox proportional hazards model , combined t - cd9/i - cd9 expression was not an independent prognostic factor of dfs in left - sided crcs ( p=.199 ) ( table 2 ) . many previous in vitro and in vivo models of cancer cell lines have contributed to our understanding of the role of cd9 and the associated functional complexes with other members of the tetraspanin family and various integrins [ 14 - 16,19 ] . high expression of cd9 in tumor cells has been associated with favorable prognosis , reduced metastatic potential , and cancer growth in various tumor types , including lung , breast , prostate , stomach , pancreas , and colon carcinoma [ 4,20 - 22 ] . miyake et al . demonstrated that injection of cd9-transfected melanoma cells suppressed lung metastasis in a rodent model and ovalle et al . showed that ectopic cd9 expression in a colon cancer cell line resulted in inhibition of cell growth . in addition , mori et al . stated that reduced expression of cd9 by reverse transcription polymerase chain reaction is linked to poor prognosis in colon cancer . our data demonstrated that patients with high expression of cd9 in tumor cells ( t - cd9 [ + ] ) tended to have a longer dfs , although the difference was not statistically significant ( p=.057 ) . however , studies have yielded controversial results about cd9 expression in non - neoplastic colorectal cells . mori et al . demonstrated that cd9 mrna is consistently expressed in non - neoplastic colonic tissues adjacent to tumors and its expression level was higher in normal colonic tissues than in tumors in six normal / tumor paired samples using northern blot analysis . okochi et al . also showed that strong cd9 expression was detected in normal colonic epithelium by ihc . on the contrary , negative or weakly positive expression of cd9 these contradictory finding of the non - neoplastic cd9 expression may be attributed to differing experimental techniques such as northern blot analysis and ihc , as well as variation in the different types of cd9 antibodies . furthermore , our data has limitation in this regard , because very few normal colorectal tissue samples ( n=5 ) were included , and pairing of normal and tumor samples has not been carried out . several mechanisms have been suggested to explain the tumor suppressant function of cd9 . in conjunction with egfr , cd9 has been shown to induce apoptosis via restricted activation of p46 shc isoforms or the c - jun n - terminal kinase / stress - activated protein kinase and p38 mitogen - activated protein kinase pathways . by forming a functional complex with integrin 1 furthermore , cd9 may hinder cell proliferation through internalization of egfr , resulting in attenuation of egf - egfr induced signals . notably , the protective prognostic role of t - cd9 on dfs was demonstrated in the relatively homogenous left - sided crc group in this study with statistical significance ( p=.021 ) . however , in the right - sided tumor group , no significance of t - cd9 was observed in dfs , but this finding was considered to have limited reliability due to the small number of cases ( n=89 ) . the prognostic effect of t - cd9 is speculated to be attributed to different molecular carcinogenic mechanisms between right- and left - sided crcs . in left - sided crcs , the majority of tumors develop through a chromosomal instability pathway known as the adenoma - carcinoma sequence . in this pathway , progression to adenoma - carcinoma from normal mucosa is established by accumulation of various genomic changes including activation of proto - oncogenes such as kras and inactivation of tumor suppressor genes such as apc and tp53 . meanwhile , a large proportion of tumors with microsatellite unstable ( microsatellite instability they are caused by genetic and epigenetic alterations of mismatch repair genes and consequently exhibit a hypermutated phenotype . serrated morphology tumors and cpg island methylator phenotypes are seen more frequently in right - sided tumors than in left - sided crcs . these findings suggest that even in the same tumor , the prognostic implication of cd9 can be different depending on the underlying molecular mechanisms of carcinogenesis . extended study is required to verify the hypothesis presented above and the location based variation of cd9 roles in tumor progression . tumor - infiltrating immune cells are thought to play a substantial role in shaping the microenvironment depending on various factors such as cytokines and chemokines , towards either immunostimulatory antitumor conditions or immunoregulatory tumor promoting mileu . in survival analysis , i - cd9 ( + ) showed a tendency to be associated with a high recurrence rate in left - sided crcs ( p=.156 ) and this finding is contrary to the case of t - cd9 . in a previous study , erovic et al . described the cd9-positive peritumoral lymphocytes in squamous cell carcinoma of the head and neck . bruno et al . demonstrated that cd9-expressing tumor - infiltrating immune cells , especially tumor - associated natural killer cells , these findings are consistent with our results regarding i - cd9 . to the best of our knowledge , this is the first report to identify a differential prognostic role of cd9 expression in tumor cells and immune cells in crcs . . stated that a therapeutic agent targeting tetraspanin , especially cd9 , using monoclonal abs such as alb6 and pain13 , might be beneficial for cancer patients . however , several researchers have suggested the contradictory finding of t - cd9 in various cancer types including multiple myeloma , fibrosarcoma , and even in gastric carcinoma , showing that cd9 expression of tumor cells has a positive influence on tumor proliferation and invasion by enhancing hb - egf / egfr interaction or metalloproteinases-2 secretion . in addition , our study also demonstrated an opposite effect of t - cd9 and i - cd9 on tumor progression . therefore , future studies should focus on the varying mechanisms and the therapeutic response of i - cd9 and t - cd9 in crcs . in combined survival analysis of t - cd9 and i - cd9 , tumors with high t - cd9 expression plus low i - cd9 expression showed a tendency to have a longer dfs compared with ( 1 ) tumors with concurrent high or low expression of t - cd9 and i - cd9 , or ( 2 ) tumors with low t - cd9 expression plus high i - cd9 expression ( p=.054 ) , and a similar result was found in left - sided crcs with statistical significance ( p=.030 ) . these findings suggested that the combined evaluation of t - cd9 and i - cd9 is required to determine the comprehensive prognostic effect of cd9 in crcs . in conclusion , high expression of cd9 in tumor cells tends to be inversely related to tumor recurrence , especially in left - sided crcs . however , a different tendency on dfs was demonstrated in immune cells regardless of tumor location . additional studies are necessary , with an emphasis on differential expression of cd9 in tumor cells and immune cells .

backgroundcd9 , a member of the tetraspanin superfamily , is a tumor suppressor in many malignancies . the aim of this study was to evaluate the immunohistochemical expression of cd9 in colorectal carcinomas ( crcs ) and determine clinicopathological and prognostic significance of its expression.methodsthe cd9 expression status of 305 crcs was evaluated using a semi - quantitative scoring system in tumor cells ( t - cd9 ) and immune cells ( i - cd9 ) by classifying the results as high and low expression.resultshigh t - cd9 ( t - cd9 [ + ] ) expression was detected in 175 samples ( 57.6% ) and high i - cd9 ( i - cd9 [ + ] ) expression was detected in 265 samples ( 86.9% ) . using kaplan - meier survival analysis , the t - cd9 ( + ) group showed a tendency for better disease - free survival ( dfs ) ( p = .057 ) . in left - sided tumors , dfs was significantly longer in the t - cd9 ( + ) group ( p = .021 ) but no statistical significance was observed with right - sided tumors ( p = .453 ) . i - cd9 ( + ) crcs significantly correlated with well / moderately differentiation ( p = .014 ) . in kaplan - meier analysis , the i - cd9 ( + ) group had a tendency towards worse dfs compared to the i - cd9 ( ) group ( p = .156 ) . in combined survival analysis of t - cd9 and i - cd9 , we found that the longest dfs was among patients in the t - cd9 ( + ) /i - cd9 ( ) group , whereas the t - cd9 ( )/i - cd9 ( + ) group showed the shortest dfs ( p = .054).conclusionshigh expression of t - cd9 was associated with a favorable dfs , especially in left - sided crcs . combined evaluation of t - cd9 and i - cd9 is required to determine the comprehensive prognostic effect of cd9 in crcs .

MATERIALS AND METHODS Patients and specimens Tissue microarray and immunohistochemistry Statistical analysis RESULTS CD9 expression status in CRC Characteristics of a T-CD9 subset of CRC Characteristics of an I-CD9 subset of CRC Characteristics of a combined T-CD9/I-CD9 subset of CRC DISCUSSION

the follow - up period for patients ( from the date of surgery to the date of recurrence or the last follow - up ) ranged from 4 to 243 months and the average disease - free survival ( dfs ) was 85 months . the study was approved by the yeungnam university medical center institutional review board ( no . immunohistochemical ( ihc ) staining with rabbit monoclonal abs against cd9 ( 1:1,200 , epr2949 ; abcam , cambridge , uk ) was performed on each tma block using a benchmark xt immunostainer ( ventana medical system , tucson , az , usa ) following the manufacturer s protocol . immunoreactivity of cd9 expression was assessed in tumor cells ( t - cd9 ) and immune cells ( i - cd9 ) in stroma semiquantitatively . in cases of t - cd9 the intensity was initially scored on a scale of 0 to 3 : negative ( 0 ) , weak positive ( 1 + ) , moderately positive ( 2 + ) , and strongly positive ( 3 + ) ( fig . the extent of staining was categorized into five groups according to the percentages of positively stained cells : 0% ( 0 ) , 25% ( 1 + ) , > 25 and 50% ( 2 + ) , > 50 and 75% ( 3 + ) , and > 75% ( 4 + ) . the final t - cd9 score was determined by multiplying the extent and intensity scores and recording the final score from 0 to 12 . the t - cd9 immunoreactivity score was then dichotomized as low ( score , 0 to 3 ) or high ( score , 4 to 12 ) . in cases of i - cd9 , the percentage of stained immune cells was estimated on a 0 , 1 + , 2 + , and 3 + scale : 0% ( 0 ) , 25% ( 1 + ) , > 25 and 50% ( 2 + ) , and > 50% ( 3 + ) ( fig . the cases of i - cd9 score 0 were classified as low , and when the score was 1 to 3 , they were classified as high . dfs was defined as the period from the date of primary radical resection to the date of tumor recurrence , metastasis , death , or the last follow - up . dfs was assessed by the kaplan - meier method with the log - rank test , and multivariate analysis with the cox regression model was used to adjust variables that had been statistically significant for dfs in univariate analysis . all statistical analyses were two - sided , and statistical significance was considered to be p<.05 . the follow - up period for patients ( from the date of surgery to the date of recurrence or the last follow - up ) ranged from 4 to 243 months and the average disease - free survival ( dfs ) was 85 months . immunohistochemical ( ihc ) staining with rabbit monoclonal abs against cd9 ( 1:1,200 , epr2949 ; abcam , cambridge , uk ) was performed on each tma block using a benchmark xt immunostainer ( ventana medical system , tucson , az , usa ) following the manufacturer s protocol . immunoreactivity of cd9 expression was assessed in tumor cells ( t - cd9 ) and immune cells ( i - cd9 ) in stroma semiquantitatively . in cases of t - cd9 the intensity was initially scored on a scale of 0 to 3 : negative ( 0 ) , weak positive ( 1 + ) , moderately positive ( 2 + ) , and strongly positive ( 3 + ) ( fig . 1b the extent of staining was categorized into five groups according to the percentages of positively stained cells : 0% ( 0 ) , 25% ( 1 + ) , > 25 and 50% ( 2 + ) , > 50 and 75% ( 3 + ) , and > 75% ( 4 + ) . the final t - cd9 score was determined by multiplying the extent and intensity scores and recording the final score from 0 to 12 . the t - cd9 immunoreactivity score was then dichotomized as low ( score , 0 to 3 ) or high ( score , 4 to 12 ) . in cases of i - cd9 , the percentage of stained immune cells was estimated on a 0 , 1 + , 2 + , and 3 + scale : 0% ( 0 ) , 25% ( 1 + ) , > 25 and 50% ( 2 + ) , and > the cases of i - cd9 score 0 were classified as low , and when the score was 1 to 3 , they were classified as high . dfs was defined as the period from the date of primary radical resection to the date of tumor recurrence , metastasis , death , or the last follow - up . dfs was assessed by the kaplan - meier method with the log - rank test , and multivariate analysis with the cox regression model was used to adjust variables that had been statistically significant for dfs in univariate analysis . all statistical analyses were two - sided , and statistical significance was considered to be p<.05 . five normal sample of colorectal tissue and 305 crc samples were examined to identify cd9 expression . of the 305 crc samples , one case t - cd9 sample was excluded from evaluation because repeated sectioning of the tma block resulted in very few tumor cells remaining for evaluation . as a result , 304 cases of t - cd9 and 305 cases of i - cd9 were analyzed . among the five samples of non - neoplastic colorectal mucosae , the expression of cd-9 was negative or weakly positive in almost all epithelial cells ( fig . these weakly positive cells showed similar intensity and were classified into the low expression group . on the other hand , more than 50% of stained stromal immune cells in non - neoplastic colorectal tissues exhibited strong expression of cd9 throughout the entire samples and were classified as the high expression group . t - cd9 ( + ) was detected in 175 of 304 crcs ( 57.6% ) , and i - cd9 ( + ) was detected in 265 of 305 cases ( 86.9% ) . one hundred fifty - four cases ( 50.7% ) showed both t - cd9 ( + ) and i - cd9 ( + ) and 19 cases ( 6.3% ) exhibited both t - cd9 ( ) and i - cd9 ( ) . no statistically significant differences were noted in clinicopathological parameters including age , sex , stage , gross type , location , pt / pn categories , or frequency of angiolymphatic and perineural invasion between the t - cd9 ( + ) and t - cd9 ( ) groups ( table 1 ) . in kaplan - meier survival analysis , the t - cd9 ( + ) group showed a tendency for better dfs than the t - cd9 ( ) group , but statistical significance was not reached ( p=.057 ) ( fig . we additionally subdivided cases into two groups according to tumor location : right - sided tumors ( n=89 ) and left - sided tumors ( n=215 ) . based on the previous literature , crcs were classified into right- and left - sided tumors relative to the splenic flexure . in other words , tumors arising proximal to the splenic flexure ( cecum , ascending colon , and transverse colon ) are considered right - sided , and tumors distal to the splenic flexure ( descending colon , sigmoid colon , and rectum ) are considered left - sided . in left - sided tumors , dfs was significantly better in the t - cd9 ( + ) group compared to the t - cd9 ( ) group ( p=.021 ) ( fig . however , in right - sided tumors , no significant difference was observed between the two groups ( p=.453 ) ( fig . multivariate analysis including stage , lymphatic invasion , vascular invasion , perineural invasion and t - cd9which were significant prognostic factors in univariate analysis revealed that the stage ( p=.002 ) was still an independent prognostic predictor for dfs , whereas t - cd9 expression failed to correlate with prognosis , with no difference between t - cd9 ( + ) and t - cd9 ( ) groups in patients with left - sided crc ( p=.167 ) ( table 2 ) . i - cd9 ( + ) crcs significantly correlated with well / moderately differentiation ( p=.014 ) compared to i - cd9 ( ) crcs . in kaplan - meier survival analysis , the i - cd9 ( + ) group had a tendency towards worse dfs compared to the i - cd9 ( ) group , but no significant difference was found ( p=.156 ) ( fig . when the cases were subdivided into left- and right - sided crc groups , i - cd9 ( ) suggested a potential protective role in left - sided tumors ( p=.233 ) , but statistical significance was not reached ( fig . kaplan - meier survival analysis found the longest dfs for patients in the t - cd9 ( + ) /i - cd9 ( ) group , whereas the t - cd9 ( )/i - cd9 ( + ) group had the shortest dfs , but statistical significance was not reached ( p=.054 ) ( fig . when the patients were divided into left- and right - sided crc groups , increased survival differences between subgroups was observed in left - sided tumors ( p=.030 ) ( fig . using a cox proportional hazards model , combined t - cd9/i - cd9 expression was not an independent prognostic factor of dfs in left - sided crcs ( p=.199 ) ( table 2 ) . five normal sample of colorectal tissue and 305 crc samples were examined to identify cd9 expression . of the 305 crc samples , one case t - cd9 sample was excluded from evaluation because repeated sectioning of the tma block resulted in very few tumor cells remaining for evaluation . as a result , 304 cases of t - cd9 and 305 cases of i - cd9 were analyzed . among the five samples of non - neoplastic colorectal mucosae , the expression of cd-9 was negative or weakly positive in almost all epithelial cells ( fig . on the other hand , more than 50% of stained stromal immune cells in non - neoplastic colorectal tissues exhibited strong expression of cd9 throughout the entire samples and were classified as the high expression group . t - cd9 ( + ) was detected in 175 of 304 crcs ( 57.6% ) , and i - cd9 ( + ) was detected in 265 of 305 cases ( 86.9% ) . one hundred fifty - four cases ( 50.7% ) showed both t - cd9 ( + ) and i - cd9 ( + ) and 19 cases ( 6.3% ) exhibited both t - cd9 ( ) and i - cd9 ( ) . no statistically significant differences were noted in clinicopathological parameters including age , sex , stage , gross type , location , pt / pn categories , or frequency of angiolymphatic and perineural invasion between the t - cd9 ( + ) and t - cd9 ( ) groups ( table 1 ) . in kaplan - meier survival analysis , the t - cd9 ( + ) group showed a tendency for better dfs than the t - cd9 ( ) group , but statistical significance was not reached ( p=.057 ) ( fig . we additionally subdivided cases into two groups according to tumor location : right - sided tumors ( n=89 ) and left - sided tumors ( n=215 ) . based on the previous literature , crcs were classified into right- and left - sided tumors relative to the splenic flexure . in other words , tumors arising proximal to the splenic flexure ( cecum , ascending colon , and transverse colon ) are considered right - sided , and tumors distal to the splenic flexure ( descending colon , sigmoid colon , and rectum ) are considered left - sided . in left - sided tumors , dfs was significantly better in the t - cd9 ( + ) group compared to the t - cd9 ( ) group ( p=.021 ) ( fig . however , in right - sided tumors , no significant difference was observed between the two groups ( p=.453 ) ( fig . multivariate analysis including stage , lymphatic invasion , vascular invasion , perineural invasion and t - cd9which were significant prognostic factors in univariate analysis revealed that the stage ( p=.002 ) was still an independent prognostic predictor for dfs , whereas t - cd9 expression failed to correlate with prognosis , with no difference between t - cd9 ( + ) and t - cd9 ( ) groups in patients with left - sided crc ( p=.167 ) ( table 2 ) . i - cd9 ( + ) crcs significantly correlated with well / moderately differentiation ( p=.014 ) compared to i - cd9 ( ) crcs . in kaplan - meier survival analysis , the i - cd9 ( + ) group had a tendency towards worse dfs compared to the i - cd9 ( ) group , but no significant difference was found ( p=.156 ) ( fig . when the cases were subdivided into left- and right - sided crc groups , i - cd9 ( ) suggested a potential protective role in left - sided tumors ( p=.233 ) , but statistical significance was not reached ( fig . , no statistical significance was observed between the two groups ( p=.643 ) ( fig . kaplan - meier survival analysis found the longest dfs for patients in the t - cd9 ( + ) /i - cd9 ( ) group , whereas the t - cd9 ( )/i - cd9 ( + ) group had the shortest dfs , but statistical significance was not reached ( p=.054 ) ( fig . when the patients were divided into left- and right - sided crc groups , increased survival differences between subgroups was observed in left - sided tumors ( p=.030 ) ( fig . using a cox proportional hazards model , combined t - cd9/i - cd9 expression was not an independent prognostic factor of dfs in left - sided crcs ( p=.199 ) ( table 2 ) . many previous in vitro and in vivo models of cancer cell lines have contributed to our understanding of the role of cd9 and the associated functional complexes with other members of the tetraspanin family and various integrins [ 14 - 16,19 ] . high expression of cd9 in tumor cells has been associated with favorable prognosis , reduced metastatic potential , and cancer growth in various tumor types , including lung , breast , prostate , stomach , pancreas , and colon carcinoma [ 4,20 - 22 ] . stated that reduced expression of cd9 by reverse transcription polymerase chain reaction is linked to poor prognosis in colon cancer . our data demonstrated that patients with high expression of cd9 in tumor cells ( t - cd9 [ + ] ) tended to have a longer dfs , although the difference was not statistically significant ( p=.057 ) . demonstrated that cd9 mrna is consistently expressed in non - neoplastic colonic tissues adjacent to tumors and its expression level was higher in normal colonic tissues than in tumors in six normal / tumor paired samples using northern blot analysis . also showed that strong cd9 expression was detected in normal colonic epithelium by ihc . on the contrary , negative or weakly positive expression of cd9 these contradictory finding of the non - neoplastic cd9 expression may be attributed to differing experimental techniques such as northern blot analysis and ihc , as well as variation in the different types of cd9 antibodies . notably , the protective prognostic role of t - cd9 on dfs was demonstrated in the relatively homogenous left - sided crc group in this study with statistical significance ( p=.021 ) . however , in the right - sided tumor group , no significance of t - cd9 was observed in dfs , but this finding was considered to have limited reliability due to the small number of cases ( n=89 ) . the prognostic effect of t - cd9 is speculated to be attributed to different molecular carcinogenic mechanisms between right- and left - sided crcs . in left - sided crcs , the majority of tumors develop through a chromosomal instability pathway known as the adenoma - carcinoma sequence . serrated morphology tumors and cpg island methylator phenotypes are seen more frequently in right - sided tumors than in left - sided crcs . these findings suggest that even in the same tumor , the prognostic implication of cd9 can be different depending on the underlying molecular mechanisms of carcinogenesis . extended study is required to verify the hypothesis presented above and the location based variation of cd9 roles in tumor progression . in survival analysis , i - cd9 ( + ) showed a tendency to be associated with a high recurrence rate in left - sided crcs ( p=.156 ) and this finding is contrary to the case of t - cd9 . demonstrated that cd9-expressing tumor - infiltrating immune cells , especially tumor - associated natural killer cells , these findings are consistent with our results regarding i - cd9 . to the best of our knowledge , this is the first report to identify a differential prognostic role of cd9 expression in tumor cells and immune cells in crcs . stated that a therapeutic agent targeting tetraspanin , especially cd9 , using monoclonal abs such as alb6 and pain13 , might be beneficial for cancer patients . however , several researchers have suggested the contradictory finding of t - cd9 in various cancer types including multiple myeloma , fibrosarcoma , and even in gastric carcinoma , showing that cd9 expression of tumor cells has a positive influence on tumor proliferation and invasion by enhancing hb - egf / egfr interaction or metalloproteinases-2 secretion . in addition , our study also demonstrated an opposite effect of t - cd9 and i - cd9 on tumor progression . therefore , future studies should focus on the varying mechanisms and the therapeutic response of i - cd9 and t - cd9 in crcs . in combined survival analysis of t - cd9 and i - cd9 , tumors with high t - cd9 expression plus low i - cd9 expression showed a tendency to have a longer dfs compared with ( 1 ) tumors with concurrent high or low expression of t - cd9 and i - cd9 , or ( 2 ) tumors with low t - cd9 expression plus high i - cd9 expression ( p=.054 ) , and a similar result was found in left - sided crcs with statistical significance ( p=.030 ) . these findings suggested that the combined evaluation of t - cd9 and i - cd9 is required to determine the comprehensive prognostic effect of cd9 in crcs . in conclusion , high expression of cd9 in tumor cells tends to be inversely related to tumor recurrence , especially in left - sided crcs . however , a different tendency on dfs was demonstrated in immune cells regardless of tumor location . additional studies are necessary , with an emphasis on differential expression of cd9 in tumor cells and immune cells .

big data , that is , massive bodies of digital data collected from all sorts of sources that are too large , raw , or unstructured for analysis using conventional relational database techniques , is the buzzword of the day for the research community , businesses , and most recently , government . almost 90% of the global data existing today has been created during the past two years , as 2.5 quintillion bytes of data are generated every day . even though businesses are leading big data applications , the public sector has begun to be very active , particularly in the search for effective uses of big data with the aim of serving citizens better and overcoming national challenges such as skyrocketing healthcare costs , job creation , natural disasters , terrorism , and other concerns . for instance , many business reports and white papers focusing on healthcare have insisted that , if properly applied , big data could be used to guarantee public health , determine and implement appropriate treatment paths for patients , support clinical improvement , monitor the safety of healthcare systems , assure managerial control , and promote health system accountability to the public . however , several researchers have argued that it would be somewhat difficult to ensure that big data plays a central role in a health system 's ability to secure improved health for its users . in particular , they are concerned that big data entails many new challenges regarding its complexity , security , and privacy risks , as well as the need for new technologies and human skills . regarding these various perspectives , the main purpose of this study is to explore whether the big data can effectively reduce healthcare concerns such as the selection of appropriate treatment paths , improvement of healthcare systems , and so on . we discuss the current healthcare problems in developed and developing countries . in the third section , we review the main attributes of big data in order to understand the potential of practical applications of big data to the healthcare environment . in the fourth section , we present the current practices and technologies related to big data in the healthcare environment . in the fifth section , we insightfully discuss the different attributes and interpretations of big data between the business sector and the medical sector since the two environments have to adopt different approaches and practices . this comparative analysis provides more insight into how big data can be effectively implemented in the medical environment and be used to improve healthcare - related concerns . in the last section , we discuss the results and elaborate on the implications , limitations , and the possibilities for further study . healthcare is one of the top social and economic issues in many countries , such as the united states , the uk , south korea , and even middle - income countries . in the united states , although healthcare expenditures are the highest of any developed country , at 15.3% of gdp , such expenditures do not improve health outcomes . regarding this issue , many researchers have found that the united states does not spend healthcare money efficiently , arguing that the rising cost of medical care and health insurance is impacting the livelihood of many americans . according to the commonwealth fund biennial health insurance survey in 2007 , nearly 50 million americans did not have health insurance , while another 25 million were underinsured - the underinsured are those who have health insurance but still struggle to pay their healthcare bills . figure 1 indicates the percentages of persons in families with certain financial burdens related to medical care in the united states . in the period january - june 2011 , one in every five persons was a member of a household experiencing problems in paying its medical bills , while one in four persons was in a family paying medical bills over time . notably , one in 10 persons was in a family that had medical bills they were unable to pay at all . the national health service ( nhs ) in the uk provides public healthcare to all permanent residents ( about 58 million people ) . healthcare coverage is free at the point of need and is paid for by general taxation . around 8.4% of the uk 's gross domestic product ( i.e. , approximately 0.18984 trillion gbp ) is spent on healthcare . however , while the nhs has remained the dominant provider of healthcare in the uk , a growing number of people cover their healthcare by private health insurance . in recent years , public healthcare in the uk has faced major problems following a cut in the health budget ( of around $ 29 billion ) by the nhs in 2010 . south korea has one of the most advanced information technology ( it ) infrastructures in the world , and the application of it in health systems is rapidly progressing from computerization to information systems , ubiquitous systems , and smart systems . however , a major problem concerning healthcare resources lies in the regional disparities between medical services . most private medical facilities are located in urban areas , and around 90% of physicians are concentrated in cities , while ( only ) 80% of the population lives in urban areas . south korea is becoming an aging society faster than any other country , including japan . with regard to the increase in the elderly population , there has been an increase in medical expenditure for chronic degenerative diseases , which has become a large social burden . in recent years , the south korean government has attempted to reduce the financial burden through comprehensive health care reforms encompassing the expansion of healthcare facilities and the introduction of the long - term care insurance program . the healthcare system in japan has led to national debt and could bankrupt the country . therefore , many researches argue that the japanese healthcare system is very inefficient and wasteful . for instance , patients often stay in the hospital much longer than they need to , undergoing a lot of unnecessary tests simply because the health insurance will pay for all of it . thirty percent of the healthcare budget is spent on drugs , compared to 11% in the united states . an additional concern about the healthcare system in japan is that the country does not have a system of family doctors to provide continuous , comprehensive , person - centered care in the community . many developing countries are faced with healthcare concerns , such as health - financing reforms and effective analysis of healthcare - related information . in particular , many low and middle - income countries are looking at health - financing reforms because they have failed to deliver care through their own public system . their rigid and bureaucratic systems have not been able to motivate healthcare - related staff or improve efficiency in the delivery of services . their governments need to access and analyze information about the available resources in the private and public sectors in order to formulate the necessary health - financing reforms . before discussing big data practice in the healthcare environment , it is important to look at what the main attributes of big data are . today 's big data era is based on the following stages in the evolution of it - enabled decision support systems : 1 ) data processing in the 1960s , 2 ) information applications in the 1970s-1980s , 3 ) decision support models in the 1990s , 4 ) data warehousing and mining in the 2000s , and 5 ) emergence of the big data era ( after 2010 ) . the big data era is still in its early stage as most of the technologies and analytical applications emerged around 2010 . many white papers and articles have described the attributes of and challenges posed by big data using the ' three v 's ' , namely , volume , velocity , and variety . in particular , volume is the primary attribute of big data since organizations generate terabytes or petabytes of data while conducting their business and complying with government regulations . variety describes a chief characteristic of today 's data , i.e. , data comes in various forms : structured data ( traditional databases , like sql ) ; semi - structured data ( data that have tags and markers , without a formal structure like database ) ; and unstructured data ( i.e. , unorganized data ) . velocity refers to the enormous frequency with which today 's data is generated , delivered , and processed . in other words , since big data is so large , it is hard to manage , and even more difficult to extract value out of , as conventional information technologies are not effective in managing it . since the primary concept of big data has evolved to imply not only the size of the data , but also the process of creating value out of it , big data , which has become synonymous with business intelligence ( bi ) , business analytics , and data mining , has brought about a shift in bi from reporting and decision support to prediction and next - move decisions . for instance , hadoop , an open - source platform , is the most widely applied technology . according to business reports , hodoop helps solve such problems as storage and access , the management of overheads associated with large data sets , and the operation of very fast parallel processing . however , hadoop is challenging for many businesses , especially small and medium - sized firms , as its applications require expertise and experience that are not yet widely available and for which the relevant work may need to be outsourced . finding the right talents to analyze big data is one of the biggest challenges for business organizations as the skills required are not simple or just technology - oriented . furthermore , searching for competent data scientists ( professionals with skills in data mining , visualization , analysis , manipulation , and discovery ) is too difficult and expensive for most organizations . there are other commercially available technologies for processing big data , one of which is the cassandra database , a dynamo - based tool that can store two million columns in a single row , thus allowing the utilization of a large amount of data without requiring prior knowledge of data formatting . thus , another challenge for business organizations is to make the right decision about which of the technologies is best for them , i.e. , either open - source technology such as hadoop , or commercial implementations such as cassandra , cloudera , hortonworks , mapr , and so forth . there are other big data issues involving integration and costs as well as data security and compliance . healthcare is the best example , showing how to create , store , and manage the flow of big data in medical service organizations where compliance issues arise . most of the data technologies today , including hadoop and cassandra , do not have sufficient security tools . in the next section , we will look at the current practices of big data in the healthcare environment in order to understand how big data can be used in the healthcare environment more effectively . physicians have traditionally used their judgment when making treatment decisions , but in the last few years there has been a move toward evidence - based medicine . evidence - based medicine involves systematically reviewing clinical data and making treatment decisions based on the best available information . in recent years , aggregating individual medical data sets into big data algorithms provides the most robust evidence that helps patients , physicians , and other healthcare stakeholders identify value and opportunities . thus , an era of open information ( e.g. , big data ) in healthcare is now under way . in march 2012 , the obama administration launched a $ 200 million " big data research and development initiative , " the main aim of which is to transform the use of big data for scientific discovery and biomedical research among other areas , with the participation of several federal departments and agencies such as the white house office of science and technology policy , the national science foundation ( nsf ) , the national institute of health ( nih ) , the department of defense , health and human services , and various other agencies and organizations . the main objectives of this initiative are the following : 1 ) to advance state - of - the - art core technologies of the big data era ; 2 ) to accelerate the pace of discovery in science and engineering , strengthen national security , and transform teaching and learning ; and 3 ) to expand the workforce needed to develop and use big data technologies . for instance , the nih has accumulated 200 terabytes of data on human genetic variations in a cloud system , amazon web services , to enable researchers to access and analyze a huge body of data without the need for their own supercomputing capability . the nsf joined the nih in starting the core techniques and technologies for advancing big data science & engineering ( bigdata ) to advance the core scientific and technological means of managing , analyzing , visualizing , and extracting useful information from large , diverse , distributed , and heterogeneous datasets . in south korea , a big data task - force was created to play the leading role in utilizing big data and building the necessary infrastructures . the goals of this organization are to establish a pan - governmental big data network and analysis systems , promote data convergence between the government and the private sector , build a public data diagnosis system , improve the laws for the system , produce talented professionals and retrain them , guarantee the privacy and security of personal information , develop big data infrastructure technologies , and develop big data management and analytical technologies . the korean bio - information center plans to operate the national dna management system which , by integrating massive dna and various types of medical patient information , will provide customized diagnosis and medical treatment to patients . in recent times , the healthcare industry has used big data analytics to better detect diseases and aid medical research . for instance , hiv researchers in the european union worked with ibm , applying big data tooling to perform clinical genomic analysis . by assisting hiv researchers in optimizing therapies for patients and participating in the euresist project , ibm big data tooling played a key role in helping researchers understand clinical data from numerous countries in order to discover treatments based on accumulated empirical data . the obama administration proposed " health 2.0 " to manage patients , medical institutions , medical insurance , and government efficiently . in " health 2.0 " , it technologies and networking patients , medical institutions , and medical insurance , are applied to healthcare to cut down medical service costs , and establish more convenient policies . pillbox 's objective is to provide accurate information about specific medicines a user wants to know about . a pillbox user describes the numbers or letters written on a pill along with its color , size , and shape and obtains the information about its effects . pillbox service is designed to be user - friendly even for the elderly , or those who are unfamiliar with internet . pillbox service is expected to reduce the cost of identifying pills and their effects , offer information about medicine using big data , and help maintain a clean medical system by checking the sale of medicine and medical records . an increasing number and variety of organizations , including healthcare - related companies , are beginning to apply big data to address multiple healthcare challenges , such as supporting research ( e.g. , genomics ) , transforming data to information , and assisting providers to improve patient care . for instance , genomics has been at the cutting edge of the big data revolution in the life sciences field . genome health solutions applies its expertise and network of physicians and technology providers to integrate personal genomics and streamline care delivery to make possible a new standard of care aimed at improving outcomes for patients with cancer and other diseases . dnanexus provides a cloud - based , community - inspired , collaborative , and scalable data technology platform that contributes to next - generation sequencing data management , analysis , and visualization . in particular , dnanexus enables customers to store , manage , analyze , and visualize next - generation dna sequencing data through a web - based cloud service model . regarding the rapidly rising flood of healthcare data , transforming data to information is an important stepping stone to enabling data - driven healthcare . predixion software uses cloud - based predictive software to explain patterns in hospital datasets to reduce readmissions and prevent hospital - acquired conditions . practice fusion is a free , cloud - based electronic medical record platform for medical practices that aggregates population data across multiple sites to improve clinical research and public health analysis . as many white papers and researchers have insisted , an era of open information in healthcare is now under way ( e.g. , ) . in particular , many consulting firms ( e.g. , ibm , sas , and mckinsey ) have already experienced a decade of progress in digitizing medical records , as pharmaceutical companies and other organizations have aggregated years of research and development data in electronic databases . in particular , many governments and other public healthcare stakeholders have accelerated the move toward transparency by making decades of stored data usable , searchable , and actionable by the healthcare sector as well . however , as we looked at the practical application of big data in the healthcare environment , we found that healthcare big data has different attributes and values and poses different challenges compared to the business sector . the real difference of healthcare data is its scale and scope , which have been growing steadily for years . each government agency or department , or healthcare stakeholder typically has its own warehouse ( a so - called " silo " ) of confidential or public healthcare - related information . security , the primary attribute of big data for governments or healthcare stakeholders , describes the extra care needed in using healthcare data where security , privacy , authority , and legitimacy issues are concerned . the attribute of ' variety ' of healthcare data , as for business organizations , refers to the existence of data in all forms : structured , semi - structured , and unstructured . however , healthcare data have one main difference from business data , i.e. , the majority of healthcare data are structured ( e.g. , electronic health records ) rather than semi - structured or unstructured . although the purpose of big data management is similar , a better understanding of the problems , the values sought , and the challenges involved differ considerably between business firms and healthcare organizations . business firms use big data to deal with customers ' needs and behavior patterns , develop unique core competencies , and create innovative products or services , whereas governments and healthcare stakeholders use big data and predictive analytics to search for sustainable solutions to such issues as tracking public health , determining and implementing appropriate treatment paths for patients , supporting clinical improvements , monitoring the safety of healthcare systems , assuring managerial control , and promoting health system accountability to the citizens . choosing and implementing the right technologies to extract value , and finding skilled personnel are constant challenges involving big data for both businesses and healthcare . however , the challenges for healthcare are more severe as they necessarily involve breaking down the healthcare - related silos for integration , establishing sufficient capacity for the control towers ( e.g. , the federal data center in the united states ) , and implementing regulations on security and compliance . a summary of the comparisons of big data with regard to differences in the attributes , values , and challenges faced by business organizations ( the " three v 's " ) and the healthcare sector ( the " two s 's and one v " [ variety ] ) is shown in figure 2 . given the differences in their business models and dataset attributes , the big data application projects implemented and/or being initiated by the healthcare side differ considerably from those in business . today , the big data issue looms large over many healthcare stakeholders in developed and developing countries . everyone seems to have realized that the capability to manage and create value from today 's large stream of data , from various sources and in many forms ( structured / stored , semi - structured / tagged ; unstructured / in - motion ) , represents the new competitive differentiation . as most governments and healthcare stakeholders are currently operating big data projects or are in the planning stage , they need a step - by - step approach to setting the right goals and entertaining realistic expectations regarding big data . their success will depend on the ability to develop technical capabilities to effectively integrate and analyze information - using new technologies ( e.g. , hadoop ) , develop the right support systems ( such as the establishment of big data control towers ) , and support effective decision making through analytics . by providing an overview of the current state of big data applications in the healthcare environment , this study has explored the current challenges that governments and healthcare stakeholders are facing , and the opportunities presented by big data . such insights could also help follower countries or healthcare stakeholders in their plans for deploying big data in the resolution of healthcare issues . the advantage for such follower countries and healthcare stakeholders is that they can possibly leapfrog the leaders ' big data applications by conducting a careful analysis of the leaders ' successes and failures , as well as exploiting the expected future opportunities in mobile services . this paper offers the following observations and insights - for follower countries or healthcare stakeholders - concerning the current state of big data applications in the medical sector . first , all big data projects in leading countries ' governments and healthcare industries have similar general common goals , such as the provision of easy and equal access to public services , better citizens ' healthcare services , and the improvement of medical - related concerns . however , each government or healthcare stakeholder has its own priorities , opportunities , and threats , based on its country 's unique environment ( e.g. , healthcare expenditures in the united states , the inefficient and wasteful healthcare system in japan , regional disparities in the healthcare resources in korea , etc . ) which big data projects must address ( e.g. , ) . second , for medical data that cuts across departmental boundaries , a top - down approach is needed to effectively manage and integrate big data . governments and healthcare stakeholders should establish big data control towers to integrate accumulated datasets , whether structured or unstructured , from each silo . additionally , governments and healthcare stakeholders need to establish an advanced analytics agency , which will be tasked with developing strategies on how big data could be best managed through new technology platforms and analytics as well as how to secure skilled professional staff to use the new tools and techniques . third , real - time analysis of in - motion big data should be carried out , while protecting privacy and security . thus , governments and healthcare stakeholders should explore new technological playgrounds , such as cloud computing , advanced analytics , security technologies , legislation , etc . for instance , because the volume of healthcare data will eventually amount to 15 zettabytes worth of information ( e.g. , one zettabyte is equal to 1000 exabytes , and one exabyte is the equivalent of 1 million terabytes ) , government and healthcare - related stakeholders should establish technological playgrounds such as cloud computing technology . fourth , with regard to the improvement of healthcare systems , leading big data governments appear to have different goals and priorities ; therefore , they use different sets of data management systems , technologies , and analytics . while such information is not readily available in the literature , the main concerns with big data applications among these countries and companies converge on the following : security , speed , interoperability , analytics capabilities , and the lack of competent professionals . fifth , governments and healthcare stakeholders should collaborate with " ict big brothers " such as ibm , sas , emc , and other entities that possess a great deal of expertise and technologies . for instance , amazon web services already hosts many public datasets , such as the united states and japanese census data , in addition to large amounts of genome and medical data . big data medicine is still largely unproven , but that is not stopping several medical centers and analytics vendors from jumping in with both feet . finally , this study is limited in that the practical applications of big data for investigating healthcare issues have not yet been fully demonstrated due to the dearth of practice . with regard to future study , practitioners and researchers should carefully look at and accumulate information with regard to the practical applications of big data in order to determine the best ways of using big data in healthcare issues .

objectivesthe main purpose of this study was to explore whether the use of big data can effectively reduce healthcare concerns , such as the selection of appropriate treatment paths , improvement of healthcare systems , and so on.methodsby providing an overview of the current state of big data applications in the healthcare environment , this study has explored the current challenges that governments and healthcare stakeholders are facing as well as the opportunities presented by big data.resultsinsightful consideration of the current state of big data applications could help follower countries or healthcare stakeholders in their plans for deploying big data to resolve healthcare issues . the advantage for such follower countries and healthcare stakeholders is that they can possibly leapfrog the leaders ' big data applications by conducting a careful analysis of the leaders ' successes and failures and exploiting the expected future opportunities in mobile services.conclusionsfirst , all big data projects undertaken by leading countries ' governments and healthcare industries have similar general common goals . second , for medical data that cuts across departmental boundaries , a top - down approach is needed to effectively manage and integrate big data . third , real - time analysis of in - motion big data should be carried out , while protecting privacy and security .

I. Introduction II. Healthcare Problems in Developed and Middle-Income Countries III. Attributes of Big Data and Challenges IV. Big Data Applications in the Healthcare Environment V. Different Attributes of Big Data between Business Sector and Medical Sector VI. Conclusion

big data , that is , massive bodies of digital data collected from all sorts of sources that are too large , raw , or unstructured for analysis using conventional relational database techniques , is the buzzword of the day for the research community , businesses , and most recently , government . almost 90% of the global data existing today has been created during the past two years , as 2.5 quintillion bytes of data are generated every day . even though businesses are leading big data applications , the public sector has begun to be very active , particularly in the search for effective uses of big data with the aim of serving citizens better and overcoming national challenges such as skyrocketing healthcare costs , job creation , natural disasters , terrorism , and other concerns . for instance , many business reports and white papers focusing on healthcare have insisted that , if properly applied , big data could be used to guarantee public health , determine and implement appropriate treatment paths for patients , support clinical improvement , monitor the safety of healthcare systems , assure managerial control , and promote health system accountability to the public . however , several researchers have argued that it would be somewhat difficult to ensure that big data plays a central role in a health system 's ability to secure improved health for its users . in particular , they are concerned that big data entails many new challenges regarding its complexity , security , and privacy risks , as well as the need for new technologies and human skills . regarding these various perspectives , the main purpose of this study is to explore whether the big data can effectively reduce healthcare concerns such as the selection of appropriate treatment paths , improvement of healthcare systems , and so on . we discuss the current healthcare problems in developed and developing countries . in the third section , we review the main attributes of big data in order to understand the potential of practical applications of big data to the healthcare environment . in the fourth section , we present the current practices and technologies related to big data in the healthcare environment . in the fifth section , we insightfully discuss the different attributes and interpretations of big data between the business sector and the medical sector since the two environments have to adopt different approaches and practices . this comparative analysis provides more insight into how big data can be effectively implemented in the medical environment and be used to improve healthcare - related concerns . in the last section , we discuss the results and elaborate on the implications , limitations , and the possibilities for further study . healthcare is one of the top social and economic issues in many countries , such as the united states , the uk , south korea , and even middle - income countries . in the united states , although healthcare expenditures are the highest of any developed country , at 15.3% of gdp , such expenditures do not improve health outcomes . according to the commonwealth fund biennial health insurance survey in 2007 , nearly 50 million americans did not have health insurance , while another 25 million were underinsured - the underinsured are those who have health insurance but still struggle to pay their healthcare bills . figure 1 indicates the percentages of persons in families with certain financial burdens related to medical care in the united states . in the period january - june 2011 , one in every five persons was a member of a household experiencing problems in paying its medical bills , while one in four persons was in a family paying medical bills over time . the national health service ( nhs ) in the uk provides public healthcare to all permanent residents ( about 58 million people ) . around 8.4% of the uk 's gross domestic product ( i.e. however , while the nhs has remained the dominant provider of healthcare in the uk , a growing number of people cover their healthcare by private health insurance . in recent years , public healthcare in the uk has faced major problems following a cut in the health budget ( of around $ 29 billion ) by the nhs in 2010 . south korea has one of the most advanced information technology ( it ) infrastructures in the world , and the application of it in health systems is rapidly progressing from computerization to information systems , ubiquitous systems , and smart systems . however , a major problem concerning healthcare resources lies in the regional disparities between medical services . most private medical facilities are located in urban areas , and around 90% of physicians are concentrated in cities , while ( only ) 80% of the population lives in urban areas . with regard to the increase in the elderly population , there has been an increase in medical expenditure for chronic degenerative diseases , which has become a large social burden . in recent years , the south korean government has attempted to reduce the financial burden through comprehensive health care reforms encompassing the expansion of healthcare facilities and the introduction of the long - term care insurance program . the healthcare system in japan has led to national debt and could bankrupt the country . for instance , patients often stay in the hospital much longer than they need to , undergoing a lot of unnecessary tests simply because the health insurance will pay for all of it . thirty percent of the healthcare budget is spent on drugs , compared to 11% in the united states . an additional concern about the healthcare system in japan is that the country does not have a system of family doctors to provide continuous , comprehensive , person - centered care in the community . many developing countries are faced with healthcare concerns , such as health - financing reforms and effective analysis of healthcare - related information . their rigid and bureaucratic systems have not been able to motivate healthcare - related staff or improve efficiency in the delivery of services . their governments need to access and analyze information about the available resources in the private and public sectors in order to formulate the necessary health - financing reforms . before discussing big data practice in the healthcare environment , it is important to look at what the main attributes of big data are . today 's big data era is based on the following stages in the evolution of it - enabled decision support systems : 1 ) data processing in the 1960s , 2 ) information applications in the 1970s-1980s , 3 ) decision support models in the 1990s , 4 ) data warehousing and mining in the 2000s , and 5 ) emergence of the big data era ( after 2010 ) . the big data era is still in its early stage as most of the technologies and analytical applications emerged around 2010 . many white papers and articles have described the attributes of and challenges posed by big data using the ' three v 's ' , namely , volume , velocity , and variety . in particular , volume is the primary attribute of big data since organizations generate terabytes or petabytes of data while conducting their business and complying with government regulations . , data comes in various forms : structured data ( traditional databases , like sql ) ; semi - structured data ( data that have tags and markers , without a formal structure like database ) ; and unstructured data ( i.e. velocity refers to the enormous frequency with which today 's data is generated , delivered , and processed . in other words , since big data is so large , it is hard to manage , and even more difficult to extract value out of , as conventional information technologies are not effective in managing it . since the primary concept of big data has evolved to imply not only the size of the data , but also the process of creating value out of it , big data , which has become synonymous with business intelligence ( bi ) , business analytics , and data mining , has brought about a shift in bi from reporting and decision support to prediction and next - move decisions . according to business reports , hodoop helps solve such problems as storage and access , the management of overheads associated with large data sets , and the operation of very fast parallel processing . finding the right talents to analyze big data is one of the biggest challenges for business organizations as the skills required are not simple or just technology - oriented . furthermore , searching for competent data scientists ( professionals with skills in data mining , visualization , analysis , manipulation , and discovery ) is too difficult and expensive for most organizations . there are other commercially available technologies for processing big data , one of which is the cassandra database , a dynamo - based tool that can store two million columns in a single row , thus allowing the utilization of a large amount of data without requiring prior knowledge of data formatting . thus , another challenge for business organizations is to make the right decision about which of the technologies is best for them , i.e. , either open - source technology such as hadoop , or commercial implementations such as cassandra , cloudera , hortonworks , mapr , and so forth . there are other big data issues involving integration and costs as well as data security and compliance . healthcare is the best example , showing how to create , store , and manage the flow of big data in medical service organizations where compliance issues arise . in the next section , we will look at the current practices of big data in the healthcare environment in order to understand how big data can be used in the healthcare environment more effectively . in recent years , aggregating individual medical data sets into big data algorithms provides the most robust evidence that helps patients , physicians , and other healthcare stakeholders identify value and opportunities . in march 2012 , the obama administration launched a $ 200 million " big data research and development initiative , " the main aim of which is to transform the use of big data for scientific discovery and biomedical research among other areas , with the participation of several federal departments and agencies such as the white house office of science and technology policy , the national science foundation ( nsf ) , the national institute of health ( nih ) , the department of defense , health and human services , and various other agencies and organizations . the main objectives of this initiative are the following : 1 ) to advance state - of - the - art core technologies of the big data era ; 2 ) to accelerate the pace of discovery in science and engineering , strengthen national security , and transform teaching and learning ; and 3 ) to expand the workforce needed to develop and use big data technologies . the nsf joined the nih in starting the core techniques and technologies for advancing big data science & engineering ( bigdata ) to advance the core scientific and technological means of managing , analyzing , visualizing , and extracting useful information from large , diverse , distributed , and heterogeneous datasets . in south korea , a big data task - force was created to play the leading role in utilizing big data and building the necessary infrastructures . the goals of this organization are to establish a pan - governmental big data network and analysis systems , promote data convergence between the government and the private sector , build a public data diagnosis system , improve the laws for the system , produce talented professionals and retrain them , guarantee the privacy and security of personal information , develop big data infrastructure technologies , and develop big data management and analytical technologies . in recent times , the healthcare industry has used big data analytics to better detect diseases and aid medical research . for instance , hiv researchers in the european union worked with ibm , applying big data tooling to perform clinical genomic analysis . by assisting hiv researchers in optimizing therapies for patients and participating in the euresist project , ibm big data tooling played a key role in helping researchers understand clinical data from numerous countries in order to discover treatments based on accumulated empirical data . the obama administration proposed " health 2.0 " to manage patients , medical institutions , medical insurance , and government efficiently . in " health 2.0 " , it technologies and networking patients , medical institutions , and medical insurance , are applied to healthcare to cut down medical service costs , and establish more convenient policies . a pillbox user describes the numbers or letters written on a pill along with its color , size , and shape and obtains the information about its effects . pillbox service is expected to reduce the cost of identifying pills and their effects , offer information about medicine using big data , and help maintain a clean medical system by checking the sale of medicine and medical records . an increasing number and variety of organizations , including healthcare - related companies , are beginning to apply big data to address multiple healthcare challenges , such as supporting research ( e.g. , genomics ) , transforming data to information , and assisting providers to improve patient care . for instance , genomics has been at the cutting edge of the big data revolution in the life sciences field . dnanexus provides a cloud - based , community - inspired , collaborative , and scalable data technology platform that contributes to next - generation sequencing data management , analysis , and visualization . in particular , dnanexus enables customers to store , manage , analyze , and visualize next - generation dna sequencing data through a web - based cloud service model . regarding the rapidly rising flood of healthcare data , transforming data to information is an important stepping stone to enabling data - driven healthcare . practice fusion is a free , cloud - based electronic medical record platform for medical practices that aggregates population data across multiple sites to improve clinical research and public health analysis . , ibm , sas , and mckinsey ) have already experienced a decade of progress in digitizing medical records , as pharmaceutical companies and other organizations have aggregated years of research and development data in electronic databases . in particular , many governments and other public healthcare stakeholders have accelerated the move toward transparency by making decades of stored data usable , searchable , and actionable by the healthcare sector as well . however , as we looked at the practical application of big data in the healthcare environment , we found that healthcare big data has different attributes and values and poses different challenges compared to the business sector . the real difference of healthcare data is its scale and scope , which have been growing steadily for years . each government agency or department , or healthcare stakeholder typically has its own warehouse ( a so - called " silo " ) of confidential or public healthcare - related information . security , the primary attribute of big data for governments or healthcare stakeholders , describes the extra care needed in using healthcare data where security , privacy , authority , and legitimacy issues are concerned . the attribute of ' variety ' of healthcare data , as for business organizations , refers to the existence of data in all forms : structured , semi - structured , and unstructured . , the majority of healthcare data are structured ( e.g. although the purpose of big data management is similar , a better understanding of the problems , the values sought , and the challenges involved differ considerably between business firms and healthcare organizations . business firms use big data to deal with customers ' needs and behavior patterns , develop unique core competencies , and create innovative products or services , whereas governments and healthcare stakeholders use big data and predictive analytics to search for sustainable solutions to such issues as tracking public health , determining and implementing appropriate treatment paths for patients , supporting clinical improvements , monitoring the safety of healthcare systems , assuring managerial control , and promoting health system accountability to the citizens . choosing and implementing the right technologies to extract value , and finding skilled personnel are constant challenges involving big data for both businesses and healthcare . however , the challenges for healthcare are more severe as they necessarily involve breaking down the healthcare - related silos for integration , establishing sufficient capacity for the control towers ( e.g. , the federal data center in the united states ) , and implementing regulations on security and compliance . a summary of the comparisons of big data with regard to differences in the attributes , values , and challenges faced by business organizations ( the " three v 's " ) and the healthcare sector ( the " two s 's and one v " [ variety ] ) is shown in figure 2 . given the differences in their business models and dataset attributes , the big data application projects implemented and/or being initiated by the healthcare side differ considerably from those in business . today , the big data issue looms large over many healthcare stakeholders in developed and developing countries . everyone seems to have realized that the capability to manage and create value from today 's large stream of data , from various sources and in many forms ( structured / stored , semi - structured / tagged ; unstructured / in - motion ) , represents the new competitive differentiation . as most governments and healthcare stakeholders are currently operating big data projects or are in the planning stage , they need a step - by - step approach to setting the right goals and entertaining realistic expectations regarding big data . their success will depend on the ability to develop technical capabilities to effectively integrate and analyze information - using new technologies ( e.g. , hadoop ) , develop the right support systems ( such as the establishment of big data control towers ) , and support effective decision making through analytics . by providing an overview of the current state of big data applications in the healthcare environment , this study has explored the current challenges that governments and healthcare stakeholders are facing , and the opportunities presented by big data . such insights could also help follower countries or healthcare stakeholders in their plans for deploying big data in the resolution of healthcare issues . the advantage for such follower countries and healthcare stakeholders is that they can possibly leapfrog the leaders ' big data applications by conducting a careful analysis of the leaders ' successes and failures , as well as exploiting the expected future opportunities in mobile services . this paper offers the following observations and insights - for follower countries or healthcare stakeholders - concerning the current state of big data applications in the medical sector . first , all big data projects in leading countries ' governments and healthcare industries have similar general common goals , such as the provision of easy and equal access to public services , better citizens ' healthcare services , and the improvement of medical - related concerns . however , each government or healthcare stakeholder has its own priorities , opportunities , and threats , based on its country 's unique environment ( e.g. , healthcare expenditures in the united states , the inefficient and wasteful healthcare system in japan , regional disparities in the healthcare resources in korea , etc . ) which big data projects must address ( e.g. second , for medical data that cuts across departmental boundaries , a top - down approach is needed to effectively manage and integrate big data . governments and healthcare stakeholders should establish big data control towers to integrate accumulated datasets , whether structured or unstructured , from each silo . additionally , governments and healthcare stakeholders need to establish an advanced analytics agency , which will be tasked with developing strategies on how big data could be best managed through new technology platforms and analytics as well as how to secure skilled professional staff to use the new tools and techniques . third , real - time analysis of in - motion big data should be carried out , while protecting privacy and security . thus , governments and healthcare stakeholders should explore new technological playgrounds , such as cloud computing , advanced analytics , security technologies , legislation , etc . for instance , because the volume of healthcare data will eventually amount to 15 zettabytes worth of information ( e.g. , one zettabyte is equal to 1000 exabytes , and one exabyte is the equivalent of 1 million terabytes ) , government and healthcare - related stakeholders should establish technological playgrounds such as cloud computing technology . fourth , with regard to the improvement of healthcare systems , leading big data governments appear to have different goals and priorities ; therefore , they use different sets of data management systems , technologies , and analytics . while such information is not readily available in the literature , the main concerns with big data applications among these countries and companies converge on the following : security , speed , interoperability , analytics capabilities , and the lack of competent professionals . fifth , governments and healthcare stakeholders should collaborate with " ict big brothers " such as ibm , sas , emc , and other entities that possess a great deal of expertise and technologies . for instance , amazon web services already hosts many public datasets , such as the united states and japanese census data , in addition to large amounts of genome and medical data . big data medicine is still largely unproven , but that is not stopping several medical centers and analytics vendors from jumping in with both feet . finally , this study is limited in that the practical applications of big data for investigating healthcare issues have not yet been fully demonstrated due to the dearth of practice . with regard to future study , practitioners and researchers should carefully look at and accumulate information with regard to the practical applications of big data in order to determine the best ways of using big data in healthcare issues .

nearly all licensed vaccines confer protection against infectious diseases by stimulating the production of pathogen - specific abs by b cells . understanding the nature of a successful ab response is therefore a fundamental step to providing new tools for the design of novel or better vaccines . the isolation and characterization of the ab repertoire produced by antigen - specific b cells has acquired a central importance in the last decade to unravel the response to vaccine antigens . dissecting the basic mechanisms that define the dynamics of the ab responses to vaccination and deepening the knowledge of the correlates of vaccine - induced protection or biological signatures of responsiveness are becoming fundamental in the development of novel vaccines . both memory b cells ( mbcs ) and plasmablasts ( peaking at day 8 after vaccination ) have been used to generate naturally derived antigen - specific mabs . mbcs were shown to be more suitable for this kind of application because of their capability to secrete abs after ebv immortalization and in the presence of a tlr9 ligand and/or allogeneic irradiated mononuclear cells ( traggiai et al . , 2004 ) . usually , total peripheral blood lymphocytes or sorted igg mbcs are cultured and the released abs can be screened for antigen specificity and/or functionality . more recently , it has been discovered that even single plasmablasts can be cultured without immortalization , and they can produce sufficient amounts of abs to allow screening for ab specificity and function ( jin et al . , 2009 ; corti et al . this approach has been particularly successful in isolating nabs from individuals infected by rapidly evolving viral pathogens , leading to the identification of new target molecules that induce the most potent or broadly neutralizing response without prior knowledge of their nature . the power of the characterization of the abs produced by human b cells that were generated in vivo in response to specific infections has been proved so far for different viruses , such as influenza , hcmv , dengue , and rsv ( beltramello et al . , 2010 ; , 2010 , 2013 ; macagno et al . , 2010 ; krause et al . , 2011 ; de alwis et al . , 2012 ) two emblematic examples of how human mabs can be valuable tools to discover or design new antigens come from hcmv and rsv . most studies on hcmv have focused on the viral glycoproteins gb or ghgl as targets of nabs . by screening mabs from an immune donor based on their capacity to neutralize infection in vitro , these abs recognize multiple antigenic sites on a pentameric complex of gh / gl / pul128/pul130/pul131a , which was not previously known to be the target of nabs ( macagno et al . , 2010 ) . in a follow - up study , a pentamer - based vaccine was reported to elicit very high nab titers in mice ( kabanova et al . , 2014 ) , providing evidence of the power of the reverse vaccinology 2.0 approach for the identification and design of improved hcmv vaccine candidates . furthermore , independent structural and immunological studies have also demonstrated the antigenic potential of the hcmv pentamer in mice ( wen et al . , 2014 ; ciferri et al . , a second example comes from the analysis of the nab response to the f protein of rsv . initially , the postfusion f protein of rsv was chosen as a candidate to develop a vaccine because it is a very stable protein and is recognized by a neutralizing mab ( palivizumab ) that is used to prevent rsv infection in newborns ( the impact - rsv study group , 1998 ) . however rsv - specific human b cells transduced with bcl-6 and bcl - xl were used as a source to identify abs that effectively neutralized the virus . by screening b cell culture supernatants , a nab called d25many times more potent than palivizumab was isolated and shown to completely prevent viral replication in an in vivo rsv infection model in cotton rats ( kwakkenbos et al . , 2010 ) . in a subsequent study , it was shown that d25 and two additional highly potent rsv - specific nabs were specific for the prefusion form of rsv f ( mclellan et al . , 2013b ) . furthermore , by analyzing many different mabs isolated from an rsv sero - positive individual , corti et al . ( 2013 ) have also shown that the most potent nabs recognize the prefusion form of the f antigen , suggesting that the prefusion protein could be considered as the most effective vaccine antigen . these examples of isolation and characterization of bnabs induced by infection have highlighted how understanding the mechanisms leading to the elicitation of a protective response can aid the design of more effective vaccines . however , the identification of mabs of the desired specificity usually requires the interrogation of thousands of b cells , with labor - intensive culture and screening steps of the b cell culture supernatants that lead to the isolation of a few dozen abs . moreover , this approach does not allow the gain of substantial information on the shaping of the ig gene repertoire induced by the response to a given pathogen or vaccine . a milestone in understanding the nature and generation of successful ab responses has been the development of ab repertoire sequencing technologies for the identification and generation of recombinant human mabs . the first description of the single - cell rt - pcr method showed the possibility of isolating the ig heavy ( vh ) and light ( vl ) variable chain genes from single b cells , followed by the expression of chimeric murine and rabbit mabs ( babcook et al . , 1996 ) . another hallmark in the field of human immunology has been the demonstration that cloning and expression of human mabs could be achieved through the recovery of the ig sequences from single b cells derived from the bone marrow and blood of healthy donors and by transfection of producer cells with the vh and vl chain genes ( wardemann et al . , furthermore , the possibility of using fluorescently labeled antigens to sort antigen - specific b cells by flow cytometry has facilitated more focused analyses of the ig gene repertoire usage and of the corresponding ab binding and/or functional capability toward targeted antigen - specific and often rare b cell subpopulations after vaccination or infection ( tiller et al . although this methodology allows for sequencing and expression of the abs encoded by a small number of b cells and individuals , it has brought numerous immunological insights to the development of the b cell response to antigens of a different nature . for example , it has been demonstrated that the b cell responses to polysaccharides , such as streptococcus pneumoniae polysaccharides , show high clonality with a limited ig gene usage and can be different in diverse age groups ( kolibab et al . , 2005 ; smith et al . , , greater repertoire diversity has been highlighted between different individuals and even within a single individual at different time points after vaccination with protein antigens , such as influenza hemagglutinin ( ha ) and tetanus toxoid ( tt ; wrammert et al . 2010 ) . however , it is mainly the research in the hiv field that has demonstrated how the characterization of bnabs , isolated from immune donors , and the knowledge of their pathway of generation can provide new insights into how to induce them through new vaccination strategies . novel recombinant and soluble hiv-1 envelope ( env ) proteins have been used as baits to identify and capture env ( gp160)specific b cells by flow cytometry in selected hiv - infected donors ( scheid et al . single - cell ab cloning techniques have successfully generated hundreds of hiv - specific abs and , among them , dozens of new extremely potent next - generation bnabs ( doria - rose and connors , 2009 ; klein et al . the real challenge now is to take advantage of our understanding of the generation of bnabs to design new immunogens able to induce the affinity maturation of b cell lineages expressing bnabs . essentially , this means being able to orchestrate a fine - tuned interplay between the host immune system and a tailored antigen ( or antigens ) that should prime the b cell precursors in an effective way ( discussed further in the section a vacciny strategy targeting germline abs ) . both of the aforementioned methods have two limitations for the identification of the best protective abs : first , the required ebv transformation of b cells can have a bias on the secretion of mabs ; and second , in vitro expressed synthetic abs might not always faithfully reflect their natural functional capability . to overcome these limits , burtion , poignard , and co - workers have isolated hiv - specific human mabs from mbcs expanded in vitro with the addition of feeder cells and conditioned medium generated from mitogen - stimulated human t cells and screened the supernatants for neutralization activity ( walker et al . a modified high - throughput approach has also recently been developed based on the isolation of mbcs from pbmcs and expansion in vitro without the need for activated t cell supernatants , allowing secretion of sufficient amounts of abs in culture to be screened in binding and functional assays ( huang et al . , 2013 ) . the main advantage of this technique is provided by the possibility of isolating and directly characterizing the abs produced by individually cloned b cells without the need for cloning and expression of all the recombinant mabs and , more importantly , maintaining the original natural properties of binding and functionality . after characterization of the binding and/or functional capacities of the abs , the ig gene sequences can be recovered . in turn , repertoire analyses to understand the origin and evolution of the abs of interest can be performed , and further cloning and expression steps for deeper structural characterizations can be performed ( fig . recently , through this new approach , broad and potent hiv nabs have been discovered in the pool of antigen - specific mbcs , highlighting new vulnerability sites on the hiv env antigen ( huang et al . , 2014 ) . this efficient new methodology , applicable both to preselected antigen - specific single - sorted mbcs or to b cell subpopulations of unknown specificity , will enable the identification of new target antigens or epitopes involved in a protective ab response ( fig . beginning at center left , a flow path representation of how human b cell repertoire analyses enable the identification of protective abs from vaccinated or infected subjects . ( top inset ) single b cell sorting and culturing in the presence of feeder cells allow direct screening and selection of naturally produced abs with desired functionality and recovery of the corresponding ig gene sequence . this approach is one of the most recent that allows for the interrogation of single - sorted b cells through direct screening of ab functionality . in turn , repertoire analyses to understand the origin and evolution of the abs of interest can be performed ( huang et al . , 2013 ) . starting from the recovered sequences , further expression of the recombinant abs of interest in the most appropriate system allows for a fine - tuned characterization of their properties . ( center right ) structural characterization of such abs bound to their target antigen ( ag ) allows for the detailed definition of the protective epitope . a molecular detail of an antigen ab ( fab ) complex as revealed by a cocrystal structure identifying a protective epitope is represented as an example . the protective epitope ( red shape ) can then be engineered for presentation as an optimized immunogen in a novel format ( for example , by mounting the epitope in an oriented multicopy array on a nanoparticle [ orange octagon ] , because nanoparticles can increase an epitope - focused immune response ; lpez - sagaseta et al . , ( bottom inset ) the new antigen can be developed with the best formulation or delivery system to then be tested in humans . overall , the single b cell cloning approaches used to isolate mabs have been demonstrated to be extremely powerful for the identification and characterization of key protective antigens but have the drawback of being low - throughput technologies , revealing only a very limited slice of the full ab repertoire . therefore , using these techniques to analyze the impact that a vaccine antigen or a vaccine formulation can have in inducing specific repertoire signatures shared by different individuals may lead to erroneous and misleading conclusions . recent advances in next - generation sequencing technology have enabled the sequencing of ig genes from millions of b cells simultaneously , giving a high - resolution characterization of the ab sequence repertoire ( reddy and georgiou , 2011 ) . nevertheless , the ability of this approach to recognize the antigen - specific ab repertoire inside the totality of the individual ab repertoire and evaluate specific changes induced by a vaccine or pathogen still remains questionable . one approach is to computationally search the total repertoire data for ab sequences that are shared among individuals that have been exposed to the same antigen through infection or vaccination , identifying a three recent studies found that conserved cdr3 sequences were produced in patients recovering from acute dengue infection ( parameswaran et al . , 2013 ) , or during the immune response to pandemic influenza h1n1 vaccination ( jackson et al . , 2014 ) and hib - polysaccharide vaccination ( trck et al . , 2015 ) . although the convergent repertoire demonstrates changes in the large - scale structural features of the repertoire , the antigen specificity of the identified ab sequences always needs experimental confirmation through the expression , purification , and functional testing of recombinant abs . however , only a small subset of abs are usually expressed and investigated after the exhaustive interrogation of the repertoire , missing the possibility of having a full picture of the properties and functionality of all abs directed against one specific antigen . to start from an enriched antigen - specific b cell population , most studies have used pbmcs isolated at day 8 after immunization , when there is the peak of circulating plasmablasts . yet , this approach entails a loss of information on the longitudinal evolution of the b cell memory repertoire and eventually of bnabs and their maintenance in the long - term memory population . finally , in recent studies , the sequencing of paired vh / vl ig gene repertoire by next - generation sequencing technology has been combined with novel proteomic methods mining the antigen - specific ab repertoire that comprises the human serum polyclonal response ( wine et al . , 2015 ) . the new perspective offered by combining the analysis of the b cell and ab repertoire induced by tt vaccination has highlighted that the anti - tt serum igg repertoire is composed of a limited number of ab clonotypes ( 80100 ) , whereas the b cell receptor repertoire diversity in the memory and plasmablast compartments is orders of magnitude greater than that of the serological repertoire ( lavinder et al . , 2014 ) . this observation suggests that most peripheral b cell encoded abs are unlikely to be present in detectable amounts as soluble proteins , leaving unanswered questions regarding the nature and dynamics of the elements regulating the serological memory . collectively , all of the technologies that allow the dissection of the human ab response to pathogens are increasing our understanding of how protection is induced and can drive the design of more efficacious antigens for the development of vaccines to prevent current and future pathogenic threats . the incredible antigenic variability of hiv env initially led to the belief that bnabs would be nearly impossible to elicit , and that the only way to tackle hiv efficiently would be by generating t cell mediated immunity . however , this viewpoint was dramatically impacted by landmark discoveries reported in 1994 ; namely , the generation of two new recombinant mabs : mab b12 from a combinatorial fab phage display library ( burton et al . , 1991 ) and the human mab 2f5 isolated from an hiv-1positive volunteer ( conley et al . , 1994 ) , both of which were shown to be broadly neutralizing against panels of hiv strains . in particular , mab b12 binds the conserved cd4 binding site ( cd4bs ) on the env gp120 subunit and was shown to neutralize 5075% of primary hiv isolates tested ( burton et al . , 1994 ; binley et al . , 2004 ) . however , a clear limit of b12 was the somewhat low potency of this mab , which required up to 25 g / ml for neutralization of most hiv strains in the united states . moreover , mab b12 showed a very limited effect in controlling viremia in vivo , even when combined with 4e10 , a distinct bnab with similar potency ( poignard et al . , 1999 ; cardoso et al . , 2005 ) ; in more recent analyses , b12 neutralized only 3060% of diverse hiv strains ( walker et al . , 2011 ) . these findings again led researchers to believe that ab - based therapies and vaccines would be useless for hiv control , and the development of vaccines based on env components lost some momentum . the hiv field changed completely around 2009/2010 with the identification of second generation bnabs . first , using a neutralization screening of supernatants from thousands of mbcs from an hiv - infected donor , two more potent human mabs ( pg9 and pg16 ) targeting a broadly neutralizing epitope were described as able to neutralize 7080% of the tested hiv strains with an ic50 < 50 g / ml and 5060% with an ic50 < 1 g / ml ( walker et al . , 2009 ) . then , by using antigen - specific human b cell sorting and engineered probe env antigens that better exposed conserved epitopes , powerful new bnabs ( e.g. , mabs of the group called vrc01 ) targeting the conserved cd4bs on the env gp120 subunit were identified , and one of them was found able to neutralize up to 90% of hiv isolates at an ic50 < 50 g / ml and 70% at an ic50 < 1 g / ml , with an overall low geometric mean ic50 of 0.3 g / ml ( wu et al . , 2010 ) . subsequently , the ability to use single - cell technologies allowed for the identification of several hundred anti - hiv human mabs , some of which were bnabs with a median ic50 as low as 0.02 g / ml , > 1,000-fold better than the recombinant b12 ab isolated in 1994 ( scheid et al . , 2011 ; walker et al . , 2011 ; burton and mascola , 2015 ) . epitope mapping studies using the new abs showed that in addition to the original cd4bs epitope , several other regions of hiv env were able to induce bnabs . the new abs also showed remarkable therapeutic effects in vivo : in rhesus monkeys chronically infected with pathogenic simian human immunodeficiency virus shiv - sf162p3 , they were able to rapidly reduce plasma viremia to undetectable levels and keep it at bay for months ( barouch et al . , 2013 ) . similarly , therapeutic benefits were observed by the suppression of viremia in shiv - infected macaques ( shingai et al . , 2013 ) . excitingly , one such therapeutic bnab , 3bnc117 , which targets the cd4bs epitope on gp120 , was tested in phase 1 clinical trials and was recently reported to reduce plasma viremia in chronically infected humans ( caskey et al . , 2015 ) . likewise , a phase 1 study using infusion of a vrc01 class ab showed promising results , reducing plasma viremia and suppressing neutralization - sensitive strains in a subset of hiv-1infected subjects ( lynch et al . , 2015 ) . the new findings described in the previous paragraph may completely change the approach to hiv prevention and therapy , and today , ab - mediated protection is believed to be a promising approach to hiv control . the immunological and structural information available is being used to design vaccine antigens , notably including the emerging class of recombinant env sosip ( soluble gp140 stabilized by disulphide bridges and by an ile to pro mutation ) immunogens stabilized in native - like conformations and able to induce nabs against neutralization - resistant ( tier 2 ) hiv in rabbits and , to a lesser extent , macaques ( sanders et al . , 2015 ) . alternatively , such information could be used to produce a cocktail of therapeutic abs to be delivered simultaneously by passive immunization . delivery of bnab - encoding genes by gene therapy vectored immunoprophylaxis has also been shown to be an efficient method to prevent hiv infection in humanized mice , and it will be interesting to see if this strategy can be translated to protect humans against hiv ( balazs et al . , 2012 , 2014 ) . the quest for an rsv vaccine has met many challenges that have inspired research breakthroughs , illustrating how information from human immunology and structural biology studies can be used in combination to instruct vaccine antigen design . rsv is the main viral cause of severe respiratory tract disease in children worldwide ( nair et al . the membrane - anchored fusion glycoprotein f present on the surface of this virus is the major target of nabs ( anderson et al . , 2010 ) . however , although an approved therapeutic mab treatment ( palivizumab ) targeting f has existed for nearly two decades ( the impact - rsv study group , 1998 ) , attempts to develop an rsv vaccine using recombinant f antigen have been hampered by its poor behavior in solution and its tendency to undergo large conformational changes . early epitope - focused rational antigen designs were enabled by the cocrystal structure of the fab region of an rsv - neutralizing mab ( motavizumab , an affinity - enhanced form of palivizumab ) bound to its target f epitope prepared as a synthetic 24-residue peptide that adopted a helix - turn - helix motif in the crystal ( mclellan et al . , 2010 ) . however , initial attempts to design an immunogen comprised of a scaffold protein presenting this f epitope with the observed helix - turn - helix conformation were only partially successful : after immunization , conformation - specific anti - f abs were elicited but lacked the ability to neutralize rsv ( mclellan et al . , 2011 ) . ( 2014 ) reported a proof - of - principle study , wherein they developed a new structure - based approach for the computational design and optimization of improved biophysically robust scaffold proteins displaying the desired rsv peptide epitope in a conformationally faithful manner that was able to elicit rsv nabs in nonhuman primates ( nhps ) . furthermore , when the rsv peptide epitope scaffold antigen was mounted in multicopy ordered arrays on the surface of a virus - like particle by linking to the hepatitis b core antigen , at least half of the nhps raised rsv - neutralizing activity comparable to that induced by natural human infection . thus , careful manipulations to properly present a structurally optimized form of a single well - defined protective epitope resulted in the generation of the desired immune response , demonstrating the feasibility of this novel immune - focusing approach . nevertheless , variability of the immune response in a diverse human population , together with the risk of the emergence of naturally occurring epitope escape mutants , makes a vaccine approach relying on a single protective epitope potentially less robust compared with immunization strategies based on a full - length antigen , such as the entire ectodomain of the rsv f protein , which would potentially offer a broader immune response . the trimeric f protein of rsv undergoes a major conformational change from a relatively compact meta - stable prefusion state to a more elongated highly stable postfusion state ( mclellan et al . , 2013c ) . because of its exposure on the virion surface , the prefusion structure was an obvious candidate vaccine antigen , but the spontaneous conversion of recombinant prefusion f to the post - fusion state discouraged initial efforts in this direction . however , although an engineered postfusion f antigen elicited nabs in animals ( swanson et al . , ( 2012 ) using immunized rabbit sera and sero - positive human sera reported that abs targeting the prefusion f protein accounted for most of the rsv - neutralizing activity . the latter highlighted the importance of renewed attempts to make a stable and efficacious prefusion f antigen , which was facilitated by the discovery and structural characterization of new mouse and human rsv - neutralizing mabs specific for prefusion f. crucially , a cocrystal structure reported by mclellan et al . ( 2013b ) showed how the potently rsv - neutralizing human fab d25 binds and traps f in a previously unobserved prefusion conformation . the structure revealed a large quaternary epitope on the prefusion f trimer apex , defining a major site of vulnerability on a novel antigenic structure . ( 2013a ) were able to stabilize the prefusion f antigen in a trimeric conformation by replacing its transmembrane region with a trimerization domain and by inserting ad hoc mutations to create a novel disulfide bridge and a core structure with improved hydrophobic packing . this new prefusion antigen was able to elicit much higher levels of rsv - neutralizing activity compared with the postfusion f antigen in mouse and nhp models . although polyinosinic : polycytidylic acid adjuvant was used in combination with the engineered f antigen to stimulate an appropriate neutralizing response and no information was provided regarding their potency in the absence of such a strong adjuvant , this work provides an elegant preclinical demonstration of the feasibility of a structure - based vaccine strategy driven by the analysis of the human natural protective immune response and focused on mimicry of a large conformational epitope . indeed , this study provides hope that a vaccine containing a prefusion - stabilized f protein would induce a response protective against rsv infection in humans , and clinical trials are ongoing . yet another important area where human immunology is informing the design of novel immunogens is in the search for broadly protective vaccine antigens to protect against influenza ( flu ) , a persistent global public health threat ( lambert and fauci , 2010 ) . traditional flu vaccines made using ha and neuraminidase antigens purified from egg - grown influenza virus are efficacious when the vaccine antigens are genotypically matched against the circulating strains and subtypes . however , the frequent occurrence of genetic variation via antigenic drift and reassortment implies that such flu vaccines need to be revised annually to elicit protective responses . indeed , the bulk of the immune response triggered either by the vaccine or by natural infection is against the head of ha , which is highly diverse between distinct strains , and therefore , only minimal cross - protection from one strain to another is typically achieved . consequently , flu vaccine efficiency can be highly variable depending on how much the ha of the circulating flu strains matches the ha in the vaccine strains . moreover , all presently available seasonal flu vaccines are probably unable to prevent infection with a new emerging strain of highly pathogenic flu virus to which the human population has not been previously exposed , such as the h5n1 or h7n9 strains . because the latter circulates among avian populations , there is the risk that zoonosis to humans may trigger a deadly pandemic infection . therefore , an important goal is the development of a potent and universally protective flu vaccine to generate high titers of bnabs against influenza virus . such a vaccine should ideally induce an immune response against one or more conserved epitopes in the viral proteins to prevent infection or disease from all or nearly all possible circulating strains of the flu virus . the ha stem region is indeed highly conserved among different strains and subtypes of influenza virus , but this region is poorly immunogenic when in the full - length ha protein contained in conventional vaccines . therefore , conventional flu vaccines are not able to elicit a powerful anti - stem ha ab response . however , the appropriateness of the ha stem region as a component in a universal flu vaccine was recently confirmed by the finding that bnabs against this region of ha can be selected from human subjects after infection or vaccination ( kashyap et al . , 2008 ; throsby et al . , 2008 ; ekiert et al . , 2009 , 2011 ; sui et al . , 2009 ; corti et al . , these observations have driven experimental approaches aimed at improving the immune response against the stem ( hai et al . , 2012 ; schneemann et al . , 2012 ; kanekiyo et al . , this aim is complicated because , as for rsv f ( see rsv section above ) , the native ha is a meta - stable membrane glycoprotein that undergoes large conformational changes . to overcome this challenge , kanekiyo et al . ( 2013 ) used a structure - based design to generate a genetic fusion encoding ha from one h1 strain linked to the bacterial protein ferritin . ferritin polypeptide spontaneously self - assembled into 24-mer nanoparticles , each displaying eight trimeric h1 subtype ha spikes projecting off the ferritin nanoparticle scaffold , oriented such that the immune system could encounter the ordered arrays of ha in a native - like conformation . when tested in preclinical in vivo models , this elegant multivalent immunogen presenting structurally intact ha trimers was effective in eliciting higher titers of nabs compared with a similarly adjuvanted trivalent inactivated vaccine ( tiv ) in both mice and ferrets , providing broader coverage against unmatched h1n1 viruses . analysis of the specificity of the immune response revealed that the nabs generated by ha ferritin nanoparticles were directed against both the sensitive receptor binding site in the ha head and the conserved ha stem region , and the latter was responsible for half of the observed heterosubtype neutralizations . although it could not be considered a real universal flu vaccine , this ha nanoparticle based vaccine proved that a potent and broad neutralizing response against the ha stem region can be induced by the use of an appropriate immunogen . however , a potent cross - protective response was observed only in the presence of strong adjuvants such as ribi or mf59 . such adjuvants may work by improving the development of t follicular helper cells and the germinal center antigen - specific b cell response ( mastelic gavillet et al . , 2015 ) , thus helping the selection of low - abundance b cell clones recognizing poorly immunogenic epitopes such as those of the ha stem region , as experimentally proved for other model antigens ( victora et al . , 2010 ) . the identification of human mabs able to bind conserved epitopes in the ha stem region of several flu strains belonging to different subtypes can be considered the first step toward the discovery of an ideal immunogen for a universal flu vaccine . these abs are precious tools that can be used both for cocrystal structure studies to precisely identify the neutralizing epitopes in the ha stem region and for binding assays to isolate the most appropriate immunogen to induce a broad protection . two different groups have recently taken advantage of such anti ha stem abs and the cognate structurally derived epitope mapping information to rationally design and screen a series of novel immunogens , leading to selection of a stable immunogen containing the appropriate conformational epitopes from the ha stem ( impagliazzo et al . , 2015 ; yassine et al . , 2015 ) . a team led by nabel and graham have again taken advantage of ferritin nanoparticles to present a refined ha antigen comprised of a stem - only immunogen , which was rationally designed and stabilized in a trimeric headless state ( yassine et al . , 2015 ) . in the study , the design strategy was driven by prior human immunology studies and detailed structural knowledge that showed that the immunogenically subdominant stem region of ha contains highly conserved protective conformational epitopes recognized by human mabs with multisubtype ha recognition profiles broader than those targeting the more variable head region ( throsby et al . , 2008 ; ekiert et al . , 2009 , 2011 ; sui et al . , 2009 ; new structural studies by x - ray crystallography and cryoelectron microscopy informed an iterative design pathway that resulted in seven generations of h1 subtype ha - stabilized stem immunogens ( termed h1-ss ) , with the last designed form presenting the desired trimeric structure and the epitope recognized by the broadly neutralizing human mabs . when this recombinant antigen was fused to ferritin , it assembled on the nanoparticle surfaces ( termed h1-ss - np ) , which were confirmed to possess the desired antigenic features . more importantly , in preclinical immunization studies , h1-ss - np induced a higher ab response against the ha stem compared with the classical trivalent inactivated flu vaccine , with cross - reactivity not only to many h1 strains but also to distant subtypes from both group 1 ( h2 , h5 , and h9 ) and group 2 ( h3 and h7 ) . although neutralization could be observed only for the homologous and two other heterologous h1 strains , vaccination with h1-ss - np resulted in the complete protection of mice and partial protection of ferrets against lethal viral challenge using the h5n1 heterosubtype . the latter finding was further supported by passive transfer of immunoglobulins from the immunized mice , which protected naive mice against h5n1 challenge ( yassine et al . , 2015 ) , suggesting that anti ha stem abs can have a protective effect via a different mechanism than virus neutralization . ( 2015 ) , who used ingenious structure - based design methods combined with a library approach to generate minimally sized trimeric stem only ha immunogens ( termed mini - has ) , which were soluble and conformationally stable and satisfied the prerequisite to structurally and antigenically mimic the highly conserved stem region of full - length ha , as revealed by their capacity to bind anti ha stem bnabs . described a lead candidate , mini - ha # 4900 , that raised broad and protective immune responses , completely protecting mice in lethal heterologous and heterosubtypic challenge models . furthermore , in an nhp model , when compared with tiv , this immunogen elicited higher ab titers against homologous as well as a variety of heterologous and heterosubtypic strains that bound ha in the stem - neutralizing epitope and triggered potent ab - dependent cellular cytotoxicity activity and were able to neutralize an h5n1 heterosubtypic strain . however , the capacity of lead mini - ha candidates to reduce fever in nhps after sublethal challenge with a heterologous h1n1 strain was only comparable to that of tiv , whereas no reduction of tracheal viral load could be observed in mini - ha immunized animals , leaving some doubts about the superiority of this novel vaccine in this animal model and , thus , about its potential as a universal flu vaccine for humans . nevertheless , these small ha - stem constructs can be considered good starting points for a vaccine candidate raising a broadly protective immune response against group 1 flu viruses , which may be extendable to group 2 . finally , it should be noted that both the structurally designed mini - has and the h1-ss - np described in the previous paragraph were reported to elicit broadly reactive abs against the ha stem when administered in the presence of ribi and matrix m , respectively . again , these studies appear to suggest that a strong adjuvant may be required to obtain an appropriate response to conserved subdominant epitopes in the ha stem region , most likely acting by enhancing t follicular helper cells in the germinal center and therefore driving selection of scarce b cells specific for these epitopes . collectively , such studies are fine demonstrations of how structure - based antigen designs can be used to obtain an improved immune response specifically directed toward a target epitope identified by human immunology studies and validated by structural biology studies . clinical studies in humans will be required to fully demonstrate the power of these synergistic approaches . the incredible antigenic variability of hiv env initially led to the belief that bnabs would be nearly impossible to elicit , and that the only way to tackle hiv efficiently would be by generating t cell mediated immunity . however , this viewpoint was dramatically impacted by landmark discoveries reported in 1994 ; namely , the generation of two new recombinant mabs : mab b12 from a combinatorial fab phage display library ( burton et al . , 1991 ) and the human mab 2f5 isolated from an hiv-1positive volunteer ( conley et al . , 1994 ) , both of which were shown to be broadly neutralizing against panels of hiv strains . in particular , mab b12 binds the conserved cd4 binding site ( cd4bs ) on the env gp120 subunit and was shown to neutralize 5075% of primary hiv isolates tested ( burton et al . , 1994 ; binley et al . , 2004 ) . however , a clear limit of b12 was the somewhat low potency of this mab , which required up to 25 g / ml for neutralization of most hiv strains in the united states . moreover , mab b12 showed a very limited effect in controlling viremia in vivo , even when combined with 4e10 , a distinct bnab with similar potency ( poignard et al . , 1999 ; cardoso et al . , 2005 ) ; in more recent analyses , b12 neutralized only 3060% of diverse hiv strains ( walker et al . , 2011 ) . these findings again led researchers to believe that ab - based therapies and vaccines would be useless for hiv control , and the development of vaccines based on env components lost some momentum . the hiv field changed completely around 2009/2010 with the identification of second generation bnabs . first , using a neutralization screening of supernatants from thousands of mbcs from an hiv - infected donor , two more potent human mabs ( pg9 and pg16 ) targeting a broadly neutralizing epitope were described as able to neutralize 7080% of the tested hiv strains with an ic50 < 50 g / ml and 5060% with an ic50 < 1 g / ml ( walker et al . , 2009 ) . then , by using antigen - specific human b cell sorting and engineered probe env antigens that better exposed conserved epitopes , powerful new bnabs ( e.g. , mabs of the group called vrc01 ) targeting the conserved cd4bs on the env gp120 subunit were identified , and one of them was found able to neutralize up to 90% of hiv isolates at an ic50 < 50 g / ml and 70% at an ic50 < 1 g / ml , with an overall low geometric mean ic50 of 0.3 g / ml ( wu et al . , 2010 ) . subsequently , the ability to use single - cell technologies allowed for the identification of several hundred anti - hiv human mabs , some of which were bnabs with a median ic50 as low as 0.02 g / ml , > 1,000-fold better than the recombinant b12 ab isolated in 1994 ( scheid et al . , 2011 ; walker et al . , 2011 ; burton and mascola , 2015 ) . epitope mapping studies using the new abs showed that in addition to the original cd4bs epitope , several other regions of hiv env were able to induce bnabs . the new abs also showed remarkable therapeutic effects in vivo : in rhesus monkeys chronically infected with pathogenic simian human immunodeficiency virus shiv - sf162p3 , they were able to rapidly reduce plasma viremia to undetectable levels and keep it at bay for months ( barouch et al . , 2013 ) . similarly , therapeutic benefits were observed by the suppression of viremia in shiv - infected macaques ( shingai et al . , 2013 ) . excitingly , one such therapeutic bnab , 3bnc117 , which targets the cd4bs epitope on gp120 , was tested in phase 1 clinical trials and was recently reported to reduce plasma viremia in chronically infected humans ( caskey et al . , 2015 ) . likewise , a phase 1 study using infusion of a vrc01 class ab showed promising results , reducing plasma viremia and suppressing neutralization - sensitive strains in a subset of hiv-1infected subjects ( lynch et al . , 2015 ) . the new findings described in the previous paragraph may completely change the approach to hiv prevention and therapy , and today , ab - mediated protection is believed to be a promising approach to hiv control . the immunological and structural information available is being used to design vaccine antigens , notably including the emerging class of recombinant env sosip ( soluble gp140 stabilized by disulphide bridges and by an ile to pro mutation ) immunogens stabilized in native - like conformations and able to induce nabs against neutralization - resistant ( tier 2 ) hiv in rabbits and , to a lesser extent , macaques ( sanders et al . , 2015 ) . alternatively , such information could be used to produce a cocktail of therapeutic abs to be delivered simultaneously by passive immunization . delivery of bnab - encoding genes by gene therapy vectored immunoprophylaxis has also been shown to be an efficient method to prevent hiv infection in humanized mice , and it will be interesting to see if this strategy can be translated to protect humans against hiv ( balazs et al . , 2012 , 2014 ) . the quest for an rsv vaccine has met many challenges that have inspired research breakthroughs , illustrating how information from human immunology and structural biology studies can be used in combination to instruct vaccine antigen design . rsv is the main viral cause of severe respiratory tract disease in children worldwide ( nair et al . , 2010 ) and also afflicts elderly and immunocompromised adults ( falsey et al . , 2005 ) . the membrane - anchored fusion glycoprotein f present on the surface of this virus is the major target of nabs ( anderson et al . , 2010 ) . however , although an approved therapeutic mab treatment ( palivizumab ) targeting f has existed for nearly two decades ( the impact - rsv study group , 1998 ) , attempts to develop an rsv vaccine using recombinant f antigen have been hampered by its poor behavior in solution and its tendency to undergo large conformational changes . early epitope - focused rational antigen designs were enabled by the cocrystal structure of the fab region of an rsv - neutralizing mab ( motavizumab , an affinity - enhanced form of palivizumab ) bound to its target f epitope prepared as a synthetic 24-residue peptide that adopted a helix - turn - helix motif in the crystal ( mclellan et al . , 2010 ) . however , initial attempts to design an immunogen comprised of a scaffold protein presenting this f epitope with the observed helix - turn - helix conformation were only partially successful : after immunization , conformation - specific anti - f abs were elicited but lacked the ability to neutralize rsv ( mclellan et al . , 2011 ) . 2014 ) reported a proof - of - principle study , wherein they developed a new structure - based approach for the computational design and optimization of improved biophysically robust scaffold proteins displaying the desired rsv peptide epitope in a conformationally faithful manner that was able to elicit rsv nabs in nonhuman primates ( nhps ) . furthermore , when the rsv peptide epitope scaffold antigen was mounted in multicopy ordered arrays on the surface of a virus - like particle by linking to the hepatitis b core antigen , at least half of the nhps raised rsv - neutralizing activity comparable to that induced by natural human infection . thus , careful manipulations to properly present a structurally optimized form of a single well - defined protective epitope resulted in the generation of the desired immune response , demonstrating the feasibility of this novel immune - focusing approach . nevertheless , variability of the immune response in a diverse human population , together with the risk of the emergence of naturally occurring epitope escape mutants , makes a vaccine approach relying on a single protective epitope potentially less robust compared with immunization strategies based on a full - length antigen , such as the entire ectodomain of the rsv f protein , which would potentially offer a broader immune response . the trimeric f protein of rsv undergoes a major conformational change from a relatively compact meta - stable prefusion state to a more elongated highly stable postfusion state ( mclellan et al . , 2013c ) . because of its exposure on the virion surface , the prefusion structure was an obvious candidate vaccine antigen , but the spontaneous conversion of recombinant prefusion f to the post - fusion state discouraged initial efforts in this direction . however , although an engineered postfusion f antigen elicited nabs in animals ( swanson et al . , 2011 ) , an alternative study by magro et al . ( 2012 ) using immunized rabbit sera and sero - positive human sera reported that abs targeting the prefusion f protein accounted for most of the rsv - neutralizing activity . the latter highlighted the importance of renewed attempts to make a stable and efficacious prefusion f antigen , which was facilitated by the discovery and structural characterization of new mouse and human rsv - neutralizing mabs specific for prefusion f. crucially , a cocrystal structure reported by mclellan et al . ( 2013b ) showed how the potently rsv - neutralizing human fab d25 binds and traps f in a previously unobserved prefusion conformation . the structure revealed a large quaternary epitope on the prefusion f trimer apex , defining a major site of vulnerability on a novel antigenic structure . ( 2013a ) were able to stabilize the prefusion f antigen in a trimeric conformation by replacing its transmembrane region with a trimerization domain and by inserting ad hoc mutations to create a novel disulfide bridge and a core structure with improved hydrophobic packing . this new prefusion antigen was able to elicit much higher levels of rsv - neutralizing activity compared with the postfusion f antigen in mouse and nhp models . although polyinosinic : polycytidylic acid adjuvant was used in combination with the engineered f antigen to stimulate an appropriate neutralizing response and no information was provided regarding their potency in the absence of such a strong adjuvant , this work provides an elegant preclinical demonstration of the feasibility of a structure - based vaccine strategy driven by the analysis of the human natural protective immune response and focused on mimicry of a large conformational epitope . indeed , this study provides hope that a vaccine containing a prefusion - stabilized f protein would induce a response protective against rsv infection in humans , and clinical trials are ongoing . yet another important area where human immunology is informing the design of novel immunogens is in the search for broadly protective vaccine antigens to protect against influenza ( flu ) , a persistent global public health threat ( lambert and fauci , 2010 ) . traditional flu vaccines made using ha and neuraminidase antigens purified from egg - grown influenza virus are efficacious when the vaccine antigens are genotypically matched against the circulating strains and subtypes . however , the frequent occurrence of genetic variation via antigenic drift and reassortment implies that such flu vaccines need to be revised annually to elicit protective responses . indeed , the bulk of the immune response triggered either by the vaccine or by natural infection is against the head of ha , which is highly diverse between distinct strains , and therefore , only minimal cross - protection from one strain to another is typically achieved . consequently , flu vaccine efficiency can be highly variable depending on how much the ha of the circulating flu strains matches the ha in the vaccine strains . moreover , all presently available seasonal flu vaccines are probably unable to prevent infection with a new emerging strain of highly pathogenic flu virus to which the human population has not been previously exposed , such as the h5n1 or h7n9 strains . because the latter circulates among avian populations , there is the risk that zoonosis to humans may trigger a deadly pandemic infection . therefore , an important goal is the development of a potent and universally protective flu vaccine to generate high titers of bnabs against influenza virus . such a vaccine should ideally induce an immune response against one or more conserved epitopes in the viral proteins to prevent infection or disease from all or nearly all possible circulating strains of the flu virus . the ha stem region is indeed highly conserved among different strains and subtypes of influenza virus , but this region is poorly immunogenic when in the full - length ha protein contained in conventional vaccines . therefore , conventional flu vaccines are not able to elicit a powerful anti - stem ha ab response . however , the appropriateness of the ha stem region as a component in a universal flu vaccine was recently confirmed by the finding that bnabs against this region of ha can be selected from human subjects after infection or vaccination ( kashyap et al . , 2008 ; throsby et al . , 2008 ; ekiert et al . , 2009 , 2011 ; sui et al . , 2009 ; these observations have driven experimental approaches aimed at improving the immune response against the stem ( hai et al . this aim is complicated because , as for rsv f ( see rsv section above ) , the native ha is a meta - stable membrane glycoprotein that undergoes large conformational changes . to overcome this challenge , kanekiyo et al . ( 2013 ) used a structure - based design to generate a genetic fusion encoding ha from one h1 strain linked to the bacterial protein ferritin . ferritin polypeptide spontaneously self - assembled into 24-mer nanoparticles , each displaying eight trimeric h1 subtype ha spikes projecting off the ferritin nanoparticle scaffold , oriented such that the immune system could encounter the ordered arrays of ha in a native - like conformation . when tested in preclinical in vivo models , this elegant multivalent immunogen presenting structurally intact ha trimers was effective in eliciting higher titers of nabs compared with a similarly adjuvanted trivalent inactivated vaccine ( tiv ) in both mice and ferrets , providing broader coverage against unmatched h1n1 viruses . analysis of the specificity of the immune response revealed that the nabs generated by ha ferritin nanoparticles were directed against both the sensitive receptor binding site in the ha head and the conserved ha stem region , and the latter was responsible for half of the observed heterosubtype neutralizations . although it could not be considered a real universal flu vaccine , this ha nanoparticle based vaccine proved that a potent and broad neutralizing response against the ha stem region can be induced by the use of an appropriate immunogen . however , a potent cross - protective response was observed only in the presence of strong adjuvants such as ribi or mf59 . such adjuvants may work by improving the development of t follicular helper cells and the germinal center antigen - specific b cell response ( mastelic gavillet et al . , 2015 ) , thus helping the selection of low - abundance b cell clones recognizing poorly immunogenic epitopes such as those of the ha stem region , as experimentally proved for other model antigens ( victora et al . , 2010 ) . the identification of human mabs able to bind conserved epitopes in the ha stem region of several flu strains belonging to different subtypes can be considered the first step toward the discovery of an ideal immunogen for a universal flu vaccine . these abs are precious tools that can be used both for cocrystal structure studies to precisely identify the neutralizing epitopes in the ha stem region and for binding assays to isolate the most appropriate immunogen to induce a broad protection . two different groups have recently taken advantage of such anti ha stem abs and the cognate structurally derived epitope mapping information to rationally design and screen a series of novel immunogens , leading to selection of a stable immunogen containing the appropriate conformational epitopes from the ha stem ( impagliazzo et al . , 2015 ; yassine et al . , 2015 ) . a team led by nabel and graham have again taken advantage of ferritin nanoparticles to present a refined ha antigen comprised of a stem - only immunogen , which was rationally designed and stabilized in a trimeric headless state ( yassine et al . , 2015 ) . in the study , the design strategy was driven by prior human immunology studies and detailed structural knowledge that showed that the immunogenically subdominant stem region of ha contains highly conserved protective conformational epitopes recognized by human mabs with multisubtype ha recognition profiles broader than those targeting the more variable head region ( throsby et al . , 2008 ; ekiert et al . , 2009 , 2011 ; sui et al . , 2009 ; new structural studies by x - ray crystallography and cryoelectron microscopy informed an iterative design pathway that resulted in seven generations of h1 subtype ha - stabilized stem immunogens ( termed h1-ss ) , with the last designed form presenting the desired trimeric structure and the epitope recognized by the broadly neutralizing human mabs . when this recombinant antigen was fused to ferritin , it assembled on the nanoparticle surfaces ( termed h1-ss - np ) , which were confirmed to possess the desired antigenic features . more importantly , in preclinical immunization studies , h1-ss - np induced a higher ab response against the ha stem compared with the classical trivalent inactivated flu vaccine , with cross - reactivity not only to many h1 strains but also to distant subtypes from both group 1 ( h2 , h5 , and h9 ) and group 2 ( h3 and h7 ) . although neutralization could be observed only for the homologous and two other heterologous h1 strains , vaccination with h1-ss - np resulted in the complete protection of mice and partial protection of ferrets against lethal viral challenge using the h5n1 heterosubtype . the latter finding was further supported by passive transfer of immunoglobulins from the immunized mice , which protected naive mice against h5n1 challenge ( yassine et al . , 2015 ) , suggesting that anti ha stem abs can have a protective effect via a different mechanism than virus neutralization . ( 2015 ) , who used ingenious structure - based design methods combined with a library approach to generate minimally sized trimeric stem only ha immunogens ( termed mini - has ) , which were soluble and conformationally stable and satisfied the prerequisite to structurally and antigenically mimic the highly conserved stem region of full - length ha , as revealed by their capacity to bind anti ha stem bnabs . described a lead candidate , mini - ha # 4900 , that raised broad and protective immune responses , completely protecting mice in lethal heterologous and heterosubtypic challenge models . furthermore , in an nhp model , when compared with tiv , this immunogen elicited higher ab titers against homologous as well as a variety of heterologous and heterosubtypic strains that bound ha in the stem - neutralizing epitope and triggered potent ab - dependent cellular cytotoxicity activity and were able to neutralize an h5n1 heterosubtypic strain . however , the capacity of lead mini - ha candidates to reduce fever in nhps after sublethal challenge with a heterologous h1n1 strain was only comparable to that of tiv , whereas no reduction of tracheal viral load could be observed in mini - ha immunized animals , leaving some doubts about the superiority of this novel vaccine in this animal model and , thus , about its potential as a universal flu vaccine for humans . nevertheless , these small ha - stem constructs can be considered good starting points for a vaccine candidate raising a broadly protective immune response against group 1 flu viruses , which may be extendable to group 2 . finally , it should be noted that both the structurally designed mini - has and the h1-ss - np described in the previous paragraph were reported to elicit broadly reactive abs against the ha stem when administered in the presence of ribi and matrix m , respectively . again , these studies appear to suggest that a strong adjuvant may be required to obtain an appropriate response to conserved subdominant epitopes in the ha stem region , most likely acting by enhancing t follicular helper cells in the germinal center and therefore driving selection of scarce b cells specific for these epitopes . collectively , such studies are fine demonstrations of how structure - based antigen designs can be used to obtain an improved immune response specifically directed toward a target epitope identified by human immunology studies and validated by structural biology studies . clinical studies in humans will be required to fully demonstrate the power of these synergistic approaches . another great advance in our knowledge of the human immune response to pathogens that can aid the design of new efficacious vaccines comes from the possibility of obtaining a detailed overview of the ab repertoire generated by infection . such an analysis applied to hiv - infected individuals has revealed that rare , naturally occurring anti - env bnabs are characterized by a large number of somatic mutations arising from multiple rounds of selection in response to emerging escape mutants of the hiv env glycoprotein ( mouquet et al . , 2010 ) . surprisingly , it was then found that germline ab precursors of these hiv bnabs have only very low affinity for most of the native heterologous env variants ; these observations suggested that immunogens based on natural env are inefficient in expanding the right germline b cell clones and are unable to elicit cross - neutralizing abs ( xiao et al . , 2009 ; liao et al . , 2013 ; doria - rose et al . , therefore , an alternative step - wise vaccination strategy has been recently proposed based on priming with an engineered antigen designed to target the germline precursors of hiv bnabs followed by a sequential boost with a series of intermediate antigens , which would progressively match the conformation of the natural env protein . although a complete demonstration of the validity of the germline - targeting vaccine approach has not been achieved yet , promising supporting data have been published in recent months . it was previously shown that a group of hiv cross - neutralizing abs from different donors , collectively called vrc01 class abs , targeting the cd4bs of hiv env derive from the same germline variable heavy chain vh1 - 2 * 02 and a few variable light chains characterized by a short ( only five amino acids ) cdr3 . in 2013 , jardine et al . ( 2013 ) reported the use of multiple rounds of computational design and structural analysis to engineer an external outer domain ( eod ) of the env protein able to bind the germline precursors of vrc01 abs with high affinity . the selected protein , eod - gt6 , was fused to a linker from a bacterial lumazine synthase to allow the formation of a 60-mer virus - like protein nanoparticle able to activate b cells expressing either a mature vrc01 b cell receptor or its germline precursor . more recently , an improved version of the multimeric germline - targeting antigen , eod - gt8 , has been used in two independent studies to immunize knock - in mice for the human vh1 - 2 * 02 variable heavy chain ( dosenovic et al . , 2015 ; jardine et al . , 2015 both studies have shown that eod - gt8 was able to expand b cells expressing human transgenic heavy chain combined to a mouse light chain with a five amino acids long cdr3 resembling naturally occurring light chains of human vrc01 , introducing a series of somatic mutations in the vh1 - 2 * 02 gene similar to those observed in naturally occurring hiv bnabs . in contrast to eod - gt8 , native env trimers were unable to expand vh1 - 2 * 02 b cells in the same knock - in mice . importantly , in both studies , the abs generated by immunization with eod - gt8 , although similar to vrc01 , were not able to neutralize hiv infection . in a study by dosenovic ( 2015 ) , eod - gt8 and env trimers were also used to immunize a different knock - in mouse strain expressing the mature heavy chain of the human vrc01 nabs , containing all naturally raised somatic mutations that would pair with a naive chain from the mouse repertoire . in this mouse strain , eod - gt8 was not efficient in triggering nab responses , whereas env trimers triggered a robust cross - neutralizing ab response through the selection of appropriate mouse light chains . the data generated so far did not demonstrate that it is possible to generate hiv broadly cross - neutralizing abs using only germline targeting antigens . however , they suggest that a germline targeting antigen should be used to prime naive b cells encoding the germline precursors of hiv bnabs , and then native - like hiv env antigens are required to select and expand b cells expressing cross - neutralizing abs as a consequence of the somatic hypermutation process . additional work using multiple intermediate env antigens is required to obtain a definitive proof of concept for the feasibility of the germline - targeting vaccination approach . recent technological advances in human immunology and structural biology have provided new reagents and improved tools that allow a better understanding of the basic biological and molecular mechanisms leading to a protective human immune response to pathogens , inspiring new strategies for vaccine design . for some of these approaches ( e.g. , stabilization of rsv prefusion f antigen and discovery of the hcmv pentamer complex ) , a solid preclinical proof of principle has been obtained supporting a clinical development . however , additional preclinical work is still required to demonstrate the validity of other rationally designed vaccines ( e.g. , universal flu or hiv germline targeting antigens ) . nevertheless , further investigations of the basic mechanisms regulating the human immune response to pathogens will continue to facilitate the design of new and more efficacious vaccines against infectious diseases . the combined application of the described new tools in human immunology and structural biology with microbial genomics will establish a new multidisciplinary approach to vaccine antigen discovery and design that may be termed reverse vaccinology 2.0 .

rappuoli et al . illustrate how new breakthroughs in the field of human immunology can be combined with insights from structural and computational biology for the rational design of novel broadly protective immunogens , potentiating the development of new vaccines against infectious diseases that still present an important unmet medical need .

Human B cell technologies to identify functional Abs against infectious diseases Use of protective Abs in structure-based antigen design HIV RSV Influenza virus A vaccine strategy targeting germline Abs Concluding remarks

nearly all licensed vaccines confer protection against infectious diseases by stimulating the production of pathogen - specific abs by b cells . understanding the nature of a successful ab response is therefore a fundamental step to providing new tools for the design of novel or better vaccines . dissecting the basic mechanisms that define the dynamics of the ab responses to vaccination and deepening the knowledge of the correlates of vaccine - induced protection or biological signatures of responsiveness are becoming fundamental in the development of novel vaccines . mbcs were shown to be more suitable for this kind of application because of their capability to secrete abs after ebv immortalization and in the presence of a tlr9 ligand and/or allogeneic irradiated mononuclear cells ( traggiai et al . more recently , it has been discovered that even single plasmablasts can be cultured without immortalization , and they can produce sufficient amounts of abs to allow screening for ab specificity and function ( jin et al . by screening mabs from an immune donor based on their capacity to neutralize infection in vitro , these abs recognize multiple antigenic sites on a pentameric complex of gh / gl / pul128/pul130/pul131a , which was not previously known to be the target of nabs ( macagno et al . , 2014 ) , providing evidence of the power of the reverse vaccinology 2.0 approach for the identification and design of improved hcmv vaccine candidates . furthermore , independent structural and immunological studies have also demonstrated the antigenic potential of the hcmv pentamer in mice ( wen et al . by screening b cell culture supernatants , a nab called d25many times more potent than palivizumab was isolated and shown to completely prevent viral replication in an in vivo rsv infection model in cotton rats ( kwakkenbos et al . in a subsequent study , it was shown that d25 and two additional highly potent rsv - specific nabs were specific for the prefusion form of rsv f ( mclellan et al . a milestone in understanding the nature and generation of successful ab responses has been the development of ab repertoire sequencing technologies for the identification and generation of recombinant human mabs . another hallmark in the field of human immunology has been the demonstration that cloning and expression of human mabs could be achieved through the recovery of the ig sequences from single b cells derived from the bone marrow and blood of healthy donors and by transfection of producer cells with the vh and vl chain genes ( wardemann et al . although this methodology allows for sequencing and expression of the abs encoded by a small number of b cells and individuals , it has brought numerous immunological insights to the development of the b cell response to antigens of a different nature . for example , it has been demonstrated that the b cell responses to polysaccharides , such as streptococcus pneumoniae polysaccharides , show high clonality with a limited ig gene usage and can be different in diverse age groups ( kolibab et al . novel recombinant and soluble hiv-1 envelope ( env ) proteins have been used as baits to identify and capture env ( gp160)specific b cells by flow cytometry in selected hiv - infected donors ( scheid et al . single - cell ab cloning techniques have successfully generated hundreds of hiv - specific abs and , among them , dozens of new extremely potent next - generation bnabs ( doria - rose and connors , 2009 ; klein et al . both of the aforementioned methods have two limitations for the identification of the best protective abs : first , the required ebv transformation of b cells can have a bias on the secretion of mabs ; and second , in vitro expressed synthetic abs might not always faithfully reflect their natural functional capability . a modified high - throughput approach has also recently been developed based on the isolation of mbcs from pbmcs and expansion in vitro without the need for activated t cell supernatants , allowing secretion of sufficient amounts of abs in culture to be screened in binding and functional assays ( huang et al . after characterization of the binding and/or functional capacities of the abs , the ig gene sequences can be recovered . recently , through this new approach , broad and potent hiv nabs have been discovered in the pool of antigen - specific mbcs , highlighting new vulnerability sites on the hiv env antigen ( huang et al . this approach is one of the most recent that allows for the interrogation of single - sorted b cells through direct screening of ab functionality . in turn , repertoire analyses to understand the origin and evolution of the abs of interest can be performed ( huang et al . although the convergent repertoire demonstrates changes in the large - scale structural features of the repertoire , the antigen specificity of the identified ab sequences always needs experimental confirmation through the expression , purification , and functional testing of recombinant abs . finally , in recent studies , the sequencing of paired vh / vl ig gene repertoire by next - generation sequencing technology has been combined with novel proteomic methods mining the antigen - specific ab repertoire that comprises the human serum polyclonal response ( wine et al . the new perspective offered by combining the analysis of the b cell and ab repertoire induced by tt vaccination has highlighted that the anti - tt serum igg repertoire is composed of a limited number of ab clonotypes ( 80100 ) , whereas the b cell receptor repertoire diversity in the memory and plasmablast compartments is orders of magnitude greater than that of the serological repertoire ( lavinder et al . collectively , all of the technologies that allow the dissection of the human ab response to pathogens are increasing our understanding of how protection is induced and can drive the design of more efficacious antigens for the development of vaccines to prevent current and future pathogenic threats . moreover , mab b12 showed a very limited effect in controlling viremia in vivo , even when combined with 4e10 , a distinct bnab with similar potency ( poignard et al . these findings again led researchers to believe that ab - based therapies and vaccines would be useless for hiv control , and the development of vaccines based on env components lost some momentum . subsequently , the ability to use single - cell technologies allowed for the identification of several hundred anti - hiv human mabs , some of which were bnabs with a median ic50 as low as 0.02 g / ml , > 1,000-fold better than the recombinant b12 ab isolated in 1994 ( scheid et al . delivery of bnab - encoding genes by gene therapy vectored immunoprophylaxis has also been shown to be an efficient method to prevent hiv infection in humanized mice , and it will be interesting to see if this strategy can be translated to protect humans against hiv ( balazs et al . the quest for an rsv vaccine has met many challenges that have inspired research breakthroughs , illustrating how information from human immunology and structural biology studies can be used in combination to instruct vaccine antigen design . early epitope - focused rational antigen designs were enabled by the cocrystal structure of the fab region of an rsv - neutralizing mab ( motavizumab , an affinity - enhanced form of palivizumab ) bound to its target f epitope prepared as a synthetic 24-residue peptide that adopted a helix - turn - helix motif in the crystal ( mclellan et al . thus , careful manipulations to properly present a structurally optimized form of a single well - defined protective epitope resulted in the generation of the desired immune response , demonstrating the feasibility of this novel immune - focusing approach . the latter highlighted the importance of renewed attempts to make a stable and efficacious prefusion f antigen , which was facilitated by the discovery and structural characterization of new mouse and human rsv - neutralizing mabs specific for prefusion f. crucially , a cocrystal structure reported by mclellan et al . yet another important area where human immunology is informing the design of novel immunogens is in the search for broadly protective vaccine antigens to protect against influenza ( flu ) , a persistent global public health threat ( lambert and fauci , 2010 ) . consequently , flu vaccine efficiency can be highly variable depending on how much the ha of the circulating flu strains matches the ha in the vaccine strains . therefore , an important goal is the development of a potent and universally protective flu vaccine to generate high titers of bnabs against influenza virus . the ha stem region is indeed highly conserved among different strains and subtypes of influenza virus , but this region is poorly immunogenic when in the full - length ha protein contained in conventional vaccines . however , the appropriateness of the ha stem region as a component in a universal flu vaccine was recently confirmed by the finding that bnabs against this region of ha can be selected from human subjects after infection or vaccination ( kashyap et al . , 2012 ; schneemann et al . to overcome this challenge , kanekiyo et al . such adjuvants may work by improving the development of t follicular helper cells and the germinal center antigen - specific b cell response ( mastelic gavillet et al . the identification of human mabs able to bind conserved epitopes in the ha stem region of several flu strains belonging to different subtypes can be considered the first step toward the discovery of an ideal immunogen for a universal flu vaccine . these abs are precious tools that can be used both for cocrystal structure studies to precisely identify the neutralizing epitopes in the ha stem region and for binding assays to isolate the most appropriate immunogen to induce a broad protection . two different groups have recently taken advantage of such anti ha stem abs and the cognate structurally derived epitope mapping information to rationally design and screen a series of novel immunogens , leading to selection of a stable immunogen containing the appropriate conformational epitopes from the ha stem ( impagliazzo et al . , 2015 ; yassine et al . a team led by nabel and graham have again taken advantage of ferritin nanoparticles to present a refined ha antigen comprised of a stem - only immunogen , which was rationally designed and stabilized in a trimeric headless state ( yassine et al . in the study , the design strategy was driven by prior human immunology studies and detailed structural knowledge that showed that the immunogenically subdominant stem region of ha contains highly conserved protective conformational epitopes recognized by human mabs with multisubtype ha recognition profiles broader than those targeting the more variable head region ( throsby et al . , 2008 ; ekiert et al . , 2009 , 2011 ; sui et al . although neutralization could be observed only for the homologous and two other heterologous h1 strains , vaccination with h1-ss - np resulted in the complete protection of mice and partial protection of ferrets against lethal viral challenge using the h5n1 heterosubtype . nevertheless , these small ha - stem constructs can be considered good starting points for a vaccine candidate raising a broadly protective immune response against group 1 flu viruses , which may be extendable to group 2 . collectively , such studies are fine demonstrations of how structure - based antigen designs can be used to obtain an improved immune response specifically directed toward a target epitope identified by human immunology studies and validated by structural biology studies . moreover , mab b12 showed a very limited effect in controlling viremia in vivo , even when combined with 4e10 , a distinct bnab with similar potency ( poignard et al . these findings again led researchers to believe that ab - based therapies and vaccines would be useless for hiv control , and the development of vaccines based on env components lost some momentum . first , using a neutralization screening of supernatants from thousands of mbcs from an hiv - infected donor , two more potent human mabs ( pg9 and pg16 ) targeting a broadly neutralizing epitope were described as able to neutralize 7080% of the tested hiv strains with an ic50 < 50 g / ml and 5060% with an ic50 < 1 g / ml ( walker et al . , mabs of the group called vrc01 ) targeting the conserved cd4bs on the env gp120 subunit were identified , and one of them was found able to neutralize up to 90% of hiv isolates at an ic50 < 50 g / ml and 70% at an ic50 < 1 g / ml , with an overall low geometric mean ic50 of 0.3 g / ml ( wu et al . subsequently , the ability to use single - cell technologies allowed for the identification of several hundred anti - hiv human mabs , some of which were bnabs with a median ic50 as low as 0.02 g / ml , > 1,000-fold better than the recombinant b12 ab isolated in 1994 ( scheid et al . , 2011 ; walker et al . the new findings described in the previous paragraph may completely change the approach to hiv prevention and therapy , and today , ab - mediated protection is believed to be a promising approach to hiv control . delivery of bnab - encoding genes by gene therapy vectored immunoprophylaxis has also been shown to be an efficient method to prevent hiv infection in humanized mice , and it will be interesting to see if this strategy can be translated to protect humans against hiv ( balazs et al . the quest for an rsv vaccine has met many challenges that have inspired research breakthroughs , illustrating how information from human immunology and structural biology studies can be used in combination to instruct vaccine antigen design . early epitope - focused rational antigen designs were enabled by the cocrystal structure of the fab region of an rsv - neutralizing mab ( motavizumab , an affinity - enhanced form of palivizumab ) bound to its target f epitope prepared as a synthetic 24-residue peptide that adopted a helix - turn - helix motif in the crystal ( mclellan et al . the latter highlighted the importance of renewed attempts to make a stable and efficacious prefusion f antigen , which was facilitated by the discovery and structural characterization of new mouse and human rsv - neutralizing mabs specific for prefusion f. crucially , a cocrystal structure reported by mclellan et al . yet another important area where human immunology is informing the design of novel immunogens is in the search for broadly protective vaccine antigens to protect against influenza ( flu ) , a persistent global public health threat ( lambert and fauci , 2010 ) . consequently , flu vaccine efficiency can be highly variable depending on how much the ha of the circulating flu strains matches the ha in the vaccine strains . therefore , an important goal is the development of a potent and universally protective flu vaccine to generate high titers of bnabs against influenza virus . such a vaccine should ideally induce an immune response against one or more conserved epitopes in the viral proteins to prevent infection or disease from all or nearly all possible circulating strains of the flu virus . however , the appropriateness of the ha stem region as a component in a universal flu vaccine was recently confirmed by the finding that bnabs against this region of ha can be selected from human subjects after infection or vaccination ( kashyap et al . such adjuvants may work by improving the development of t follicular helper cells and the germinal center antigen - specific b cell response ( mastelic gavillet et al . the identification of human mabs able to bind conserved epitopes in the ha stem region of several flu strains belonging to different subtypes can be considered the first step toward the discovery of an ideal immunogen for a universal flu vaccine . these abs are precious tools that can be used both for cocrystal structure studies to precisely identify the neutralizing epitopes in the ha stem region and for binding assays to isolate the most appropriate immunogen to induce a broad protection . two different groups have recently taken advantage of such anti ha stem abs and the cognate structurally derived epitope mapping information to rationally design and screen a series of novel immunogens , leading to selection of a stable immunogen containing the appropriate conformational epitopes from the ha stem ( impagliazzo et al . , 2015 ; yassine et al . in the study , the design strategy was driven by prior human immunology studies and detailed structural knowledge that showed that the immunogenically subdominant stem region of ha contains highly conserved protective conformational epitopes recognized by human mabs with multisubtype ha recognition profiles broader than those targeting the more variable head region ( throsby et al . , 2009 , 2011 ; sui et al . although neutralization could be observed only for the homologous and two other heterologous h1 strains , vaccination with h1-ss - np resulted in the complete protection of mice and partial protection of ferrets against lethal viral challenge using the h5n1 heterosubtype . ( 2015 ) , who used ingenious structure - based design methods combined with a library approach to generate minimally sized trimeric stem only ha immunogens ( termed mini - has ) , which were soluble and conformationally stable and satisfied the prerequisite to structurally and antigenically mimic the highly conserved stem region of full - length ha , as revealed by their capacity to bind anti ha stem bnabs . furthermore , in an nhp model , when compared with tiv , this immunogen elicited higher ab titers against homologous as well as a variety of heterologous and heterosubtypic strains that bound ha in the stem - neutralizing epitope and triggered potent ab - dependent cellular cytotoxicity activity and were able to neutralize an h5n1 heterosubtypic strain . nevertheless , these small ha - stem constructs can be considered good starting points for a vaccine candidate raising a broadly protective immune response against group 1 flu viruses , which may be extendable to group 2 . again , these studies appear to suggest that a strong adjuvant may be required to obtain an appropriate response to conserved subdominant epitopes in the ha stem region , most likely acting by enhancing t follicular helper cells in the germinal center and therefore driving selection of scarce b cells specific for these epitopes . collectively , such studies are fine demonstrations of how structure - based antigen designs can be used to obtain an improved immune response specifically directed toward a target epitope identified by human immunology studies and validated by structural biology studies . another great advance in our knowledge of the human immune response to pathogens that can aid the design of new efficacious vaccines comes from the possibility of obtaining a detailed overview of the ab repertoire generated by infection . , 2009 ; liao et al . , 2013 ; doria - rose et al . in 2013 , jardine et al . more recently , an improved version of the multimeric germline - targeting antigen , eod - gt8 , has been used in two independent studies to immunize knock - in mice for the human vh1 - 2 * 02 variable heavy chain ( dosenovic et al . , 2015 ; jardine et al . , 2015 both studies have shown that eod - gt8 was able to expand b cells expressing human transgenic heavy chain combined to a mouse light chain with a five amino acids long cdr3 resembling naturally occurring light chains of human vrc01 , introducing a series of somatic mutations in the vh1 - 2 * 02 gene similar to those observed in naturally occurring hiv bnabs . nevertheless , further investigations of the basic mechanisms regulating the human immune response to pathogens will continue to facilitate the design of new and more efficacious vaccines against infectious diseases .

the uncertainty quantification of pk parameters from preclinical experiments to clinical has been extensively investigated in the literature . prediction methods for clearance is by far the most studied case because of its importance for systemic drug elimination and oral bioavailability . clearance is defined as the volume of blood , from which the drug is removed , per unit of time . reported evaluations of in vivo clearance prediction - methods are often based on relatively small datasets ( up to 50 compounds ) , although a few investigations are based on larger datasets ( up to 400 compounds).9,12,17,20 the main reason for evaluation - data scarcity is that many compounds are only administered by the oral route to humans , making unambiguous distinction of in vivo clearance and bioavailability impossible . although some studies show that simple allometric prediction from a monkey performs best,12,17,21 there are typically cost and ethical reasons against using higher species in preclinical research . it has been suggested , albeit using smaller datasets , that the rat and dog data may perform equally well.6,13 allometry may indeed work well for compounds excreted renally , or whose clearance is limited by liver blood flow , but may not be applicable for low - clearance compounds limited by cytochrome p450 enzyme activity.13,29 in some studies , methods using correction factors , such as the rule of exponents method or fraction ( unbound ) corrected intercept method have been shown to predict better than allometry or at least decrease the risk of overprediction.6,8,12 including a correction for species differences in intrinsic clearance could also possibly decrease the prediction error of allometric methods.30 however , as reported by the publication using the largest dataset,12 the best allometric methods only predict approximately 60% of compounds within twofold of the human clearance . in vitro - in vivo extrapolation methods ( hepatocytes or microsomes ) for hepatic clearance predictions have varying success rates reported in the literature , ranging from 2090% of compounds predicted within twofold.6,13,14,22,3134 the variation may be due to the differences in experimental setups and in experimental / empirical correction factors ( see fagerholm14 for an extensive review ) . it has been reported that in vitro - in vivo extrapolation , using a regression - line correction established in the utilized hepatocyte assays , may be the method of choice because it will correct for systematic errors in the experimental setup.31 overall , it seems reasonable to suggest that the uncertainty in clearance predictions falls in the order of a factor three for high - performance methods ( this can , for example , be approximated by a lognormal distribution with 95% chance of falling within threefold from the predicted point estimate ) . volume of distribution at steady state ( vss ) is defined as the proportionality factor between the total amount of drugs in the body at steady state and plasma ( or blood ) concentration . similar to clearance , vss can be predicted using several different methods with allometry historically being most used . indeed , allometry may be a reasonable method to predict vss because of the physiological aspects governing this parameter , such as organ weights and volumes . there is little consensus of the best method for predicting vss.7,11,13,15,18 two publications7,11 suggest that the semimechanistic oie - tozer equation predicts relatively well if nonhuman in vivo data are available . to confidently predict vss , especially when in vivo data are scarce , it is important to consider the physicochemical properties of the compound and that they conform to the assumptions made in the model . one way to investigate the predictive power of a method is to apply the method between preclinical species.7 similar to clearance , it seems reasonable to suggest that the vss predictions will fall within threefold of the true value . the absorption of drugs from the gut into the systemic circulation is a complex dynamic process.35,36 the main determinants for the extent and kinetics of this process are dose , solubility / dissolution rate , and permeability . however , gut metabolism , stability of the drug in the intestine , fasted / fed state , and mass transfer may also affect the dynamics . bioavailability ( f ) is defined as : 1where fa denotes fraction absorbed , fg denotes fraction that escapes gut metabolism , and fh denotes fraction that escapes hepatic metabolism . absorption is often predicted using the biopharmaceutics classification system , which categorizes the candidate drugs on the basis of their solubility and permeability . compounds with high solubility and high permeability ( biopharmaceutics classification system i ) have an almost complete and rapid absorption . however , for compounds with low permeability or low solubility , or both ( biopharmaceutics classification system ii iv ) , it is more difficult to accurately predict the absorption process , and compounds in these classes may display a large variation in both extent and kinetics between patients and administrations.37,38 in vitro methods are routinely used to get a qualitative understanding of absorption properties.3941 translating fa and fg from animal to human is difficult because of interspecies differences in intestinal physiology as well as enzymatic activity.42,43 for example , bioavailability in dogs is often higher than in humans , possibly because of a more leaky intestine paracellular pathway,44,45 and monkeys often have lower bioavailability because of a higher enzymatic activity in the gut.46 physiologically based pharmacokinetic models are reported to underpredict bioavailability , but the number of investigated compounds is limited ( 8 per biopharmaceutics classification system class).19 most drug - discovery programs actively strive to develop compounds with sufficiently high solubility and permeability in order to reduce the uncertainty and risk of variation in exposure . for such compounds , it is reasonable to assume that absorption can be controlled by formulation . in the three case studies presented here , the working hypothesis has been that the compounds will have good absorption properties in humans . this has been based on in vitro ( permeability , crystalline solubility ) combined with in vivo animal pk studies . naturally , if solubility and permeability properties are less favorable , or gut metabolism is suspected , one should account for uncertainty in absorption parameters ( using the same principles as outlined for cl and vss ) . as mentioned earlier , differences in pd effect can be attributed to differences in metabolism between species ( i.e. , active metabolites ) , leading to different pharmacological / toxicological responses.1,47 another example is species differences in receptor distribution , which have been reported for the opioid receptors in the brain48 and in histamine receptor subtypes in bronchi.49 differences in relative receptor density between species can be found both for serotonergic and dopaminergic receptors.50,51 cardiac glycosides ( e.g. , digoxin and related compounds ) are an example of compounds with a large difference in affinity between species , in the order of 100-fold lower affinity in rodents compared with humans.52 further , translational modeling of disease progression is not straightforward , but can sometimes be scaled with the expected lifespan of the species.53 taking all these factors into account , uncertainty in pd can influence the dose prediction significantly , especially for a first - in - class compound targeting an unprecedented class of target or pathway . on the other hand , confidence increases when clinical data on the same target / pathway have been generated , the preclinical disease model has been shown to translate to human , or the system pharmacology of the target is understood , or a combination of those.5457 the human dose is often predicted using relatively simple models , which can be represented by closed - form expressions . as a base case , the dose prediction for a compound with assumed one - compartment kinetics , systemic clearance cl , a desired average steady - state concentration ( css ) , bioavailability f , and dosing interval is calculated as : 2 alternatively , for a compound with a desired coverage above a minimum effective concentration ( mec ) , the predicted dose is calculated as : 3where ka denotes the absorption rate - constant . for nonlinear models with no closed - form expression for dose , simulation - based methods are needed . to investigate the uncertainty of the dose prediction , pk and pd parameters can be represented by empirical distributions based on available information on translational performance . specifically , for a pk parameter , the distribution around the point estimate reflects the uncertainty of each method , as well as potential variability in input data . the shape of the distribution can be chosen based on reasonable assumptions or inferred empirically from compounds tested in man . a log - normal distribution is useful to avoid negative values ( exemplified in figure 1d ) . alternatives to log - normal distributions include fuzzy numbers or other interval analyses that are common in engineering applications58 and that have also been used in physiologically based pharmacokinetic applications2,23 ( exemplified in figure 1e ) . in those probabilistic frameworks , it is straightforward to include correlations between parameters . however , there is presently not much information in the literature on potential correlations of pk / pd parameter uncertainties . in the case studies , no correlations were incorporated in the monte - carlo simulations . in the next step , the model and the parameter distributions are fed into a monte - carlo simulation to calculate the distribution of the dose prediction . basically , random samples for each parameter are drawn from the chosen distribution ( the number of samples depends on the problem , and is typically greater than 1,000 ) . the dose is calculated for each set of parameters , resulting in an empirical dose distribution . monte - carlo simulations using closed - form dose expressions , like eqs . 2 and 3 , are computationally feasible and can typically be generated within milliseconds on a standard computer . for more complex pk / pd models , the calculation may be significantly slower , because each dose calculation involves an optimization ( e.g. , to assure that plasma concentration is above a certain threshold at a specific timepoint ) , and each evaluation step in the optimization requires numerical integration of the ordinary differential equations of the model . it is often expedient to present the resulting dose distribution as a cumulative probability plot ( which describes on the y - axis the probability that the dose is less than or equal to the value on the x - axis , cf . science communication in general and uncertainty communication in particular are actively discussed in the environmental science,5961 as well as in other areas , like radiotherapy.62,63 we mention the following main challenges in order to effectively communicate uncertainty in human dose - predictions : communication of results based on modeling and simulation is hard and requires careful preparation . such preparation is often performed iteratively and involves complete listing of modeling assumptions , translation of model equations into words or overview figures , choice of simulations and interpretation of results , and reflection on consequences of the modeling predictions . note in particular that formulas and technical terms are easy to list but difficult to interpret for scientists without adequate training in mathematics.60 human dose - predictions tend to focus on pk properties of the drug , and not so much on pd properties . however , translating the pd model from animal pharmacology to the human situation is based on assumptions and associated with uncertainties.1,23,24 therefore , no or low emphasis on pd uncertainty may underestimate overall uncertainty / risk and , hence , bias decision - makers . it is easy to mistake variability for uncertainty , and a consequent language is essential to avoid this problem . such preparation is often performed iteratively and involves complete listing of modeling assumptions , translation of model equations into words or overview figures , choice of simulations and interpretation of results , and reflection on consequences of the modeling predictions . note in particular that formulas and technical terms are easy to list but difficult to interpret for scientists without adequate training in mathematics.60 human dose - predictions tend to focus on pk properties of the drug , and not so much on pd properties . however , translating the pd model from animal pharmacology to the human situation is based on assumptions and associated with uncertainties.1,23,24 therefore , no or low emphasis on pd uncertainty may underestimate overall uncertainty / risk and , hence , bias decision - makers . it is easy to mistake variability for uncertainty , and a consequent language is essential to avoid this problem . fundamental communication elements are words , numbers , and visualizations . concerning words , it is important to be aware of variation in interpretation of phrases of likelihood ( e.g. , likely and probable).64,65 to elucidate the meaning of such words , it is recommended to define a consistent language according to numerical levels of confidence as exemplified by the verbal quantifiers in table 1 . the number of levels of such a table depends on the amount and quality of data , the type of research question , and the expected level of granularity of the response . it is also wise to strive for a simple language and avoid technical jargon.61 concerning numbers , they can be directly used to communicate , for example , confidence levels , and there is empirical evidence that including both verbal and numerical characterization improves communication.59 communication by visualization is central in science to convey causal relationships , temporal trends , data , etc.27 probability words as quantitative subjective probability judgments mapping of probability words into quantitative subjective probability judgments , used by the intergovernmental panel on climate change third assessment59,65 based on recommendations developed by moss & schneider.69 successful communication depends on several factors : identify the science relevant to decision - making ; separate need - to - know science from nice - to - know science . determine the decision - maker 's background knowledge in the subject area . design communication to fill the critical gaps between what people know and need to know . collect systematic feedback provided by empirical evaluation , and use the information to evaluate the adequacy of the communication and to revise it accordingly.60 identify the science relevant to decision - making ; separate need - to - know science from nice - to - know science . determine the decision - maker 's background knowledge in the subject area . design communication to fill the critical gaps between what people know and need to know . collect systematic feedback provided by empirical evaluation , and use the information to evaluate the adequacy of the communication and to revise it accordingly.60 the case studies that we show in the next section represent the final outcome of this iterative approach ( points 13 above ) . the feedback ( step 3 ) was continuously collected from various industrial and academic stakeholders during approximately one year and is summarized in the discussion section . we have chosen to communicate by visual means complemented with verbal quantifiers ( exemplified in table 1 ) . specifically , we have condensed complex information from multiple sources into a single uncomplicated plot that captures the uncertainty in human dose - prediction , and , in that way , pinpoints the need - to - know science . we present three case studies of how uncertainty in preclinical human dose - prediction can be quantified and communicated . in these case studies , clearance was estimated from in vitro and in vivo data using two different scaling methods . first , clearance was predicted from human hepatocyte data using a regression correction method based on 36 marketed drugs reflecting the same principles as in sohlenius - sternbeck et al.31 this study reports that 66% of compounds have a predicted intrinsic clearance within twofold . second , clearance was also scaled from in vivo data obtained from rats and dogs using the liver blood flow method.17 using a clinical dataset of 18 compounds , this method predicts in vivo clearance within twofold for 67% of the compounds and within threefold for 94% of the compounds.6 the uncertainty distribution for each clearance method was represented by a log - normal distribution with 95% of samples falling within threefold of the predicted point estimate . variability in input data to the scaling methods was considered negligible in comparison to the translational uncertainty . if two predictions are available for the same parameter , it is common to take the average value of the predictions as the point estimate . for each iteration of the monte - carlo simulation , one sample is drawn from each of the two distributions , and the average value is used . in this way , two ( similar ) predictions of cl , for example , result in a consensus prediction with less uncertainty than each individual prediction . volumes of distribution for all three case studies were predicted using the semi - mechanistic oie - tozer equation.66 for this method , 78% of the predictions were within twofold and 89% within threefold of the in vivo result using a dataset of 18 compounds.7 for a larger dataset of 400 compounds , 67% of the predictions were within twofold.11 unbound vss for each series in the case studies was consistent across species ( rat and dog ; i.e. , the slope of a simple unbound allometric plot fell between 0.8 and 1.2 ) , which further adds confidence in prediction . vss was assumed log - normally distributed with 95% of samples falling within threefold of the predicted point estimate . similar to clearance predictions , variability in input data to the scaling method was considered negligible in comparison to the translational uncertainty . for all case studies , it was assumed that rate of absorption was rapid ( ka = 1h ) , unless a scenario with an absorption - modifying formulation was simulated . in addition , the fraction of the dose absorbed was assumed high ( fa = 1 ) and gut metabolism negligible ( fg = 1 ) . hence , the major contributor to f was first - pass metabolism in the liver and f was calculated as : 4where lbf is the liver blood flow and cl is the systemic blood clearance . the target audiences for communication of the three case studies have been pk / pd - modelers , medicinal chemists , bioscientists , and governance bodies . the first case study considers five compounds ( cmp 1a1e ) , that were evaluated in the lead - optimization phase of a drug - discovery project . the aim was to support ranking of the compounds for subsequent progression into toxicological studies . for each compound , 3 . however , because of an estimated short half - life ( 34 hours ) of the compounds , small changes in pk parameters had a large impact on the dose prediction , thereby increasing overall uncertainty . the project team was considering an extended release ( er ) formulation in order to generate a flatter concentration - time profile and to decrease dependence on elimination half - life and thereby obtain a more clinically realistic dose level . during these discussions , it became apparent that understanding and efficiently communicating uncertainty in the dose predictions were pivotal to progress the project . parameters and distributions were generated as described in the uncertainty quantification section ( table 2 ; example in figure 1d ) . predictions for cl using methods based on in vitro data ( in vitro - in vivo extrapolation from hepatocytes ) and in vivo data ( rat / dog liver blood flow17 ) were in a similar range and did not diverge more than twofold for any of the compounds . the point estimate of mec was derived from potency measures in a whole blood in vitro assay ( table 2 ) . the in vitro assay potency results fell within twofold of the potency seen ex vivo in human whole blood , for two reference compounds with reported clinical data , targeting the same pathway . hence , the uncertainty in pd was set to let 95% of the samples fall within twofold of the point estimate . the absorption constant , ka , was fixed because it is a parameter that can be largely controlled by formulation choices as long as the compounds are judged to show properties suitable for er formulation . specifically , ka was kept fixed at 1 h and 0.1 h when simulating immediate release ( ir ) and er formulations . parameters for case study 1 cl , clearance ; lbf , liver blood flow ; mec , minimum effective concentration ; vss , volume of distribution at steady state ; heps , hepatocytes . point estimates and 95% uncertainty ranges . ranking of the compounds was communicated by a cumulative probability distribution of the daily dose for each compound , all summarized into a single plot combined with verbal quantifiers ( figure 2a ) . we next investigated the effect of an er formulation using the same communication layout ( figure 2b ) . as already pointed out , the uncertainty around the point estimate of the ir dose was large because of the short half - life of the compounds . for example , the 90% confidence interval for cmp 1e ranges from 10 mg to more than 100,000 mg . the confidence interval for an er preparation is significantly narrower and ranges from 450 mg . cmp 1b was predicted to have a somewhat longer half - life compared with the others , and , consequently , this compound exhibits the steepest slope in figure 2a . along the same line of reasoning , the cumulative probability curves for the er formulation generally show much steeper slopes , compared to the corresponding curves for the ir formulation . the tendency of the compounds to fall into two groups in the er scenario illustrates a substantial difference in potency between the compounds and it was deemed likely that cmp 1c1e would have a daily dose below 100 mg . based on the information in figure 2a , b and table 2 , the project team concluded that the point estimates for an ir dose were low enough to facilitate once daily dosing for the best compounds . however , the uncertainty in these dose estimates was relatively large and the lead - optimization program was planned to account for er feasibility . figure 2c efficiently communicated this rationale to decision - makers for one of the top compounds . in summary , the first case study demonstrates how proper uncertainty quantification and communication support compound selection in the lead - optimization phase of a drug - discovery project . it also shows how different administration schedules and formulations can be analyzed within this framework . the second case study considers five compounds ( cmp 2a2e ) , that were evaluated in the lead - optimization phase of another drug - discovery project . the project team explored a first - in - class target , and , in the current phase of the project , it was not known if the css ( eq . the goal was to select one compound for further toxicological and pharmacological profiling , taking both uncertainty in model and choice of dosing schedule into account . pk parameters and their uncertainties were quantified as described in the uncertainty quantification section . for pd , therefore , the mec and css for each compound were predicted from in vitro potency data , and threefold translational uncertainty was assumed around the predicted point estimates ( table 3 ) . dose predictions for the case of css - driven effect show that cmp 2a2d are likely to have a human dose < 500 mg ( figure 3a ) . however , taking the more conservative mec prediction into account reveals a pronounced separation with cmp 2a as the preferred compound because of its relatively long half - life ( 14 hours compared to 36 hours for the other compounds ; figure 3b ) . the plots in figure 3a , b illustrated the effect of the long half - life on the predicted human dose , and facilitated decision - making in the project . cmp 2a is likely to have a human dose < 500 mg , whereas the other compounds have a medium likelihood of a dose > 1,000 mg . parameter ranges ( table 3 ) , compounds ( cmps ) 2a2d are likely to have a human dose below 500 mg , and cmp 2e is likely to have a dose below 1,000 mg for a model with steady - state concentration ( css)-driven effect ( a ) . if the effect is driven by the minimal effective concentration , cmp 2a is likely to have a human dose below 500 mg , whereas the others are not ( b ) ; and assuming this model is medium likelihood that cmp 2a can be administered once daily ( c ) . parameters for case study 2 cl , clearance ; heps , hepatocytes ; mec , minimum effective concentration ; vss , volume of distribution at steady state . taking also other factors than the human dose - prediction into account , the project team prioritized cmp 2a and b and was interested in understanding the influence of once daily vs. twice daily dosing schedules . simulations indicate that cmp 2a may be feasible for once daily administration while cmp 2b is not ( figure 3c ) . hence , cmp 2a stands out as superior to cmp 2b based on the human dose - prediction . this compound was assumed to be least sensitive for different dosing approaches and was selected for further profiling . in summary , the second case study adds to the first by demonstrating how model uncertainty ( css - driven vs. mec - driven effect ) influences dose quantification and uncertainty level for different dosing schedules , and how these aspects can be quantified and efficiently communicated . the third case study applies a pk / pd - link model containing a one - compartment pk model ( first order absorption , linear elimination ) connected to a receptor - occupancy model with elementary kinetics defined as : 5where c denotes drug plasma concentration , rtot denotes the total receptor concentration , rc denotes the drug - receptor complex and kon and koff are kinetic parameters ( the dissociation constant is derived as kd = koff / kon ) . for this case study , the human dose was predicted by requiring a certain level of receptor occupancy ( e.g. , 50% ) over a certain period of the day ( e.g. , 16 hours ) . the aim was to predict the required human dose and its uncertainty for a candidate drug , cmp 3a , in order to assure a certain level of receptor occupancy . human pk parameters were predicted from empirical scaling from both in vitro ( hepatocytes ) and in vivo ( mouse / rat / dog ) data ( table 4 ) . parameters for case study 3 cl , clearance ; cmp , compound ; heps , hepatocytes ; lbf , liver blood flow ; pk , pharmacokinetic ; ro , receptor occupancy ; vss , volume of distribution at steady state ; pd , pharmacodynamic . point estimates and 95% uncertainty ranges for six parameters based on domain knowledge , data , and literature . for pd parameters , the required receptor occupancy level was observed at 24 hours in rodents ( rat 37% , mouse 63% ) and translated to the same average level , 50% , but at 16 hours in humans . the difference in coverage ( 24 hours in rodents compared to 16 hours in humans ) reflects well - established species differences in the particular disease area . the uncertainty for human translation was represented by a log - normal distribution with 95% chance of falling in the range of 3763% . furthermore , human point - estimates for receptor - occupancy parameters kd and koff were obtained from pk / pd analysis of mouse data ( with kd protein - binding corrected ) . uncertainty in determination of kd and we assume that , with 95% confidence , kd and koff in humans independently varies by a factor of three to corresponding mouse parameters . in summary , uncertainty in six key pk and pd parameters influenced the human dose prediction ( table 4 ) . using a pk / pd - link model , we algorithmically searched for the minimal required human dose for each combination of receptor - occupancy coverage and dosing schedule . this calculation was defined as an optimization problem : find the minimal predicted dose that ensures a certain level of receptor occupancy , given a certain number of doses per day , a certain coverage time , and the predicted human pk parameters . a simple half - interval search was used to find the minimal dose . here , we took advantage of a flexible and efficient matlab library to ensure computational feasibility.67 then , the monte - carlo simulation was used to integrate uncertainty from all specified sources and to predict empirical distributions for dose and cmax ( figure 4 ) . data indicate that it is likely that the human dose is < 200 mg when uncertainty in both pk and pd are integrated ( figure 4a ) . understanding of human pk would significantly reduce the overall uncertainty ; here simulated for the case with pk parameters fixed at the predicted point estimates ( figure 4a ) . in order to design preclinical safety studies and interpret in vitro safety screens , it is pivotal to have a good understanding of expected exposure levels in humans . the monte - carlo simulation outputs such predictions as well , and data indicate that it is likely that cmax is < 0.4 m taking pk / pd uncertainty into account ( figure 4b ) . overall , data indicate that pd uncertainty is important to consider , in particular when key biomarkers are not accessible in at least one of the species , and when change in disease state is not well understood . parameter ranges ( table 4 ) , it is likely that the human dose of compound ( cmp ) 3a is < 150 mg ( black solid line ) ( a ) ; it is likely that cmax in humans is < 0.5 m ( black solid line ) ( b ) . in both a and b , the red steeper curve illustrates the information gain if human pharmacokinetic ( pk ) was known ( here assumed known at the predicted point estimate ) and only uncertainty in pharmacodynamic ( pd ) contributed to overall uncertainty . in summary , the third case study adds to the two others first by demonstrating how arbitrary ( nonlinear ) pk / pd model structures , as well as uncertainty in several pd parameters can be considered . the analysis can be connected to a dose optimization step and still be computationally feasible when no closed - form dose expression can be analytically derived . the third case study also shows how other parameters , like cmax , can be communicated in the same way as dose . the first case study considers five compounds ( cmp 1a1e ) , that were evaluated in the lead - optimization phase of a drug - discovery project . the aim was to support ranking of the compounds for subsequent progression into toxicological studies . for each compound , 3 . however , because of an estimated short half - life ( 34 hours ) of the compounds , small changes in pk parameters had a large impact on the dose prediction , thereby increasing overall uncertainty . the project team was considering an extended release ( er ) formulation in order to generate a flatter concentration - time profile and to decrease dependence on elimination half - life and thereby obtain a more clinically realistic dose level . during these discussions , it became apparent that understanding and efficiently communicating uncertainty in the dose predictions were pivotal to progress the project . parameters and distributions were generated as described in the uncertainty quantification section ( table 2 ; example in figure 1d ) . predictions for cl using methods based on in vitro data ( in vitro - in vivo extrapolation from hepatocytes ) and in vivo data ( rat / dog liver blood flow17 ) were in a similar range and did not diverge more than twofold for any of the compounds . the point estimate of mec was derived from potency measures in a whole blood in vitro assay ( table 2 ) . the in vitro assay potency results fell within twofold of the potency seen ex vivo in human whole blood , for two reference compounds with reported clinical data , targeting the same pathway . hence , the uncertainty in pd was set to let 95% of the samples fall within twofold of the point estimate . the absorption constant , ka , was fixed because it is a parameter that can be largely controlled by formulation choices as long as the compounds are judged to show properties suitable for er formulation . specifically , ka was kept fixed at 1 h and 0.1 h when simulating immediate release ( ir ) and er formulations . parameters for case study 1 cl , clearance ; lbf , liver blood flow ; mec , minimum effective concentration ; vss , volume of distribution at steady state ; heps , hepatocytes . point estimates and 95% uncertainty ranges . the parameter t1/2 is derived from cl and vss . ranking of the compounds was communicated by a cumulative probability distribution of the daily dose for each compound , all summarized into a single plot combined with verbal quantifiers ( figure 2a ) . we next investigated the effect of an er formulation using the same communication layout ( figure 2b ) . as already pointed out , the uncertainty around the point estimate of the ir dose was large because of the short half - life of the compounds . for example , the 90% confidence interval for cmp 1e ranges from 10 mg to more than 100,000 mg . the confidence interval for an er preparation is significantly narrower and ranges from 450 mg . cmp 1b was predicted to have a somewhat longer half - life compared with the others , and , consequently , this compound exhibits the steepest slope in figure 2a . along the same line of reasoning , the cumulative probability curves for the er formulation generally show much steeper slopes , compared to the corresponding curves for the ir formulation . the tendency of the compounds to fall into two groups in the er scenario illustrates a substantial difference in potency between the compounds and it was deemed likely that cmp 1c1e would have a daily dose below 100 mg . based on the information in figure 2a , b and table 2 , the project team concluded that the point estimates for an ir dose were low enough to facilitate once daily dosing for the best compounds . however , the uncertainty in these dose estimates was relatively large and the lead - optimization program was planned to account for er feasibility . figure 2c efficiently communicated this rationale to decision - makers for one of the top compounds . in summary , the first case study demonstrates how proper uncertainty quantification and communication support compound selection in the lead - optimization phase of a drug - discovery project . it also shows how different administration schedules and formulations can be analyzed within this framework . the second case study considers five compounds ( cmp 2a2e ) , that were evaluated in the lead - optimization phase of another drug - discovery project . the project team explored a first - in - class target , and , in the current phase of the project , it was not known if the css ( eq . the goal was to select one compound for further toxicological and pharmacological profiling , taking both uncertainty in model and choice of dosing schedule into account . pk parameters and their uncertainties were quantified as described in the uncertainty quantification section . for pd , therefore , the mec and css for each compound were predicted from in vitro potency data , and threefold translational uncertainty was assumed around the predicted point estimates ( table 3 ) . dose predictions for the case of css - driven effect show that cmp 2a2d are likely to have a human dose < 500 mg ( figure 3a ) . however , taking the more conservative mec prediction into account reveals a pronounced separation with cmp 2a as the preferred compound because of its relatively long half - life ( 14 hours compared to 36 hours for the other compounds ; figure 3b ) . the plots in figure 3a , b illustrated the effect of the long half - life on the predicted human dose , and facilitated decision - making in the project . cmp 2a is likely to have a human dose < 500 mg , whereas the other compounds have a medium likelihood of a dose > 1,000 mg . parameter ranges ( table 3 ) , compounds ( cmps ) 2a2d are likely to have a human dose below 500 mg , and cmp 2e is likely to have a dose below 1,000 mg for a model with steady - state concentration ( css)-driven effect ( a ) . if the effect is driven by the minimal effective concentration , cmp 2a is likely to have a human dose below 500 mg , whereas the others are not ( b ) ; and assuming this model is medium likelihood that cmp 2a can be administered once daily ( c ) . parameters for case study 2 cl , clearance ; heps , hepatocytes ; mec , minimum effective concentration ; vss , volume of distribution at steady state . taking also other factors than the human dose - prediction into account , the project team prioritized cmp 2a and b and was interested in understanding the influence of once daily vs. twice daily dosing schedules . simulations indicate that cmp 2a may be feasible for once daily administration while cmp 2b is not ( figure 3c ) . hence , cmp 2a stands out as superior to cmp 2b based on the human dose - prediction . this compound was assumed to be least sensitive for different dosing approaches and was selected for further profiling . in summary , the second case study adds to the first by demonstrating how model uncertainty ( css - driven vs. mec - driven effect ) influences dose quantification and uncertainty level for different dosing schedules , and how these aspects can be quantified and efficiently communicated . the third case study applies a pk / pd - link model containing a one - compartment pk model ( first order absorption , linear elimination ) connected to a receptor - occupancy model with elementary kinetics defined as : 5where c denotes drug plasma concentration , rtot denotes the total receptor concentration , rc denotes the drug - receptor complex and kon and koff are kinetic parameters ( the dissociation constant is derived as kd = koff / kon ) . for this case study , the human dose was predicted by requiring a certain level of receptor occupancy ( e.g. , 50% ) over a certain period of the day ( e.g. , 16 hours ) . the aim was to predict the required human dose and its uncertainty for a candidate drug , cmp 3a , in order to assure a certain level of receptor occupancy . human pk parameters were predicted from empirical scaling from both in vitro ( hepatocytes ) and in vivo ( mouse / rat / dog ) data ( table 4 ) . parameters for case study 3 cl , clearance ; cmp , compound ; heps , hepatocytes ; lbf , liver blood flow ; pk , pharmacokinetic ; ro , receptor occupancy ; vss , volume of distribution at steady state ; pd , pharmacodynamic . point estimates and 95% uncertainty ranges for six parameters based on domain knowledge , data , and literature . for pd parameters , the required receptor occupancy level was observed at 24 hours in rodents ( rat 37% , mouse 63% ) and translated to the same average level , 50% , but at 16 hours in humans . the difference in coverage ( 24 hours in rodents compared to 16 hours in humans ) reflects well - established species differences in the particular disease area . the uncertainty for human translation was represented by a log - normal distribution with 95% chance of falling in the range of 3763% . furthermore , human point - estimates for receptor - occupancy parameters kd and koff were obtained from pk / pd analysis of mouse data ( with kd protein - binding corrected ) . uncertainty in determination of kd and koff from mouse data we assume that , with 95% confidence , kd and koff in humans independently varies by a factor of three to corresponding mouse parameters . in summary , uncertainty in six key pk and pd parameters influenced the human dose prediction ( table 4 ) . using a pk / pd - link model , we algorithmically searched for the minimal required human dose for each combination of receptor - occupancy coverage and dosing schedule . this calculation was defined as an optimization problem : find the minimal predicted dose that ensures a certain level of receptor occupancy , given a certain number of doses per day , a certain coverage time , and the predicted human pk parameters . a simple half - interval search was used to find the minimal dose . here , we took advantage of a flexible and efficient matlab library to ensure computational feasibility.67 then , the monte - carlo simulation was used to integrate uncertainty from all specified sources and to predict empirical distributions for dose and cmax ( figure 4 ) . data indicate that it is likely that the human dose is < 200 mg when uncertainty in both pk and pd are integrated ( figure 4a ) . understanding of human pk would significantly reduce the overall uncertainty ; here simulated for the case with pk parameters fixed at the predicted point estimates ( figure 4a ) . in order to design preclinical safety studies and interpret in vitro safety screens , it is pivotal to have a good understanding of expected exposure levels in humans . the monte - carlo simulation outputs such predictions as well , and data indicate that it is likely that cmax is < 0.4 m taking pk / pd uncertainty into account ( figure 4b ) . overall , data indicate that pd uncertainty is important to consider , in particular when key biomarkers are not accessible in at least one of the species , and when change in disease state is not well understood . parameter ranges ( table 4 ) , it is likely that the human dose of compound ( cmp ) 3a is < 150 mg ( black solid line ) ( a ) ; it is likely that cmax in humans is < 0.5 m ( black solid line ) ( b ) . in both a and b , the red steeper curve illustrates the information gain if human pharmacokinetic ( pk ) was known ( here assumed known at the predicted point estimate ) and only uncertainty in pharmacodynamic ( pd ) contributed to overall uncertainty . in summary , the third case study adds to the two others first by demonstrating how arbitrary ( nonlinear ) pk / pd model structures , as well as uncertainty in several pd parameters can be considered . the analysis can be connected to a dose optimization step and still be computationally feasible when no closed - form dose expression can be analytically derived . the third case study also shows how other parameters , like cmax , can be communicated in the same way as dose . in the case studies , uncertainty in human dose - prediction is quantified by a systematic integration of information from various uncertain sources , and reported as a single uncomplicated plot complemented with verbal quantifiers that facilitates communication and enables informed decisions . the approach allows drug - project teams to evaluate various scaling scenarios or models , to identify the most influencing parameters , to rank compounds ( e.g. , compounds with similar predicted dose point - estimates but different uncertainty ) , and to properly assess the risk before the first time in humans . other uncertainty considerations , such as safety aspects , commercial feasibility , cost of goods , and competitive landscape , can be directly connected to the framework as they often use the predicted human dose as one of the input parameters ( cf . the same reasoning can be applied to the predicted cmax or area under the curve distribution or other derived parameters . the empirical calculations can be performed efficiently , not only for closed - form expressions , but also for nonlinear ordinary differential equation systems . in this way , calculations can be instantly revised as soon as new data are generated , or a different scenario is to be tested . taken together , the approach exemplified by the three case studies represents a useful way of quantifying and communicating uncertainty in human dose - predictions . however , in our experience , such communications always raise questions ( e.g. , how is ranking influenced by variability / uncertainty , and what is the chance of having a dose less than a certain amount ) . in particular , this is the case when uncertainties differ between compounds ( because of different types , amounts , or quality of input data ) , or a combination of those . a monte - carlo approach can address these and other questions . there are several ways of communicating the result of a dose prediction based on a probabilistic approach . a concentration - time plot ( figure 1c ) is one obvious alternative to the cumulative probability plot of dose advocated in this tutorial ( figure 2a ) . first , we have experienced that many audiences misinterpret the uncertainty in the concentration - time plot as population variability . second , the concentration - time plot gets crowded when more than one compound or scenario , or both , are compared ( figure 1c shows just one compound and would be hard to interpret if all compounds 1a1e were included , as in figure 2a ) . nevertheless , the concentration - time plot remains a useful complement to the dose distribution . in case study 1 , the need to investigate er feasibility of the compound series was successfully communicated by figure 2 . it was also apparent that an er formulation was likely to generate a dose below 100 mg / day for any of the three compounds 1c e . the plots were used to illustrate the risk with the ir once daily approach and gain confidence in the chosen mitigation plan using an er preparation . in addition , figure 2c efficiently communicates that an er formulation would potentially be superior to twice daily administration for one of the compounds . indeed , the high uncertainty associated with once daily ir formulation ( figure 2a ) illustrates the importance of communicating uncertainties ; only communicating point estimates gives a false sense of security . in this and the two other case studies , the compounds show good absorption properties and bioavailability was derived from cl only . however , we acknowledge that a more conservative approach with uncertainty in fa can be argued . generally , for low permeability compounds or compounds predicted to be highly metabolized in the gut , the bioavailability estimate should take fa and fg into consideration . plausible uncertainty distributions for these parameters must then be proposed in the same way as for other parameters . the method used for human dose - prediction is drug - project specific and should be chosen based on the pharmacological understanding of the target . case study 2 shows the impact of model choice on the distribution of the predicted dose ( css vs. mec - driven approach ) . of course , model averaging can be considered in situations where information is lacking and a single model can not be selected . commonly , uncertainty in clearance and bioavailability , and sometimes volume of distribution and rate of absorption , strongly influences overall uncertainty in the prediction . uncertainty in pd parameters can have a large impact on dose , as seen in case study 3 , but is even more project - specific compared to pk parameters . uncertainty in pd can be decreased by , for example , understanding the target and its biological pathway , using preclinical animal models with known translational properties , or taking advantage of published clinical data for compounds with the same target or compounds already approved for the indication . the same type of communication can be used to inform about remaining uncertainty in pd when pk is known from a clinical phase i study ( cf . furthermore , for early toxicological studies , the uncertainty in predicted exposure levels ( cmax and area under the curve ) , can be used to better assess and communicate uncertainty in safety margins . in order to quantify uncertainty in the dose prediction , it is useful to list the contributing parameters and then assess the uncertainty of each parameter , as well as correlations between those measures if independence can not be assumed . naturally , it is not uncommon that compounds in a series are supported by various amounts of data ( e.g. , clearance can be predicted from either hepatocyte data , or animal data , or both ) . however , such differences in input data can be directly incorporated in the uncertainty calculation . notably , for cases where the predictions of a parameter vary substantially between two or more methods , it is advisable to further investigate the underlying reason because one of the methods may be more suitable for the compound series . however , in principle , the computational framework also works for this case . in general , it is hard to determine the translational uncertainty in scaling methods and pd relationships , and to assign or infer reasonable distributions representing those uncertainties . however , there is lack of consensus in ranking these methods , partly because datasets are limited and differ in size . different physicochemical properties for compounds may also influence which methods are best suited for a certain compound class . as a rule - of - thumb , literature data on translation of pk parameters indicate that good methods predict 8090% of the compounds within threefold from the true parameter value.622 however , these evaluations of methods are based on data for compounds that have reached the clinical phase and have been reported in the literature ; potential bias in any direction can not be excluded . in our case studies , we have assumed log - normally distributed uncertainty with 95% probability to be within threefold from the point estimate . it is recommended to increase confidence in prediction by using different types of data ( e.g. , in vitro and in vivo , more than one scaling method , and information from other compounds with similar properties ) . in our experience , decision - makers in drug discovery gain a lot from having a rough indication of the uncertainty levels . integrating information in the way proposed here quantifies the overall uncertainty ( although not being exact ) in a manner that is easy to understand . therefore , we believe it is worthwhile doing this analysis when uncertainty levels can be captured in at least the right order of magnitude . fundamental to science communication is to determine how much and what information the audience needs to make an informed decision ; what is considered good communication for one audience may not suit another . communicating modeling and simulation can often be difficult , especially when trying to describe the impact of assumptions and uncertainty to people not used to working with prediction models or translational aspects , or both . also , it is challenging to find the right balance between communication of the point estimate and the uncertainty . hence , finding the right communication strategy requires iterative trials with intermediate collection and analysis of feedback from the audience . generally , the reception of the communications has been very positive from various academic and industrial stakeholders from different scientific backgrounds . for example , medicinal chemists emphasize the value of rough uncertainty quantifications during the design - make - test - analyze process . negative feedback during the process was mainly related to the three challenges in order to effectively communicate uncertainty in human dose - predictions listed in the uncertainty communication section . to overcome these challenges , we have strived to avoid technical jargon and minimize the usage of equations in our presentation . furthermore , as pointed out by pidgeon and fischhoff , scientists from a different field can get an exaggerated sense of scientific uncertainty when uncertainties that are most difficult to understand or model are discussed in depth , whereas uncertainties of commonly accepted methods ( e.g. , a certain pk scaling method ) are never brought up . the approach used in the case studies is designed to put the same emphasis on all contributing factors to the overall scaling uncertainty in order to avoid this problem . the hardest communication problem has been related to the distinction between variability for uncertainty ( e.g. , the uncertainty distribution of cl may be incorrectly interpreted as the population variability of cl ) . to overcome this problem , we have strived to use a consequent language ( table 1 ) , focused on dose distribution plots and not concentration - time plots ( as discussed above ) . another important factor for successful communication is to allow the audience to take active part in the reasoning behind the prediction . by using efficient software , users can revisit and change the assumptions , or simulate a different scenario , and get an instant reply . communication should be designed specifically for the target audience ( see point 1 in the numbered list in section uncertainty communication ) . in our experience , the cumulative probability plots are useful both for modelers and nonmodelers ( e.g. , decision - makers ) . however , the underlying assumptions and calculation steps should be motivated more rigorously for modelers compared to nonmodelers . in this way , we can balance the right to know and full disclosure with need - to - know . however , the number of iterations can be reduced by a well chosen starting point . the approach outlined by three case studies in this tutorial can be such a useful starting point for communicating human dose - predictions in pharmaceutical research organizations . in summary , the three case studies have been communicated by three means ( figures 4 ; tables24 ) : a single plot showing the cumulative probability of predicted dose levels , allowing several compounds or scenarios , or both , to be simultaneously evaluated . a summary table of underlying assumptions , detailing which parameters were considered uncertain and to what extent . a headline interpreting the presented data and summarizing them into the key finding ( e.g. , it is likely that the human dose is < 100 mg ) . a single plot showing the cumulative probability of predicted dose levels , allowing several compounds or scenarios , or both , to be simultaneously evaluated . a summary table of underlying assumptions , detailing which parameters were considered uncertain and to what extent . a headline interpreting the presented data and summarizing them into the key finding ( e.g. , it is likely that the human dose is < 100 mg ) . together , these three pieces fit into a single slide in presentation format ( e.g. , powerpoint ) . simple because the data is easily generated , revised , and summarized into a single slide , efficient because the audience is provided the information that they need in a form they can use , and sound because we have strived to transparently acknowledge main sources of scientific uncertainty .

human dose - prediction is fundamental for ranking lead - optimization compounds in drug discovery and to inform design of early clinical trials . this tutorial describes how uncertainty in such predictions can be quantified and efficiently communicated to facilitate decision - making . using three drug - discovery case studies , we show how several uncertain pieces of input information can be integrated into one single uncomplicated plot with key predictions , including their uncertainties , for many compounds or for many scenarios , or both .

Uncertainty quantification Uncertainty communication CASE STUDIES Case study 1 Case study 2 Case study 3 DISCUSSION CONFLICT OF INTEREST

it has been suggested , albeit using smaller datasets , that the rat and dog data may perform equally well.6,13 allometry may indeed work well for compounds excreted renally , or whose clearance is limited by liver blood flow , but may not be applicable for low - clearance compounds limited by cytochrome p450 enzyme activity.13,29 in some studies , methods using correction factors , such as the rule of exponents method or fraction ( unbound ) corrected intercept method have been shown to predict better than allometry or at least decrease the risk of overprediction.6,8,12 including a correction for species differences in intrinsic clearance could also possibly decrease the prediction error of allometric methods.30 however , as reported by the publication using the largest dataset,12 the best allometric methods only predict approximately 60% of compounds within twofold of the human clearance . it has been reported that in vitro - in vivo extrapolation , using a regression - line correction established in the utilized hepatocyte assays , may be the method of choice because it will correct for systematic errors in the experimental setup.31 overall , it seems reasonable to suggest that the uncertainty in clearance predictions falls in the order of a factor three for high - performance methods ( this can , for example , be approximated by a lognormal distribution with 95% chance of falling within threefold from the predicted point estimate ) . however , for compounds with low permeability or low solubility , or both ( biopharmaceutics classification system ii iv ) , it is more difficult to accurately predict the absorption process , and compounds in these classes may display a large variation in both extent and kinetics between patients and administrations.37,38 in vitro methods are routinely used to get a qualitative understanding of absorption properties.3941 translating fa and fg from animal to human is difficult because of interspecies differences in intestinal physiology as well as enzymatic activity.42,43 for example , bioavailability in dogs is often higher than in humans , possibly because of a more leaky intestine paracellular pathway,44,45 and monkeys often have lower bioavailability because of a higher enzymatic activity in the gut.46 physiologically based pharmacokinetic models are reported to underpredict bioavailability , but the number of investigated compounds is limited ( 8 per biopharmaceutics classification system class).19 most drug - discovery programs actively strive to develop compounds with sufficiently high solubility and permeability in order to reduce the uncertainty and risk of variation in exposure . for such compounds , it is reasonable to assume that absorption can be controlled by formulation . naturally , if solubility and permeability properties are less favorable , or gut metabolism is suspected , one should account for uncertainty in absorption parameters ( using the same principles as outlined for cl and vss ) . , digoxin and related compounds ) are an example of compounds with a large difference in affinity between species , in the order of 100-fold lower affinity in rodents compared with humans.52 further , translational modeling of disease progression is not straightforward , but can sometimes be scaled with the expected lifespan of the species.53 taking all these factors into account , uncertainty in pd can influence the dose prediction significantly , especially for a first - in - class compound targeting an unprecedented class of target or pathway . on the other hand , confidence increases when clinical data on the same target / pathway have been generated , the preclinical disease model has been shown to translate to human , or the system pharmacology of the target is understood , or a combination of those.5457 the human dose is often predicted using relatively simple models , which can be represented by closed - form expressions . as a base case , the dose prediction for a compound with assumed one - compartment kinetics , systemic clearance cl , a desired average steady - state concentration ( css ) , bioavailability f , and dosing interval is calculated as : 2 alternatively , for a compound with a desired coverage above a minimum effective concentration ( mec ) , the predicted dose is calculated as : 3where ka denotes the absorption rate - constant . in the case studies , no correlations were incorporated in the monte - carlo simulations . science communication in general and uncertainty communication in particular are actively discussed in the environmental science,5961 as well as in other areas , like radiotherapy.62,63 we mention the following main challenges in order to effectively communicate uncertainty in human dose - predictions : communication of results based on modeling and simulation is hard and requires careful preparation . note in particular that formulas and technical terms are easy to list but difficult to interpret for scientists without adequate training in mathematics.60 human dose - predictions tend to focus on pk properties of the drug , and not so much on pd properties . note in particular that formulas and technical terms are easy to list but difficult to interpret for scientists without adequate training in mathematics.60 human dose - predictions tend to focus on pk properties of the drug , and not so much on pd properties . it is also wise to strive for a simple language and avoid technical jargon.61 concerning numbers , they can be directly used to communicate , for example , confidence levels , and there is empirical evidence that including both verbal and numerical characterization improves communication.59 communication by visualization is central in science to convey causal relationships , temporal trends , data , etc.27 probability words as quantitative subjective probability judgments mapping of probability words into quantitative subjective probability judgments , used by the intergovernmental panel on climate change third assessment59,65 based on recommendations developed by moss & schneider.69 successful communication depends on several factors : identify the science relevant to decision - making ; separate need - to - know science from nice - to - know science . collect systematic feedback provided by empirical evaluation , and use the information to evaluate the adequacy of the communication and to revise it accordingly.60 identify the science relevant to decision - making ; separate need - to - know science from nice - to - know science . collect systematic feedback provided by empirical evaluation , and use the information to evaluate the adequacy of the communication and to revise it accordingly.60 the case studies that we show in the next section represent the final outcome of this iterative approach ( points 13 above ) . specifically , we have condensed complex information from multiple sources into a single uncomplicated plot that captures the uncertainty in human dose - prediction , and , in that way , pinpoints the need - to - know science . we present three case studies of how uncertainty in preclinical human dose - prediction can be quantified and communicated . in these case studies , clearance was estimated from in vitro and in vivo data using two different scaling methods . volumes of distribution for all three case studies were predicted using the semi - mechanistic oie - tozer equation.66 for this method , 78% of the predictions were within twofold and 89% within threefold of the in vivo result using a dataset of 18 compounds.7 for a larger dataset of 400 compounds , 67% of the predictions were within twofold.11 unbound vss for each series in the case studies was consistent across species ( rat and dog ; i.e. for all case studies , it was assumed that rate of absorption was rapid ( ka = 1h ) , unless a scenario with an absorption - modifying formulation was simulated . the target audiences for communication of the three case studies have been pk / pd - modelers , medicinal chemists , bioscientists , and governance bodies . the first case study considers five compounds ( cmp 1a1e ) , that were evaluated in the lead - optimization phase of a drug - discovery project . during these discussions , it became apparent that understanding and efficiently communicating uncertainty in the dose predictions were pivotal to progress the project . based on the information in figure 2a , b and table 2 , the project team concluded that the point estimates for an ir dose were low enough to facilitate once daily dosing for the best compounds . however , the uncertainty in these dose estimates was relatively large and the lead - optimization program was planned to account for er feasibility . figure 2c efficiently communicated this rationale to decision - makers for one of the top compounds . in summary , the first case study demonstrates how proper uncertainty quantification and communication support compound selection in the lead - optimization phase of a drug - discovery project . it also shows how different administration schedules and formulations can be analyzed within this framework . the second case study considers five compounds ( cmp 2a2e ) , that were evaluated in the lead - optimization phase of another drug - discovery project . pk parameters and their uncertainties were quantified as described in the uncertainty quantification section . the plots in figure 3a , b illustrated the effect of the long half - life on the predicted human dose , and facilitated decision - making in the project . if the effect is driven by the minimal effective concentration , cmp 2a is likely to have a human dose below 500 mg , whereas the others are not ( b ) ; and assuming this model is medium likelihood that cmp 2a can be administered once daily ( c ) . taking also other factors than the human dose - prediction into account , the project team prioritized cmp 2a and b and was interested in understanding the influence of once daily vs. twice daily dosing schedules . hence , cmp 2a stands out as superior to cmp 2b based on the human dose - prediction . in summary , the second case study adds to the first by demonstrating how model uncertainty ( css - driven vs. mec - driven effect ) influences dose quantification and uncertainty level for different dosing schedules , and how these aspects can be quantified and efficiently communicated . uncertainty in determination of kd and we assume that , with 95% confidence , kd and koff in humans independently varies by a factor of three to corresponding mouse parameters . in summary , uncertainty in six key pk and pd parameters influenced the human dose prediction ( table 4 ) . using a pk / pd - link model , we algorithmically searched for the minimal required human dose for each combination of receptor - occupancy coverage and dosing schedule . here , we took advantage of a flexible and efficient matlab library to ensure computational feasibility.67 then , the monte - carlo simulation was used to integrate uncertainty from all specified sources and to predict empirical distributions for dose and cmax ( figure 4 ) . data indicate that it is likely that the human dose is < 200 mg when uncertainty in both pk and pd are integrated ( figure 4a ) . the monte - carlo simulation outputs such predictions as well , and data indicate that it is likely that cmax is < 0.4 m taking pk / pd uncertainty into account ( figure 4b ) . in both a and b , the red steeper curve illustrates the information gain if human pharmacokinetic ( pk ) was known ( here assumed known at the predicted point estimate ) and only uncertainty in pharmacodynamic ( pd ) contributed to overall uncertainty . in summary , the third case study adds to the two others first by demonstrating how arbitrary ( nonlinear ) pk / pd model structures , as well as uncertainty in several pd parameters can be considered . the analysis can be connected to a dose optimization step and still be computationally feasible when no closed - form dose expression can be analytically derived . the third case study also shows how other parameters , like cmax , can be communicated in the same way as dose . the first case study considers five compounds ( cmp 1a1e ) , that were evaluated in the lead - optimization phase of a drug - discovery project . during these discussions , it became apparent that understanding and efficiently communicating uncertainty in the dose predictions were pivotal to progress the project . however , the uncertainty in these dose estimates was relatively large and the lead - optimization program was planned to account for er feasibility . figure 2c efficiently communicated this rationale to decision - makers for one of the top compounds . in summary , the first case study demonstrates how proper uncertainty quantification and communication support compound selection in the lead - optimization phase of a drug - discovery project . the second case study considers five compounds ( cmp 2a2e ) , that were evaluated in the lead - optimization phase of another drug - discovery project . the plots in figure 3a , b illustrated the effect of the long half - life on the predicted human dose , and facilitated decision - making in the project . if the effect is driven by the minimal effective concentration , cmp 2a is likely to have a human dose below 500 mg , whereas the others are not ( b ) ; and assuming this model is medium likelihood that cmp 2a can be administered once daily ( c ) . taking also other factors than the human dose - prediction into account , the project team prioritized cmp 2a and b and was interested in understanding the influence of once daily vs. twice daily dosing schedules . hence , cmp 2a stands out as superior to cmp 2b based on the human dose - prediction . in summary , the second case study adds to the first by demonstrating how model uncertainty ( css - driven vs. mec - driven effect ) influences dose quantification and uncertainty level for different dosing schedules , and how these aspects can be quantified and efficiently communicated . in summary , uncertainty in six key pk and pd parameters influenced the human dose prediction ( table 4 ) . using a pk / pd - link model , we algorithmically searched for the minimal required human dose for each combination of receptor - occupancy coverage and dosing schedule . here , we took advantage of a flexible and efficient matlab library to ensure computational feasibility.67 then , the monte - carlo simulation was used to integrate uncertainty from all specified sources and to predict empirical distributions for dose and cmax ( figure 4 ) . data indicate that it is likely that the human dose is < 200 mg when uncertainty in both pk and pd are integrated ( figure 4a ) . the monte - carlo simulation outputs such predictions as well , and data indicate that it is likely that cmax is < 0.4 m taking pk / pd uncertainty into account ( figure 4b ) . in both a and b , the red steeper curve illustrates the information gain if human pharmacokinetic ( pk ) was known ( here assumed known at the predicted point estimate ) and only uncertainty in pharmacodynamic ( pd ) contributed to overall uncertainty . in summary , the third case study adds to the two others first by demonstrating how arbitrary ( nonlinear ) pk / pd model structures , as well as uncertainty in several pd parameters can be considered . the analysis can be connected to a dose optimization step and still be computationally feasible when no closed - form dose expression can be analytically derived . the third case study also shows how other parameters , like cmax , can be communicated in the same way as dose . in the case studies , uncertainty in human dose - prediction is quantified by a systematic integration of information from various uncertain sources , and reported as a single uncomplicated plot complemented with verbal quantifiers that facilitates communication and enables informed decisions . the approach allows drug - project teams to evaluate various scaling scenarios or models , to identify the most influencing parameters , to rank compounds ( e.g. other uncertainty considerations , such as safety aspects , commercial feasibility , cost of goods , and competitive landscape , can be directly connected to the framework as they often use the predicted human dose as one of the input parameters ( cf . the empirical calculations can be performed efficiently , not only for closed - form expressions , but also for nonlinear ordinary differential equation systems . in this way , calculations can be instantly revised as soon as new data are generated , or a different scenario is to be tested . taken together , the approach exemplified by the three case studies represents a useful way of quantifying and communicating uncertainty in human dose - predictions . in particular , this is the case when uncertainties differ between compounds ( because of different types , amounts , or quality of input data ) , or a combination of those . first , we have experienced that many audiences misinterpret the uncertainty in the concentration - time plot as population variability . second , the concentration - time plot gets crowded when more than one compound or scenario , or both , are compared ( figure 1c shows just one compound and would be hard to interpret if all compounds 1a1e were included , as in figure 2a ) . in this and the two other case studies , the compounds show good absorption properties and bioavailability was derived from cl only . however , we acknowledge that a more conservative approach with uncertainty in fa can be argued . generally , for low permeability compounds or compounds predicted to be highly metabolized in the gut , the bioavailability estimate should take fa and fg into consideration . the method used for human dose - prediction is drug - project specific and should be chosen based on the pharmacological understanding of the target . commonly , uncertainty in clearance and bioavailability , and sometimes volume of distribution and rate of absorption , strongly influences overall uncertainty in the prediction . uncertainty in pd parameters can have a large impact on dose , as seen in case study 3 , but is even more project - specific compared to pk parameters . uncertainty in pd can be decreased by , for example , understanding the target and its biological pathway , using preclinical animal models with known translational properties , or taking advantage of published clinical data for compounds with the same target or compounds already approved for the indication . the same type of communication can be used to inform about remaining uncertainty in pd when pk is known from a clinical phase i study ( cf . furthermore , for early toxicological studies , the uncertainty in predicted exposure levels ( cmax and area under the curve ) , can be used to better assess and communicate uncertainty in safety margins . naturally , it is not uncommon that compounds in a series are supported by various amounts of data ( e.g. , clearance can be predicted from either hepatocyte data , or animal data , or both ) . however , such differences in input data can be directly incorporated in the uncertainty calculation . in general , it is hard to determine the translational uncertainty in scaling methods and pd relationships , and to assign or infer reasonable distributions representing those uncertainties . in our case studies , we have assumed log - normally distributed uncertainty with 95% probability to be within threefold from the point estimate . in our experience , decision - makers in drug discovery gain a lot from having a rough indication of the uncertainty levels . therefore , we believe it is worthwhile doing this analysis when uncertainty levels can be captured in at least the right order of magnitude . communicating modeling and simulation can often be difficult , especially when trying to describe the impact of assumptions and uncertainty to people not used to working with prediction models or translational aspects , or both . negative feedback during the process was mainly related to the three challenges in order to effectively communicate uncertainty in human dose - predictions listed in the uncertainty communication section . the approach used in the case studies is designed to put the same emphasis on all contributing factors to the overall scaling uncertainty in order to avoid this problem . the approach outlined by three case studies in this tutorial can be such a useful starting point for communicating human dose - predictions in pharmaceutical research organizations . in summary , the three case studies have been communicated by three means ( figures 4 ; tables24 ) : a single plot showing the cumulative probability of predicted dose levels , allowing several compounds or scenarios , or both , to be simultaneously evaluated . a summary table of underlying assumptions , detailing which parameters were considered uncertain and to what extent . , it is likely that the human dose is < 100 mg ) . a single plot showing the cumulative probability of predicted dose levels , allowing several compounds or scenarios , or both , to be simultaneously evaluated . a summary table of underlying assumptions , detailing which parameters were considered uncertain and to what extent .

recently , it was reported that the total prevalence of diabetes mellitus in the chinese adult population is 11.6% . diabetes has become the second most common cause of end - stage renal disease ( esrd ) in china . with the rapidly growing use of peritoneal dialysis ( pd ) as renal replacement therapy in china , the issue of the survival rate of diabetic nephropathy patients on pd has been raised . canusa and some other studies have suggested that a high peritoneal transport status is associated with higher mortality and technique failure in pd patients . however , some recent studies demonstrated that a high peritoneal transport status by itself was not an independent risk factor for mortality and technique failure . to our knowledge , all of the above - mentioned studies included diabetic and nondiabetic patients as study subjects . actually , many studies have confirmed that diabetes itself is an independent risk factor for mortality in pd patients . therefore , we chose patients with diabetic nephropathy as research subjects to evaluate the influence of peritoneal transport characteristics on nutritional status and clinical outcome in this specific population on pd . all patients who commenced pd between january 1 , 2005 and march 31 , 2013 in the first affiliated hospital of zhejiang university , school of medicine , were eligible for the study . inclusion criteria were as follows : ( a ) the primary cause of esrd was diabetic nephropathy with type 2 diabetes ; ( b ) age 18 years at the start of pd and survival for at least 3 months from the first pd therapy ; ( c ) a peritoneal equilibration test ( pet ) had been performed within the first 6 months of pd commencement ; ( d ) pd modality was continuous ambulatory pd ( capd ) or daytime ambulatory pd . the pd patients who transferred from hd or failed renal transplantation were excluded in this study . eventually , a total of 102 patients were enrolled in the study from all of the 1115 pd patients . dianeal pd solution ( containing 1.5% and 2.5% glucose ) was used in this study ( baxter , china ) . baseline patient demographic characteristics were collected including gender , age , body mass index , and history of cardio - cerebrovascular disease ( cvd ) . cvd was defined as myocardial infarction , angina , congestive heart failure , and cerebrovascular event . clinical and biochemical data at the initial pet of pd ( baseline ) included blood pressure , pd dose , daily transperitoneal glucose exposure , urine volume , peritoneal ultrafiltration volume , electrolytes , intact parathyroid hormone , glycated hemoglobin a1c ( hba1c ) , high - sensitivity c - reactive protein ( hscrp ) . the protein losses through peritoneal dialysate and urine were measured from the collection of 24-h peritoneal dialysate effluent and urine . adequacy of dialysis was estimated by measurement of total weekly urea clearance ( kt / v ) and total weekly creatinine clearance ( tccr ) per 1.73 m body surface area . residual renal function ( rrf ) was estimated by calculating the average residual renal clearance of urea and creatinine as described by van olden et al . peritoneal transport characteristic was evaluated by a standard pet which was performed with the 4-h dialysate / plasma creatinine ratio ( d / pcr ) used to classify a patient as high ( h ) , high average ( ha ) , low average ( la ) , or low ( l ) . according to twardowski , h was defined by d / pcr as 0.811.03 , ha as 0.650.80 , la as 0.500.64 and l as 0.340.49 . on the basis of initial pet result , patients were divided into two groups : higher transport group ( ht , including h and ha ) and lower transport group ( lt , including l and la ) . serum albumin ( alb ) , hemoglobin ( hb ) , daily protein intake ( dpi ) , normalized protein equivalent of total nitrogen appearance ( npna ) , and subjective global assessment ( sga ) were measured to determine the nutritional status during the initial pet and the last follow - up . sga scores as proposed by the canusa peritoneal dialysis study group , were coded according to a seven - point graded scale ( 12 , severe malnutrition ; 35 , mild to moderate malnutrition ; 67 , normal nutritional status ) . results are expressed as mean standard deviation for continuous data and as frequencies and percentages for categorical data . the differences between the groups were calculated by unpaired t - test , chi - square test , or mann whitney test as appropriate . a paired t - test was used to determine differences in the measured parameters between the two periods . survival curves were constructed by the kaplan meier method and compared by the log - rank test . in the analysis of patient survival , the censored events for both patient and technique survival were renal transplantation , move to another center , or still on pd at march 31 , 2014 . in addition , for patient survival , the censored data included switching to hemodialysis , and for technique survival , death as the censored data . univariate and multivariate cox proportional hazard regression models were used to analyze the risk factors for mortality and technique failure . variables with p < 0.1 in the univariate cox analysis were presented further to the multivariable cox regression analysis using backward stepwise elimination based on the likelihood ratio . data were analyzed using the software package spss for windows release 20.0 ( spss , chicago , il , usa ) . all patients who commenced pd between january 1 , 2005 and march 31 , 2013 in the first affiliated hospital of zhejiang university , school of medicine , were eligible for the study . inclusion criteria were as follows : ( a ) the primary cause of esrd was diabetic nephropathy with type 2 diabetes ; ( b ) age 18 years at the start of pd and survival for at least 3 months from the first pd therapy ; ( c ) a peritoneal equilibration test ( pet ) had been performed within the first 6 months of pd commencement ; ( d ) pd modality was continuous ambulatory pd ( capd ) or daytime ambulatory pd . the pd patients who transferred from hd or failed renal transplantation were excluded in this study . eventually , a total of 102 patients were enrolled in the study from all of the 1115 pd patients . dianeal pd solution ( containing 1.5% and 2.5% glucose ) was used in this study ( baxter , china ) . baseline patient demographic characteristics were collected including gender , age , body mass index , and history of cardio - cerebrovascular disease ( cvd ) . cvd was defined as myocardial infarction , angina , congestive heart failure , and cerebrovascular event . clinical and biochemical data at the initial pet of pd ( baseline ) included blood pressure , pd dose , daily transperitoneal glucose exposure , urine volume , peritoneal ultrafiltration volume , electrolytes , intact parathyroid hormone , glycated hemoglobin a1c ( hba1c ) , high - sensitivity c - reactive protein ( hscrp ) . the protein losses through peritoneal dialysate and urine were measured from the collection of 24-h peritoneal dialysate effluent and urine . adequacy of dialysis was estimated by measurement of total weekly urea clearance ( kt / v ) and total weekly creatinine clearance ( tccr ) per 1.73 m body surface area . residual renal function ( rrf ) was estimated by calculating the average residual renal clearance of urea and creatinine as described by van olden et al . peritoneal transport characteristic was evaluated by a standard pet which was performed with the 4-h dialysate / plasma creatinine ratio ( d / pcr ) used to classify a patient as high ( h ) , high average ( ha ) , low average ( la ) , or low ( l ) . according to twardowski , h was defined by d / pcr as 0.811.03 , ha as 0.650.80 , la as 0.500.64 and l as 0.340.49 . on the basis of initial pet result , patients were divided into two groups : higher transport group ( ht , including h and ha ) and lower transport group ( lt , including l and la ) . serum albumin ( alb ) , hemoglobin ( hb ) , daily protein intake ( dpi ) , normalized protein equivalent of total nitrogen appearance ( npna ) , and subjective global assessment ( sga ) were measured to determine the nutritional status during the initial pet and the last follow - up . sga scores as proposed by the canusa peritoneal dialysis study group , were coded according to a seven - point graded scale ( 12 , severe malnutrition ; 35 , mild to moderate malnutrition ; 67 , normal nutritional status ) . results are expressed as mean standard deviation for continuous data and as frequencies and percentages for categorical data . the differences between the groups were calculated by unpaired t - test , chi - square test , or mann whitney test as appropriate . a paired t - test was used to determine differences in the measured parameters between the two periods . survival curves were constructed by the kaplan meier method and compared by the log - rank test . in the analysis of patient survival , the censored events for both patient and technique survival were renal transplantation , move to another center , or still on pd at march 31 , 2014 . in addition , for patient survival , the censored data included switching to hemodialysis , and for technique survival , death as the censored data . univariate and multivariate cox proportional hazard regression models were used to analyze the risk factors for mortality and technique failure . variables with p < 0.1 in the univariate cox analysis were presented further to the multivariable cox regression analysis using backward stepwise elimination based on the likelihood ratio . data were analyzed using the software package spss for windows release 20.0 ( spss , chicago , il , usa ) . among the 102 patients , 65 patients were classified as higher peritoneal transporters ( ht group , including h 8 patients and ha 57 patients ) . the other 37 patients were classified as lower peritoneal transporters ( lt group , including la 32 patients and l 5 patients ) . in all of the evaluated items below , glucose exposure , 24-h dialysate protein , and tccr were significantly higher in ht group compared with those in lt group . in the meantime , ht group had lower peritoneal ultrafiltration volume than lt group . there were no significant differences between two groups for other items [ table 1 ] . baseline demographic and clinical characteristics of patients in the higher and lower transport group bmi : body mass index ; pet : peritoneal equilibration test ; cvd : cardio - cerebrovascular disease ; capd : continuous ambulatory peritoneal dialysis ; dapd : daytime ambulatory peritoneal dialysis ; ipth : intact parathyroid hormone ; hba1c : glycated hemoglobin a1c ; hscrp : high - sensitivity c - reactive protein ; rrf : residual renal function ; lt : lower peritoneal transport group ; ht : higher peritoneal transport group ; pd : peritoneal dialysis ; tccr : total clearance of creatinine . by the end of the study , 22 patients had died ( 16 in ht group , 6 in lt group ) , 28 transferred to hemodialysis , 6 had received a renal transplantation , and 3 were lost to follow - up . the most common cause of death was cardio - cerebrovascular events ( 68.2% ) . and other cause of technique failure included peritonitis ( 21.4% ) , inadequate dialysis ( 14.3% ) , catheter - related causes ( 10.8% ) , and pleuroperitoneal communication or subjective factors ( 21.4% ) . there were no significant differences between the two groups with respect to pd time , hb , baseline alb , and baseline npna ( p > 0.05 ) . however , we observed a significant difference in the levels of alb , dpi , npna , and sga between two groups over time . compared with lt group , alb , dpi , and npna meanwhile , the incidence of malnutrition by sga in ht group was significantly higher than lt group at the last follow - up [ table 2 ] . comparison of nutritional status in the higher and lower transport group over time * p<0.05 versus before values in the same group ; p<0.05 versus ht group . pet : peritoneal equilibration test ; alb : serum albumin ; hb : hemoglobin ; dpi : daily protein intake ; npna : normalized protein equivalent of total nitrogen appearance ; sga : subjective global assessment ; lt : lower peritoneal transport group ; ht : higher peritoneal transport group ; pd : peritoneal dialysis . pearson 's correlation analysis showed that the d / pcr was negatively correlated with alb and dpi [ figure 1 ] . meanwhile , the d / pcr was positively correlated with hscrp and 24-h dialysate protein [ figure 1 ] . in addition , alb was positively correlated with dpi ( r = 0.301 , p < 0.05 ) , and negatively correlated with hscrp ( r = 0.216 , p < 0.05 ) . correlation between d / pcr and alb ( a ) ; dpi ( b ) ; hscrp ( c ) and 24 h dialysate protein ( d ) . d / pcr : dialysate / plasma creatinine ratio ; alb : serum albumin ; dpi : daily protein intake ; hscrp : high - sensitivity c - reactive protein . death - censored technique survival rates at 1 , 2 , and 3 years were 91.8% , 71.0% , and 50.1% , respectively , in the ht group ; 97.3% , 90.9% , and 82.4% in the lt group , respectively . compared with lt group , death - censored technique failure was significantly increased in ht group ( log - rank p = 0.025 ) [ figure 2 ] . cumulative proportional patient survival at 1 , 2 , and 3 years were 90.2% , 77.6% , and 61.1% , respectively , in the ht group ; 94.4% , 90.8% , and 84.3% , respectively , in the lt group . a higher risk of mortality in ht group was observed than in lt group ( log - rank p = 0.047 ) [ figure 3 ] . patient survival curves for peritoneal transport characteristics . for the analysis of risk factors for death - censored technique failure , variables with p < 0.1 in the univariate cox analysis were higher peritoneal transport status , alb , rrf , and kt / v . after multivariate cox proportional hazards model analysis , higher peritoneal transport status ( hazard ratio [ hr ] : 2.311 ; 95% confidence interval [ ci ] : 1.0035.323 ) and rrf ( hr : 0.718 ; 95% ci : 0.5320.968 ) were independent predictor of death - censored technique failure [ table 3 ] . screening with univariate cox analysis , possible risk factors contributing to mortality were age , higher peritoneal transport status , alb , hb , hba1c , tccr , rrf , and npna . however , after multivariate cox proportional hazards model analysis , mortality was independently predicted by advanced age , anemia , hypoalbuminemia , and lower rrf , but not higher peritoneal transport status [ table 4 ] . predictors of death - censored technical failure by multivariate cox regression analysis ht : higher peritoneal transport status ; rrf : residual renal function ; ci : confidence interval ; se : standard error ; hr : hazard ratio . predictors of mortality by multivariate cox regression analysis ht : higher peritoneal transport status ; alb : serum albumin ; hb : hemoglobin ; rrf : residual renal function ; se : standard error ; hr : hazard ratio ; ci : confidence interval . among the 102 patients , 65 patients were classified as higher peritoneal transporters ( ht group , including h 8 patients and ha 57 patients ) . the other 37 patients were classified as lower peritoneal transporters ( lt group , including la 32 patients and l 5 patients ) . in all of the evaluated items below , glucose exposure , 24-h dialysate protein , and tccr were significantly higher in ht group compared with those in lt group . in the meantime , ht group had lower peritoneal ultrafiltration volume than lt group . there were no significant differences between two groups for other items [ table 1 ] . baseline demographic and clinical characteristics of patients in the higher and lower transport group bmi : body mass index ; pet : peritoneal equilibration test ; cvd : cardio - cerebrovascular disease ; capd : continuous ambulatory peritoneal dialysis ; dapd : daytime ambulatory peritoneal dialysis ; ipth : intact parathyroid hormone ; hba1c : glycated hemoglobin a1c ; hscrp : high - sensitivity c - reactive protein ; rrf : residual renal function ; lt : lower peritoneal transport group ; ht : higher peritoneal transport group ; pd : peritoneal dialysis ; tccr : total clearance of creatinine . by the end of the study , 22 patients had died ( 16 in ht group , 6 in lt group ) , 28 transferred to hemodialysis , 6 had received a renal transplantation , and 3 were lost to follow - up . the most common cause of death was cardio - cerebrovascular events ( 68.2% ) . and other cause of technique failure included peritonitis ( 21.4% ) , inadequate dialysis ( 14.3% ) , catheter - related causes ( 10.8% ) , and pleuroperitoneal communication or subjective factors ( 21.4% ) . the median follow - up time was 22.6 months . there were no significant differences between the two groups with respect to pd time , hb , baseline alb , and baseline npna ( p > 0.05 ) . however , we observed a significant difference in the levels of alb , dpi , npna , and sga between two groups over time . compared with lt group , alb , dpi , and npna meanwhile , the incidence of malnutrition by sga in ht group was significantly higher than lt group at the last follow - up [ table 2 ] . comparison of nutritional status in the higher and lower transport group over time * p<0.05 versus before values in the same group ; p<0.05 versus ht group . pet : peritoneal equilibration test ; alb : serum albumin ; hb : hemoglobin ; dpi : daily protein intake ; npna : normalized protein equivalent of total nitrogen appearance ; sga : subjective global assessment ; lt : lower peritoneal transport group ; ht : higher peritoneal transport group ; pd : peritoneal dialysis . pearson 's correlation analysis showed that the d / pcr was negatively correlated with alb and dpi [ figure 1 ] . meanwhile , the d / pcr was positively correlated with hscrp and 24-h dialysate protein [ figure 1 ] . in addition , alb was positively correlated with dpi ( r = 0.301 , p < 0.05 ) , and negatively correlated with hscrp ( r = 0.216 , p < 0.05 ) . correlation between d / pcr and alb ( a ) ; dpi ( b ) ; hscrp ( c ) and 24 h dialysate protein ( d ) . d / pcr : dialysate / plasma creatinine ratio ; alb : serum albumin ; dpi : daily protein intake ; hscrp : high - sensitivity c - reactive protein . death - censored technique survival rates at 1 , 2 , and 3 years were 91.8% , 71.0% , and 50.1% , respectively , in the ht group ; 97.3% , 90.9% , and 82.4% in the lt group , respectively . compared with lt group , death - censored technique failure was significantly increased in ht group ( log - rank p = 0.025 ) [ figure 2 ] . cumulative proportional patient survival at 1 , 2 , and 3 years were 90.2% , 77.6% , and 61.1% , respectively , in the ht group ; 94.4% , 90.8% , and 84.3% , respectively , in the lt group . a higher risk of mortality in ht group was observed than in lt group ( log - rank p = 0.047 ) [ figure 3 ] . for the analysis of risk factors for death - censored technique failure , variables with p < 0.1 in the univariate cox analysis were higher peritoneal transport status , alb , rrf , and kt / v . after multivariate cox proportional hazards model analysis , higher peritoneal transport status ( hazard ratio [ hr ] : 2.311 ; 95% confidence interval [ ci ] : 1.0035.323 ) and rrf ( hr : 0.718 ; 95% ci : 0.5320.968 ) were independent predictor of death - censored technique failure [ table 3 ] . screening with univariate cox analysis , possible risk factors contributing to mortality were age , higher peritoneal transport status , alb , hb , hba1c , tccr , rrf , and npna . however , after multivariate cox proportional hazards model analysis , mortality was independently predicted by advanced age , anemia , hypoalbuminemia , and lower rrf , but not higher peritoneal transport status [ table 4 ] . predictors of death - censored technical failure by multivariate cox regression analysis ht : higher peritoneal transport status ; rrf : residual renal function ; ci : confidence interval ; se : standard error ; hr : hazard ratio . predictors of mortality by multivariate cox regression analysis ht : higher peritoneal transport status ; alb : serum albumin ; hb : hemoglobin ; rrf : residual renal function ; se : standard error ; hr : hazard ratio ; ci : confidence interval . peritoneal transport characteristics are assessed primarily on the capability of peritoneal small - solute clearances . patients with higher peritoneal transport status tend to have enhanced clearance of small solutes , and easier to achieve the objective dialysis adequacy target . however , the ademex trial has demonstrated that no survival advantage was obtained with increases in peritoneal small - solute clearances within usual pd dosing regimens . to our knowledge , the role of peritoneal transport characteristics on technique failure and mortality of pd patients remains controversial . furthermore , it was reported that the survival of diabetic pd patients is inferior to nondiabetic pd patients in china . besides , diabetic patients are more prone to malnutrition relative to nondiabetic patients . we therefore performed a study to evaluate the influence of peritoneal transport characteristics on nutritional status and clinical outcome in chinese diabetic nephropathy patients on pd . we found that although the adequacy of pd assessed by tccr in ht group was higher than lt group , the alb was lower , and the incidence of malnutrition by sga was higher in ht group . the d / pcr was found having a negative correlation with alb in this cohort of pd patients . the mechanism of nutritional status affected by higher peritoneal transport characteristic in pd patients is not yet entirely clear . first , high transporters be more prone to ultrafiltration dysfunction as a result of rapid reabsorption of glucose from the dialysate . decreased ultrafiltration volume can lead to hypertension , fluid overload , inhibition of appetite , and nutrient malabsorption . and dpi was significantly lower in ht group than lt group in our follow - up . we found that the d / pcr was positively correlated with 24 h dialysate protein , and the peritoneal loss of protein was significantly higher in ht group compared with lt group . furthermore , we observed that high transporters had greater systemic exposure to glucose ( p > 0.05 ) . patients with higher peritoneal transport status are more prone to using higher glucose - containing dialysate due to less ultrafiltration . and that means more difficult to control the glucose level resulting in increased consumption of protein and a negative balance of metabolic in diabetes patients . our study demonstrated that hypoalbuminemia was a significant and independent predictor of mortality in diabetic nephropathy patients on pd ( hr : 0.870 , 95% ci : 0.7750.975 , p = 0.017 ) . hypoalbuminemia is not only an indicator of malnutrition , but also related to clinical complications and more vulnerable to micro - inflammation . it can also result in ultrafiltration dysfunction because of decreased plasma osmolality , which further affecting the prognosis of patients . recently , a 5-year clinical cohort study completed by the first affiliated hospital of sun yat - sen university demonstrated that hypoalbuminemia was an independent risk factor of mortality in chinese capd patients with diabetes . meanwhile , they found higher hba1c at the initiation of capd was also a risk factor for mortality in patients with diabetes . however , in our study , hba1c was not significantly different between the two groups of patients . the cox regression analysis also failed to prompt hba1c as an independent risk for mortality . one of the proposed mechanisms is that uremic acidosis can increase the rate of glycosylation of hba1c . meanwhile , hba1c value can be influenced by either shortening of the life span of erythrocytes or the changing proportion of young to old erythrocytes by erythropoietin use . therefore , there is a need for a much better understanding of the role of hba1c and its target in pd patients . on the kaplan meier survival analysis , we observed that death - censored technique failure and mortality were significantly increased in ht group compared with lt group . we further analyzed the independent risk factor for clinical outcomes by univariate and multivariate cox regression analyses . higher peritoneal transport status presented independently predictive power for death - censored technique failure ( hr : 2.311 ; 95% ci : 1.0035.323 ) . and the most common cause of technique failure was fluid overload in our follow - up ( 32.1% ) . our findings suggested that higher peritoneal transport status resulting in fluid overload was an important factor for technique failure in diabetic nephropathy patients on pd . diabetic pd patients had been reported to have higher peritoneal transport status and be more fluid overloaded as compared to nondiabetics . demonstrated that the diabetic pd patients have higher mesothelial loss , higher mesothelial basement membrane thickening , higher proportion of vascular wall thickening / sclerosis , and higher proportion of inflammatory infiltrate than nondiabetic pd patients . these peritoneal histological changes in diabetic pd patients can lead to higher peritoneal transport status , reducing ultrafiltration capacity and ultimately leading to increased technique failure . after adjusting for classic mortality risk factors on the multivariate cox analysis , mortality however , higher peritoneal transport status was no longer an independent predictor of mortality in this cohort of pd patients . our findings about the relationship between the peritoneal transport status and patient survival were similar to that of some earlier reports . first , yang et al . demonstrated that higher peritoneal transport is not a significant independent risk factor for mortality in patients on automated pd ( apd ) . although the australian and new zealand study showed that high transport status was independently predictive of mortality and death - censored technique failure for patients on capd , but not for those received apd . one possible explanation for these results is that the apd may possibly eliminate the consequence of an inadequate ultrafiltration due to an ht status . as such , these results suggested that the poor outcome associated with the ht status may relate more to fluid overload rather than the ht status itself . the early inherent type i which is associated with comorbidity and inflammation is a risk factor for mortality . the early inherent type ii with a large peritoneal surface area has a good prognosis , in general . and the late acquired type iii is not necessarily associated with poor outcome if fluid balance has been controlled using apd or icodextrin - based pd solution . therefore , in spite of the same high peritoneal transport characteristics , different types have different clinical outcomes . it should be noted that there are some limitations in our study . it is a single - center study and thus center - specific effects can not be excluded . and consequently , the influence of higher peritoneal transport status for all - cause mortality in diabetic nephropathy patients on pd need to be further confirmed in a larger study sample or multi - center , randomized , controlled trials . in conclusion , our findings showed that higher peritoneal transport status is associated with an increased risk for malnutrition and technical failure in diabetic nephropathy patients on pd . and although our study did not suggest higher peritoneal transport status itself a determinant factor on the mortality of this population , more effective fluid control using apd and icodextrin - based pd solutions may have important clinical benefits .

background : high peritoneal transport status was previously thought to be a poor prognostic factor in peritoneal dialysis ( pd ) patients . however , its effect on diabetic nephropathy pd patients is unclear in consideration of the adverse impact of diabetes itself . the purpose of this study was to investigate the influence of peritoneal transport characteristics on nutritional status and clinical outcome in diabetic nephropathy patients on pd.methods:one hundred and two diabetic nephropathy patients on pd were enrolled in this observational cohort study . according to the initial peritoneal equilibration test result , patients were divided into two groups : higher transport group ( ht , including high and high average transport ) and lower transport group ( lt , including low and low - average transport ) . demographic characteristics , biochemical data , dialysis adequacy , and nutritional status were evaluated . clinical outcomes were compared . risk factors for death - censored technique failure and mortality were analyzed.results:compared with lt group ( n = 37 ) , serum albumin was significantly lower and the incidence of malnutrition by subjective global assessment was significantly higher in ht group ( n = 65 ) ( p < 0.05 ) . kaplan meier analyses showed that death - censored technique failure and mortality were significantly increased in ht group compared with that in lt group . on multivariate cox analyses , higher peritoneal transport status and lower residual renal function ( rrf ) were independent predictors of death - censored technique failure when adjusted for serum albumin and total weekly urea clearance ( kt / v ) . independent predictors of mortality were advanced age , anemia , hypoalbuminemia , and lower rrf , but not higher peritoneal transport status.conclusions:higher peritoneal transport status has an adverse influence on nutrition for diabetic nephropathy patients on pd . higher peritoneal transport status is a significant independent risk factor for death - censored technique failure , but not for mortality in diabetic nephropathy patients on pd .

I M Subjects Data collection Peritoneal transport characteristics and grouping Nutrition assessment Statistical analysis R Demographic and baseline clinical data Nutritional status Correlation analysis of dialysate/plasma creatinine ratio and nutritional status Death-censored technique survival and patient survival Risk factors for death-censored technique failure and mortality D

with the rapidly growing use of peritoneal dialysis ( pd ) as renal replacement therapy in china , the issue of the survival rate of diabetic nephropathy patients on pd has been raised . canusa and some other studies have suggested that a high peritoneal transport status is associated with higher mortality and technique failure in pd patients . however , some recent studies demonstrated that a high peritoneal transport status by itself was not an independent risk factor for mortality and technique failure . actually , many studies have confirmed that diabetes itself is an independent risk factor for mortality in pd patients . therefore , we chose patients with diabetic nephropathy as research subjects to evaluate the influence of peritoneal transport characteristics on nutritional status and clinical outcome in this specific population on pd . inclusion criteria were as follows : ( a ) the primary cause of esrd was diabetic nephropathy with type 2 diabetes ; ( b ) age 18 years at the start of pd and survival for at least 3 months from the first pd therapy ; ( c ) a peritoneal equilibration test ( pet ) had been performed within the first 6 months of pd commencement ; ( d ) pd modality was continuous ambulatory pd ( capd ) or daytime ambulatory pd . eventually , a total of 102 patients were enrolled in the study from all of the 1115 pd patients . clinical and biochemical data at the initial pet of pd ( baseline ) included blood pressure , pd dose , daily transperitoneal glucose exposure , urine volume , peritoneal ultrafiltration volume , electrolytes , intact parathyroid hormone , glycated hemoglobin a1c ( hba1c ) , high - sensitivity c - reactive protein ( hscrp ) . adequacy of dialysis was estimated by measurement of total weekly urea clearance ( kt / v ) and total weekly creatinine clearance ( tccr ) per 1.73 m body surface area . residual renal function ( rrf ) was estimated by calculating the average residual renal clearance of urea and creatinine as described by van olden et al . on the basis of initial pet result , patients were divided into two groups : higher transport group ( ht , including h and ha ) and lower transport group ( lt , including l and la ) . serum albumin ( alb ) , hemoglobin ( hb ) , daily protein intake ( dpi ) , normalized protein equivalent of total nitrogen appearance ( npna ) , and subjective global assessment ( sga ) were measured to determine the nutritional status during the initial pet and the last follow - up . sga scores as proposed by the canusa peritoneal dialysis study group , were coded according to a seven - point graded scale ( 12 , severe malnutrition ; 35 , mild to moderate malnutrition ; 67 , normal nutritional status ) . univariate and multivariate cox proportional hazard regression models were used to analyze the risk factors for mortality and technique failure . variables with p < 0.1 in the univariate cox analysis were presented further to the multivariable cox regression analysis using backward stepwise elimination based on the likelihood ratio . inclusion criteria were as follows : ( a ) the primary cause of esrd was diabetic nephropathy with type 2 diabetes ; ( b ) age 18 years at the start of pd and survival for at least 3 months from the first pd therapy ; ( c ) a peritoneal equilibration test ( pet ) had been performed within the first 6 months of pd commencement ; ( d ) pd modality was continuous ambulatory pd ( capd ) or daytime ambulatory pd . eventually , a total of 102 patients were enrolled in the study from all of the 1115 pd patients . adequacy of dialysis was estimated by measurement of total weekly urea clearance ( kt / v ) and total weekly creatinine clearance ( tccr ) per 1.73 m body surface area . residual renal function ( rrf ) was estimated by calculating the average residual renal clearance of urea and creatinine as described by van olden et al . on the basis of initial pet result , patients were divided into two groups : higher transport group ( ht , including h and ha ) and lower transport group ( lt , including l and la ) . serum albumin ( alb ) , hemoglobin ( hb ) , daily protein intake ( dpi ) , normalized protein equivalent of total nitrogen appearance ( npna ) , and subjective global assessment ( sga ) were measured to determine the nutritional status during the initial pet and the last follow - up . univariate and multivariate cox proportional hazard regression models were used to analyze the risk factors for mortality and technique failure . variables with p < 0.1 in the univariate cox analysis were presented further to the multivariable cox regression analysis using backward stepwise elimination based on the likelihood ratio . among the 102 patients , 65 patients were classified as higher peritoneal transporters ( ht group , including h 8 patients and ha 57 patients ) . the other 37 patients were classified as lower peritoneal transporters ( lt group , including la 32 patients and l 5 patients ) . in all of the evaluated items below , glucose exposure , 24-h dialysate protein , and tccr were significantly higher in ht group compared with those in lt group . baseline demographic and clinical characteristics of patients in the higher and lower transport group bmi : body mass index ; pet : peritoneal equilibration test ; cvd : cardio - cerebrovascular disease ; capd : continuous ambulatory peritoneal dialysis ; dapd : daytime ambulatory peritoneal dialysis ; ipth : intact parathyroid hormone ; hba1c : glycated hemoglobin a1c ; hscrp : high - sensitivity c - reactive protein ; rrf : residual renal function ; lt : lower peritoneal transport group ; ht : higher peritoneal transport group ; pd : peritoneal dialysis ; tccr : total clearance of creatinine . by the end of the study , 22 patients had died ( 16 in ht group , 6 in lt group ) , 28 transferred to hemodialysis , 6 had received a renal transplantation , and 3 were lost to follow - up . and other cause of technique failure included peritonitis ( 21.4% ) , inadequate dialysis ( 14.3% ) , catheter - related causes ( 10.8% ) , and pleuroperitoneal communication or subjective factors ( 21.4% ) . there were no significant differences between the two groups with respect to pd time , hb , baseline alb , and baseline npna ( p > 0.05 ) . however , we observed a significant difference in the levels of alb , dpi , npna , and sga between two groups over time . compared with lt group , alb , dpi , and npna meanwhile , the incidence of malnutrition by sga in ht group was significantly higher than lt group at the last follow - up [ table 2 ] . comparison of nutritional status in the higher and lower transport group over time * p<0.05 versus before values in the same group ; p<0.05 versus ht group . pet : peritoneal equilibration test ; alb : serum albumin ; hb : hemoglobin ; dpi : daily protein intake ; npna : normalized protein equivalent of total nitrogen appearance ; sga : subjective global assessment ; lt : lower peritoneal transport group ; ht : higher peritoneal transport group ; pd : peritoneal dialysis . in addition , alb was positively correlated with dpi ( r = 0.301 , p < 0.05 ) , and negatively correlated with hscrp ( r = 0.216 , p < 0.05 ) . death - censored technique survival rates at 1 , 2 , and 3 years were 91.8% , 71.0% , and 50.1% , respectively , in the ht group ; 97.3% , 90.9% , and 82.4% in the lt group , respectively . compared with lt group , death - censored technique failure was significantly increased in ht group ( log - rank p = 0.025 ) [ figure 2 ] . cumulative proportional patient survival at 1 , 2 , and 3 years were 90.2% , 77.6% , and 61.1% , respectively , in the ht group ; 94.4% , 90.8% , and 84.3% , respectively , in the lt group . a higher risk of mortality in ht group was observed than in lt group ( log - rank p = 0.047 ) [ figure 3 ] . for the analysis of risk factors for death - censored technique failure , variables with p < 0.1 in the univariate cox analysis were higher peritoneal transport status , alb , rrf , and kt / v . after multivariate cox proportional hazards model analysis , higher peritoneal transport status ( hazard ratio [ hr ] : 2.311 ; 95% confidence interval [ ci ] : 1.0035.323 ) and rrf ( hr : 0.718 ; 95% ci : 0.5320.968 ) were independent predictor of death - censored technique failure [ table 3 ] . screening with univariate cox analysis , possible risk factors contributing to mortality were age , higher peritoneal transport status , alb , hb , hba1c , tccr , rrf , and npna . however , after multivariate cox proportional hazards model analysis , mortality was independently predicted by advanced age , anemia , hypoalbuminemia , and lower rrf , but not higher peritoneal transport status [ table 4 ] . predictors of death - censored technical failure by multivariate cox regression analysis ht : higher peritoneal transport status ; rrf : residual renal function ; ci : confidence interval ; se : standard error ; hr : hazard ratio . predictors of mortality by multivariate cox regression analysis ht : higher peritoneal transport status ; alb : serum albumin ; hb : hemoglobin ; rrf : residual renal function ; se : standard error ; hr : hazard ratio ; ci : confidence interval . among the 102 patients , 65 patients were classified as higher peritoneal transporters ( ht group , including h 8 patients and ha 57 patients ) . the other 37 patients were classified as lower peritoneal transporters ( lt group , including la 32 patients and l 5 patients ) . in all of the evaluated items below , glucose exposure , 24-h dialysate protein , and tccr were significantly higher in ht group compared with those in lt group . baseline demographic and clinical characteristics of patients in the higher and lower transport group bmi : body mass index ; pet : peritoneal equilibration test ; cvd : cardio - cerebrovascular disease ; capd : continuous ambulatory peritoneal dialysis ; dapd : daytime ambulatory peritoneal dialysis ; ipth : intact parathyroid hormone ; hba1c : glycated hemoglobin a1c ; hscrp : high - sensitivity c - reactive protein ; rrf : residual renal function ; lt : lower peritoneal transport group ; ht : higher peritoneal transport group ; pd : peritoneal dialysis ; tccr : total clearance of creatinine . by the end of the study , 22 patients had died ( 16 in ht group , 6 in lt group ) , 28 transferred to hemodialysis , 6 had received a renal transplantation , and 3 were lost to follow - up . and other cause of technique failure included peritonitis ( 21.4% ) , inadequate dialysis ( 14.3% ) , catheter - related causes ( 10.8% ) , and pleuroperitoneal communication or subjective factors ( 21.4% ) . there were no significant differences between the two groups with respect to pd time , hb , baseline alb , and baseline npna ( p > 0.05 ) . however , we observed a significant difference in the levels of alb , dpi , npna , and sga between two groups over time . compared with lt group , alb , dpi , and npna meanwhile , the incidence of malnutrition by sga in ht group was significantly higher than lt group at the last follow - up [ table 2 ] . comparison of nutritional status in the higher and lower transport group over time * p<0.05 versus before values in the same group ; p<0.05 versus ht group . pet : peritoneal equilibration test ; alb : serum albumin ; hb : hemoglobin ; dpi : daily protein intake ; npna : normalized protein equivalent of total nitrogen appearance ; sga : subjective global assessment ; lt : lower peritoneal transport group ; ht : higher peritoneal transport group ; pd : peritoneal dialysis . in addition , alb was positively correlated with dpi ( r = 0.301 , p < 0.05 ) , and negatively correlated with hscrp ( r = 0.216 , p < 0.05 ) . death - censored technique survival rates at 1 , 2 , and 3 years were 91.8% , 71.0% , and 50.1% , respectively , in the ht group ; 97.3% , 90.9% , and 82.4% in the lt group , respectively . compared with lt group , death - censored technique failure was significantly increased in ht group ( log - rank p = 0.025 ) [ figure 2 ] . a higher risk of mortality in ht group was observed than in lt group ( log - rank p = 0.047 ) [ figure 3 ] . for the analysis of risk factors for death - censored technique failure , variables with p < 0.1 in the univariate cox analysis were higher peritoneal transport status , alb , rrf , and kt / v . after multivariate cox proportional hazards model analysis , higher peritoneal transport status ( hazard ratio [ hr ] : 2.311 ; 95% confidence interval [ ci ] : 1.0035.323 ) and rrf ( hr : 0.718 ; 95% ci : 0.5320.968 ) were independent predictor of death - censored technique failure [ table 3 ] . screening with univariate cox analysis , possible risk factors contributing to mortality were age , higher peritoneal transport status , alb , hb , hba1c , tccr , rrf , and npna . however , after multivariate cox proportional hazards model analysis , mortality was independently predicted by advanced age , anemia , hypoalbuminemia , and lower rrf , but not higher peritoneal transport status [ table 4 ] . predictors of death - censored technical failure by multivariate cox regression analysis ht : higher peritoneal transport status ; rrf : residual renal function ; ci : confidence interval ; se : standard error ; hr : hazard ratio . predictors of mortality by multivariate cox regression analysis ht : higher peritoneal transport status ; alb : serum albumin ; hb : hemoglobin ; rrf : residual renal function ; se : standard error ; hr : hazard ratio ; ci : confidence interval . patients with higher peritoneal transport status tend to have enhanced clearance of small solutes , and easier to achieve the objective dialysis adequacy target . to our knowledge , the role of peritoneal transport characteristics on technique failure and mortality of pd patients remains controversial . we therefore performed a study to evaluate the influence of peritoneal transport characteristics on nutritional status and clinical outcome in chinese diabetic nephropathy patients on pd . we found that although the adequacy of pd assessed by tccr in ht group was higher than lt group , the alb was lower , and the incidence of malnutrition by sga was higher in ht group . the mechanism of nutritional status affected by higher peritoneal transport characteristic in pd patients is not yet entirely clear . and dpi was significantly lower in ht group than lt group in our follow - up . we found that the d / pcr was positively correlated with 24 h dialysate protein , and the peritoneal loss of protein was significantly higher in ht group compared with lt group . furthermore , we observed that high transporters had greater systemic exposure to glucose ( p > 0.05 ) . our study demonstrated that hypoalbuminemia was a significant and independent predictor of mortality in diabetic nephropathy patients on pd ( hr : 0.870 , 95% ci : 0.7750.975 , p = 0.017 ) . recently , a 5-year clinical cohort study completed by the first affiliated hospital of sun yat - sen university demonstrated that hypoalbuminemia was an independent risk factor of mortality in chinese capd patients with diabetes . meanwhile , they found higher hba1c at the initiation of capd was also a risk factor for mortality in patients with diabetes . on the kaplan meier survival analysis , we observed that death - censored technique failure and mortality were significantly increased in ht group compared with lt group . we further analyzed the independent risk factor for clinical outcomes by univariate and multivariate cox regression analyses . higher peritoneal transport status presented independently predictive power for death - censored technique failure ( hr : 2.311 ; 95% ci : 1.0035.323 ) . our findings suggested that higher peritoneal transport status resulting in fluid overload was an important factor for technique failure in diabetic nephropathy patients on pd . diabetic pd patients had been reported to have higher peritoneal transport status and be more fluid overloaded as compared to nondiabetics . these peritoneal histological changes in diabetic pd patients can lead to higher peritoneal transport status , reducing ultrafiltration capacity and ultimately leading to increased technique failure . after adjusting for classic mortality risk factors on the multivariate cox analysis , mortality however , higher peritoneal transport status was no longer an independent predictor of mortality in this cohort of pd patients . demonstrated that higher peritoneal transport is not a significant independent risk factor for mortality in patients on automated pd ( apd ) . although the australian and new zealand study showed that high transport status was independently predictive of mortality and death - censored technique failure for patients on capd , but not for those received apd . the early inherent type i which is associated with comorbidity and inflammation is a risk factor for mortality . therefore , in spite of the same high peritoneal transport characteristics , different types have different clinical outcomes . and consequently , the influence of higher peritoneal transport status for all - cause mortality in diabetic nephropathy patients on pd need to be further confirmed in a larger study sample or multi - center , randomized , controlled trials . in conclusion , our findings showed that higher peritoneal transport status is associated with an increased risk for malnutrition and technical failure in diabetic nephropathy patients on pd . and although our study did not suggest higher peritoneal transport status itself a determinant factor on the mortality of this population , more effective fluid control using apd and icodextrin - based pd solutions may have important clinical benefits .

wherever we look , we have hierarchies , people varying in their social status ( marmot , 2004 , p. 89 ) , and , it has been suggested , attempting to achieve status in one s social group is both ubiquitous and important in terms of the emotional and resource - based benefits it brings ( anderson , john , keltner , & kring , 2001 ) . in this paper , we describe the development of ladders to measure a number of dimensions of subjective social status among 15-year old scottish school pupils , and the relationships between these dimensions and a range of individual characteristics . subjective status has been defined as a person s sense of place within a hierarchy , which may or may not agree with objective status ( adler & stewart , 2007 ; davis , 1956 ) . our measures were adapted from the macarthur scales of subjective social status , developed in the us and subsequently used in studies of health inequalities among both adults ( e.g. adler , epel , castellazzo , & ickovics , 2000 ; operario , adler , & williams , 2004 ; ostrove , adler , kuppermann , & washington , 2000 ; singh - manoux , adler , & marmot , 2003 ) and adolescents ( goodman et al . , 2001 ; we begin by describing the macarthur scales , note evidence of , and review the literature relating to , multiple dimensions of social status in adolescence , and , finally , discuss the meaning of subjective status measurements . capture individuals sense of their place in the social ladder which takes into account standing on multiple dimensions of socio - economic status and social position it is a simple visual scale ( a picture of a ladder ) which asks respondents how they measure up to a particular comparison group . in the most commonly used ( adult ) version of the macarthur scale , respondents are asked to mark where they would place themselves on a ladder representing where people stand in the united states . the instructions note that at the top are the best off people , with most money , education and respected jobs ; at the bottom are the worst off ; the ladder thus aims to provide a summative measure of subjective socio - economic status ( ses ) . ladder , which asks respondents to define community in whatever way is most meaningful to you , the highest rung representing those with the highest standing in their community . on the basis that in adolescence , socio - economic status is ascribed by that of the family , the macarthur scale youth version society ladder asks respondents to indicate the point which best represents where your family would be . similarly , on the basis that the most salient community is that of the school , the highest rung of the community ladder is described as representing the people in your school with the most respect , the highest grades , and the highest standing . however , the range of tasks facing school pupils include both school - defined and social goals ( gorman , kim , & schimmelbusch , 2002 ; wentzel , 1999 ) , suggesting that pupil standing within the school community is likely to be located on more than one dimension . the key school - defined goal is academic attainment , pupils being differentiated formally ( streams or sets ) or via the results of tests or exams . in addition to this official , academic hierarchy , schools strive to regulate pupil behaviour and encourage particular activities , especially sport , also hierarchically arranged and recognised in the form of teams . however , within the school context , pupils also need to maintain and establish interpersonal relationships , develop social identities and a sense of belongingness . consistent with this , pilot study feedback from finnish pupils on the school community ladder suggested that respect and highest standing represented a different dimension from the self - concept literature emphases its multidimensional structure and identifies academic and non - academic self - perceptions in childhood and adolescence ( byrne & shavelson , 1996 ; marsh , barnes , cairns , & tidman , 1984 ; song & hattie , 1985 ) . thus , harter s self - perception profile measures were based on the distinction between separate domains of competence ( academic , social , sports , appearance and behaviour ) and the possibility of varying self - assessments across domains ( harter , 1982 , 1985 , 1988 ) . evidence that this multidimensional self - concept structure becomes increasingly differentiated , stable and realistic with age ( byrne & shavelson , 1996 ; cole et al . , 2001 ; marsh , trautwein , ludke , koller , & baumert , 2005 ) is consistent with the suggestion that in mid - adolescence , highly academic pupils are not necessarily one and the same as respected or high - standing pupils . a key question relates to the meaning of self - assessments and , specifically , subjective status measurements . while it might be expected that pupils have a reasonably accurate sense of their academic position , this may not extend to other dimensions . the self - concept literature makes a strong case for suggesting that self - perceptions map well onto more objective measures and/or perceptions held by others . harter found that among american 1415-year olds , self - perceived cognitive competence was strongly correlated with both teacher - perceived cognitive competence ( r = .73 ) and standardised test achievement ( r = .54 ) , while among younger pupils , correlations between self - perceived social competence and an index of friendship nominations and between self - rated physical competence and gym teacher ratings of physical competence were in the region of r = .60 ( harter , 1982 ) . similarly , in a range of studies , dimensions of self- and teacher - report self - concept were found to be substantially correlated ( marsh et al . , 1984 ) , while grade points were found to have a much stronger relationship with academic than other dimensions of self - concept ( song & hattie , 1985 ) . set against this is evidence from studies of adolescent peer relations which suggests self - reports of status should be treated with caution ( mayeux , sandstrom , & cillessen , 2008 ) . within any one status group there may be large variations in self - perceived peer experiences ( crick & ladd , 1993 ) and peer - assessed popularity does not necessarily match an adolescent s own sense of their social acceptance ( mcelhaney , antonishak , & allen , 2008 ) . there is evidence that self - reports may be more valid when acknowledging negative rather than positive attributes ( hymel & franke , 1985 ) ; further , certain children may be relatively insensitive to evidence of peer dislike and so unaware of their social status ( zakriski & coie , 1996 ) . this is further complicated by a consensus that there are distinct types of high status pupils , particularly in respect of popularity . one type , based on liking , tends to be associated with behaviours such as kindness , trustworthiness and sociability . the other , based on social reputation , impact or visibility , has been related to dominance , disruptive or aggressive behaviour ( coie , dodge , & coppotelli , 1982 ; farmer & rodkin , 1996 ; parkhurst & hopmeyer , 1998 ) . other characteristics linked with adolescent social status include appearance , material possessions , spending power , athletic ability and academic performance , the significance of each differing according to gender ( meisinger , blake , lease , palardy , & olejnik , 2007 ) . thus , many studies have demonstrated the importance of appearance ( physical attractiveness and sartorial style ) for status , particularly among girls ( de bruyn & cillessen , 2006 ; kennedy , 1990 ) . among american pre - adolescent children , higher parental ses , appearance and grooming behaviour were associated with high social status among girls , while athletic ability was the most important factor for boys ( adler , kless , & adler , 1992 ) . similarly , among american 1113-year olds , cheerleading and attractiveness were related to social status among females , while achievement on the sports field mattered for males ( eder , 1985 ; eder & kinney , 1995 ) . a study of 13-year old scottish pupils found the popularity of top girls was attributed to their spending power and appearance ( mature and street - wise ) , and although top boys were also distinguished by their good looks and brand - label clothes , their main interest was sport ( michell , 1997 ) . several studies suggest that while cognitive ability has a positive effect on social status in childhood , the associations are less consistent in adolescence ( juvonen & murdock , 1995 ; lubbers , van der werf , kuyper , & offringa , 2006 ) , and , for many adolescent males , academic achievement may be a potentially degrading stigma ( adler et al . , 1992 ) . among scottish groups who were doing well , enjoyed academic subjects , worked hard and had positive aspirations ( michell , 1997 ) . against this background , the overarching aims of this study are to describe the structure of adolescent school - based hierarchies and examine their associations with a range of individual characteristics . our questions are therefore : firstly , how do 15-year old pupils respond to a set of ladders representing different domains of subjective social status within the school community ; secondly , how are responses on the ladders inter - related ( what is the underlying factor structure ) ; and thirdly , how are the resulting factors associated with a range of variables representing other- and self report objective and behavioural characteristics ( ses and spending power ; physical appearance ; peer relationships including friendship nominations ; academic orientation and achievement ; sports participation and interest ) . given that adolescent peer relationships are shaped by gender ( giordano , 2003 ) , as evidenced by the gendered nature of most of the dimensions reviewed above , we also examine whether there are gender differences in these associations . data are drawn from the peers and levels of stress ( pals ) study , conducted among 15-year old pupils within 22 schools situated in and around glasgow city in the west of scotland . the study received approval from the relevant glasgow university ethics committee , participating local authorities and schools . the sampling scheme aimed to obtain a representative sample by selecting schools within strata based on geographical location , religious status and deprivation . subsequent analyses found that participating and non - participating schools did not differ significantly in respect of these dimensions , nor for total pupil roll or exam achievement by the end of statutory schooling ( sweeting , young , & west , 2008 ) . within selected schools , all pupils in ( scottish ) secondary 4 ( s4 ) , the final statutory school year , were invited to participate via letters sent to parents , including opt - out consent forms . during school - based sessions , pupils filled in a questionnaire in examination - type conditions , completed a brief interview and had their height and weight measured ( west , sweeting , young , & kelly , 2010 ) . the total sample comprised 1572 males and 1622 females ( 81% of the eligible sample of 3950 ) who filled in a questionnaire , of whom 3057 were interviewed and measured ( the discrepancy accounted for by pupils absent from school on the survey day who posted back their questionnaires ) . ladders relating to domains identified within the self - concept literature ( doing well at school , popular , sporty , attractive and stylish , and rebellious ) and , because of our interest in status , two further areas ( powerful and , echoing the wording in the original school community ladder , respected ) , were tested in a pilot study in which 15-year old focus group participants completed the . the majority found it an easy task and a good way to explain yourself . however , some had difficulty indicating how popular they were , since they were unsure of the reference group ( their friends or pupils in year group overall ) . the main study questionnaire therefore included seven pictures of a 10-rung ladder ( and one example ) , with the instructions the text for each ladder asked : how popular are you compared with the rest of your year group ? top = the most popular people in your year ; how well are you doing at school compared with the rest of your year group ? top = people who get the best grades ; how powerful are you compared with the rest of your year group ? top = most powerful people can get others to do what they want , in good or bad ways ; how much of a trouble - maker are you compared with the rest of your year group ? top = people who make the most trouble ; how attractive or stylish are you compared with the rest of your year group ? top = the most attractive , stylish people ; how respected are you compared with the rest of your year group ? top = people who are most respected by others ; how sporty are you compared with the rest of your year group ? top = most sporty people . the rungs were scored 110 . given strong associations between socio - economic status and both academic achievement ( uk national equality panel et al . , 2010 ) and behavioural disorders ( meltzer , gatward , goodman , & ford , 2000 ) , together with suggestions that popularity , especially that of females , is linked with parental ses , particularly visible wealth ( adler et al . , 1992 ) , our analysis included two measures of family ses . these were firstly , social class , an individual ( household ) measure reflecting both resources and culture ( bourdieu , 1984 ; carr - hill , 1990 ) ; secondly , family affluence , based on measures of household material resources on which pupils can report accurately ( currie et al . , 2008 ) and thus , potentially , representing the most visible aspect of ses for adolescents . social class was defined on the basis of the occupation of the head of the household , derived via pupil interview . valid responses were available for 85.9% of the sample ; those missing had no parent in work , were not living with parental figures , provided insufficient detail or were not interviewed . social class was coded using the uk standard occupational classification ( soc ; office of population census and surveys , 1990 ) and collapsed into three categories : non - manual ( comprising class i , professional ; class ii , managerial and technical ; and class iiinm , skilled non - manual occupations , n = 1663 ) ; skilled manual ( class iiim , n = 670 ) ; and semi or unskilled manual ( classes iv and v , n = 412 ) . family affluence ( currie et al . , 2008 ) was derived via items in respect of : number of family cars , vans or trucks ; having own ( not shared ) bedroom ; number of family computers ; and number of family holidays in the past year . it was collapsed into low ( scale scores 03 , n = 496 ) , mid ( 45 , n = 1280 ) and high ( 67 , n = 1309 ) categories . although the two measures are related , the correlation ( before collapsing ) was only moderate ( r = .325 ) . this variable was collapsed into four categories , with mean values 0.36 ( n = 647 ) , 5.38 ( n = 577 ) , 10.08 ( n = 1089 ) and 21.86 ( n = 783 ) . following a brief interview , height and weight measurements were taken in indoor clothes with no footwear . body mass index ( kg / m ) was categorised as underweight ( n = 518 ) and obese ( n = 169 ) based on standard ( international obesity task force ) definitions for children and adolescents ( cole , bellizzi , flegal , & dietz , 2000 ; cole , flegal , nicholls , & jackson , 2007 ) . average in the middle , very good at one pole and very poor at the other . ( n = 1252 ) , average ( n = 1474 ) and ( very ) poor ( n = 294 ) . interviewers were instructed to make their assessments quickly , akin to first impressions . the psychological literature on the impact of physical attractiveness on outcomes such as expectations about children s academic abilities ( clifford & walster , 1973 ) or decisions about hiring or promotion ( collins & zebrowitz , 1995 ) was incorporated into interviewer training , in order to reduce unease about this particular rating . the survey process provided the opportunity to observe large numbers of s4 pupils against which interviewers could form their judgements and it is possible that these may have altered over the course of fieldwork . since the pupil interviews and physical measures were conducted on a one - to - one basis , it was not possible to assess inter - rater reliability . however relationships between three independent ratings of approximately 900 15-year olds using a similar ( but 7-point ) scale were each in the region of spearman s rho = .3 ( macintyre & west , 1991 ) . the resulting nominations received variable was collapsed into none ( n = 223 ) , one ( n = 349 ) , two or three ( n = 1009 ) , four or five ( n = 894 ) and six or more ( n = 719 ) . in order to determine group size , friendship data were imported into the social network ucinet 6 and netdraw software packages ( borgatti , everett , & freeman , 2002 ) . newman clustering algorithm ( girvan & newman , 2002 ) applied to each school network . to ensure consistency across school networks , the sociogram / network partition with the highest q statistic was selected . finally , each school sociogram was inspected manually in conjunction with each participant s description of their own friendship group , and any ( relatively rare ) obvious misclassifications corrected . group size was collapsed into isolated pupils ( no reciprocated links , n = 449 ) , dyad ( n = 156 ) , three to five members ( n = 527 ) , six to eleven ( n = 1231 ) and twelve or more ( n = 831 ) . since our study was based on data obtained from single school year groups , we recognise that isolates may have had friends outside the s4 year group , or within the year group who did not complete the questionnaire . however , for simplicity , and in line with others ( ennett & bauman , 1993 ) we use the term isolates . the correlation between nominations received and group size ( before collapsing ) was r = .445 . for example , only half the isolates received no nominations , the remainder receiving but not reciprocating . the questionnaire asked pupils do you have a group or gang that you hang out with at school ? , with yes ( n = 2549 ) or no ( n = 603 ) response options . it also included a series of eight items , originally reported in studies of bullying in schools ( omoore , kirkham , & smith , 1997 ; whitney & smith , 1993 ) ( e.g. i ve been physically hurt , e.g. hit and kicked , no - one would talk to me ) , scored on a 4-point scale ( never = 1 to most days these were used to categorise pupils as having been victimised in any way weekly or more ( n = 398 ) , less often ( n = 1492 ) or never ( n = 1258 ) . questionnaire items about school included if i get the chance to skip school , i do ( n = 1392 ) , agree ( n = 534 ) and strongly agree ( n = 160 ) . scottish pupils take standard grade exams at the end of statutory education , and can be entered at different grade levels , credit being the highest . number of credit - level standard grades entered for , collapsed into none ( n = 648 ) , between one and four ( n = 1095 ) , five or six ( n = 601 ) and seven or eight ( n = 647 ) was used as a proxy for academic achievement . pupils were also asked about their plans on leaving school , responses collapsed into tertiary education ( higher and further , n = 2372 ) and apprenticeship , training or work ( n = 649 ) . pupils were asked how frequently they engaged in a range of leisure activities including exercise or do sports and go to watch sports matches , each coded never ( n = 281 for playing and n = 1179 for watching ) , weekly or less ( n = 1534 and n = 1660 ) and daily or most days ( n = 1329 and n = 310 ) . gender differences in ladder scores were determined via the f - statistic , associations between the ladders via pearson s correlations and gender differences in these correlations via the web - based vassarstats program for the significance of the difference between two correlation coefficients ( http://faculty.vassar.edu/lowry/rdiff.html ) . data reduction in respect of the seven ladder scores used principal components analysis with varimax rotation . since almost identical results were obtained for males and females ( detailed results available from hs ) , analysis was conducted on the whole sample . results ( scree plot and variance analysis ) suggested a three factor solution was optimum , accounting for 77% of the variance . associations between the factor scores and a range of variables , adjusted for gender , were determined via the spss procedure glm ( results shown as estimated marginal means with f , significance and partial eta - squared to show effect sizes ) . analyses were conducted separately in respect of each variable ( i.e. entering social class and gender in respect of each factor score , then family affluence score and gender , etc ) . in additional analyses , glm was used to identify gender differences in these relationships via significant interactions between gender and each independent variable . given a sample size of 1000 , a correlation as low as .081 is significant at the .01 level ( rohlf & sokal , 1969 ) . our total sample was approximately 3000 , equally split between males and females ; in addition , we report a large number of analyses . we therefore focus on correlations over .100 , rather than relying on significance levels , and , in other analyses , on significance levels of p < .01 . finally , since very similar results were obtained in analyses based on data with and without weights constructed to compensate for non - response ( sweeting et al . , 2008 ) , results based on unweighted data are shown here . responses ranged from 1 to 10 on every ladder , with very little missing data ; how sporty are you was the ladder with the highest response ( 99.1% ) , how popular are you the lowest ( 98.2% ) . in general , means ranged from 5.0 to 7.0 , but were lower for trouble - maker . males rated themselves significantly higher on each ladder , the greatest gender difference occurring for sporty and the smallest for doing well at school . all were positive , apart from that between doing well at school and trouble - maker . , attractive or stylish and respected , the weakest between doing well at school and popular , attractive or stylish , respected and sporty , and between sporty and trouble - maker . despite no clear gender differences in the pattern of relationships , correlations were significantly higher among males for sporty with popular ( ( p = .009 ) , attractive or stylish ( p < .001 ) and respected ( p < .001 ) , and for respected with popular ( p < .001 ) . correlations were significantly higher among females for doing well at school with sporty ( p = .004 ) and for powerful with trouble - maker the first factor , explaining half the total variance , included popular , powerful , respected and attractive or stylish , each loading over .800 , and the item loading most strongly on the second factor , which explained almost one - fifth of the total variance , was doing well at school ; trouble - maker had a negative loading ( .672 ) ; this factor is titled scholastic status . finally , the third factor , titled sports status , and explaining one - tenth of the total variance , was represented by the single item analyses of variance showed higher scores ( all p < .001 ) for males on the peer ( f = 82.1 ) and sports ( f = 362.0 ) factors , and for females on the scholastic factor ( f = 13.2 ) , the latter reflecting the much lower scores of females on the trouble - maker ladder , which had a negative loading on the scholastic factor . having determined the underlying factor structure of our ladder measures , the next step was to establish the correlates of these dimensions . table 4 therefore shows associations between each factor and indicators of ses and spending power ( estimated marginal means after adjusting for gender , f , significance and effect size ) . however , family affluence was positively related to each factor , and most strongly to peer . pocket money also had a strong positive relationship with peer , the significant interaction with gender indicating a stronger relationship for females ( f = 35.4 ) than males ( f = 14.0 ) . the opposite pattern was seen between pocket money and scholastic where those with the least spending power had the highest scores . as shown in table 5 , interviewer - rated attractiveness and physical maturity ratings were positively related to peer , attractiveness also being positively , although much more weakly , associated with scholastic and sports . there was a gender difference in the relationship between body mass and peer . among males , obese had the lowest and normal groups the highest peer scores . as table 6 shows , there was a strong positive linear relationship between number of friendship nominations received and peer , suggesting that this dimension was associated with an objective measure of others liking . relationships between nominations and scholastic and sports were also positive , but considerably weaker . in respect of group size , isolate status pupils had the lowest , and pupils in large groups the highest scores on each factor , the strongest relationship occurring with scholastic . peer scores were higher among those who reported having a group or gang at school , and higher scores on all three factors were associated with less frequent victimisation . each academic - related variable ( school skipping , credit - level entries and post - school plans ) was very strongly associated with scholastic , relationships with the other two factors being weaker or non - existent . school skipping was positively related to peer and negatively to scholastic and sports , while number of exam entries was positively associated with the significant gender interaction between exam entries and scholastic occurred because the pattern for males was stronger and more clearly linear than that for females . scholastic and ( weakly ) inversely associated with both peer and sports . there were a number of gender interactions in respect of associations between playing and watching sport and the three factors . thus , exercising or playing sports was positively associated with peer only among males , with watching sports matches was associated with peer ( positively ) and scholastic ( negatively ) among males only , and the positive relationship between sports watching and sports was much stronger for males . finally , examination of tables 46 shows the largest effect sizes for peer occurred in respect of pocket money ( partial eta - squared.045 ) , friendship nominations ( .027 ) and attractiveness rating ( .020 ) . as comparison , effect sizes for gender ( not shown ) in these three analyses were all in the region of .030 . for scholastic the largest effect sizes were school skipping ( .185 ) , exam entries ( .148 ) and post - school plans ( .097 ) ( effect sizes for gender all below .005 ) , while for sport they were sports playing ( .259 ) , watching ( .101 ) and body mass category ( .032 ) ; effect sizes for gender around .030 for analyses including playing or watching , .107 for the analysis including body mass category . this paper has shown that it is possible to identify different dimensions in respect of pupils subjective social status within the school community via a simple set of items which are understood by adolescents and which generate a very high response rate . the distinction between ladders representing popularity , power , receipt of respect , attractiveness or style and trouble - making , and those of academic achievement and conforming to behavioural standards , confirms that respect and highest standing the particular relationships with variables representing more objective measures suggest the dimensions identified are a reflection of peer , scholastic and sports status . our factor analysis was exploratory rather than confirmatory ; the ladders were intended to represent domains which the literature suggests may be separate ( harter , 1982 ; marsh et al . , 1984 ) . the dominant factor ( peer ) included popular , powerful , respected , attractive or stylish and , although weighting less strongly , trouble - maker . in many respects it might appear to more clearly represent status based on social reputation , impact or visibility rather than liking ( coie et al . , 1982 ; farmer & rodkin , 1996 ; parkhurst & hopmeyer , 1998 ) . pupils scoring high on this factor came from families with the most material resources and received the most pocket money , were rated more physically attractive and mature , were more likely to be in the largest friendship groups , to report belonging to a group or gang , to have no experience of victimisation and to say they would skip school . but in addition , there was a clear linear relationship between scores on this factor and friendship nominations , a strong indication that those high on self - report peer status were also liked by others . the alternative explanation , that others wanted to be friends with them , is less likely given the question wording ( think about all your friends fill out a page for each one ) . this suggests that our peer status factor may represent an amalgam of the two types of popular pupils ( liked and visible ) which other studies have found to overlap ( cillessen & rose , 2005 ) . it is possible that inclusion of additional ladders representing characteristics such as kindness or trustworthiness which other studies have shown to be more clearly associated with likeability , might have resulted in the identification of separate factors . the second factor ( scholastic ) contrasted with the other two in respect of its very strong relationships with indicators of academic orientation and achievement . the relative effect sizes suggest that of the three dimensions , self - report scholastic assessments relate most strongly to more objective measures , as might be expected given the significance and visibility of position in the academic hierarchy within the school system . this factor was also the only one to be associated with social class , reflecting the importance of links between academic engagement and cultural , over and above material , measures of ses ( reay , 2006 ) . contrasting with peer , scholastic was negatively associated with pocket money , corresponding with another study which found adolescents from higher ses backgrounds and those with less spending money were less likely to own consumer goods such as electrical items , but more likely to own books ( west , sweeting , young , & robins , 2006 ) . scholastic was positively related to friendship nominations , and , more clearly than the peer factor , to group size , but not with self - report group or gang membership , suggesting rather different types of friendship among middle groups by participants in another scottish study ; academically engaged and the kind of adolescents most adults would typically find pleasant and easy to get on with ( michell , 1997 , p. 8) . the final factor ( sports ) was very strongly weighted towards sport , and correlated with fewer variables than the other two . as an indication of its correspondence with objective measures , normal weight pupils had the highest scores on this factor , and obese pupils the lowest . like the other factors , sports status was negatively related to victimisation , highlighting the general low status of bullied pupils . gender differences evident in both the completion of the individual ladders and the correlates of the resulting three factors were consistent with literature showing the importance of athletic skills for male popularity and that academic ability is less likely to be a stigma for adolescent females ( adler et al . , 1992 ; lease , kennedy , & axelrod , 2002 ; meisinger et al . , 2007 ) . interestingly , pocket money was linked more strongly with the peer factor among females , in line with suggestions of the greater importance of material factors for female than male popularity ( adler et al . , 1992 ; meisinger et al . , 2007 ; there was also a gender difference in the relationship between body mass and peer status , underweight males and obese females having the lowest scores . double standard was also seen in a study which found that while overweight was negatively associated with others ratings of attractiveness in 1318-year old females , the same did not hold for males ( rosenblum & lewis , 1999 ) . overall , however , relationships showed more gender similarities than differences , as evidenced by the almost identical results obtained when separate factor analyses were conducted for males and females . for example , males rated themselves higher on every ladder , including doing well at school and attractive and stylish , despite that fact that females were entered for significantly more credit - level standard grade exams and were more likely to receive however , the pattern of associations which each of the three factors identified in our analyses had with a range of characteristics previously demonstrated to relate to distinct and meaningful features of adolescent social status suggests these dimensions map well onto more objective measures . an additional perspective is taken by those who point towards reciprocal and interpretivist models , citing evidence that academic self - concept affects subsequent academic interest and achievement ( marsh et al . , 2005 ) , that expecting to be liked by others is a strong predictor of success with peers ( bellmore & cillessen , 2003 ) and that correctly perceiving oneself as popular predicts increased levels of aggression in adolescents ( mayeux & cillessen , 2008 ) . the existence of different dimensions in pupils subjective social status suggests that studies using multidimensional status measures might find each to be differentially associated with other constructs . in this same study peer , scholastic and sports status are related to the stress hormone , cortisol , in different ways ( west et al . , 2010 ) . effects may also vary ( interact ) with other measures of social position , including gender and ses . one study , for example , suggested that depressive problems in adolescent males were associated with poor sports performance , but in females with not being liked ( oldehinkel , rosmalen , veenstra , dijkstra , & ormal , 2007 ) . between - school differences are also a possibility , depending on the emphasis each places on academic achievement compared with other factors such as pastoral care . thus , scholastic status might be more positively related to attributes such as leadership , and perhaps also associated with pupil well - being in schools which place a stronger emphasis on academic achievement . first is that while the ladders in our questionnaire largely represented domains identified in the self - concept literature , it is possible , as noted above , that had we included additional characteristics , alternative factors might have emerged . second is that the examination of the associations between the three dimensions and a range of other characteristics included variables which were by no means perfect . they did not include academic achievement in the form of test results or teacher ratings , nor the ratings of teachers or others of behaviour ; they relied on single measures ; we were unable to assess the reliability of the two interviewer ratings ; and the fact that the majority of variables were self - report introduces the problem of shared method variance . thus sociometric group size depends on reciprocated nominations ; perhaps scholastic pupils appeared to have larger friendship groups simply because they wrote the names of more friends . pupils within the same friendship network might have answered the group or gang question differently depending on their self - perceived peer or scholastic status . however , other - report variables were included and most self - report variables were objective ( e.g. social class , based on parental occupation ) or behavioural , rather than relating to personality or character . a third limitation is that partial effect sizes within the glm analyses were small , however those for variables most strongly related to each of the three status factors were generally larger than those in respect of gender . the original macarthur adolescent society and school community scales have produced interesting findings ( goodman et al . , 2003 , 2001 , 2007 ) . the elaboration of multiple dimensions of social hierarchy among young people , and demonstration that they correspond with more objective and/or behavioural measures , represents a first step towards the development of more robust instruments within this area .

school pupils strive to meet both school - defined and social goals , and the structure of adolescent self - concept is multidimensional , including both academic and non - academic self - perceptions . however , subjective social status within the school community has been represented as a single dimension . scottish 15-year olds participating in a school - based survey ( n = 3194 ) rated their own status , compared to their school year - group , via images of seven 10-rung ladders . these generated a very high response rate , and factor analysis distinguished three dimensions : ( 1 ) ladders representing popular , powerful , respected , attractive or stylish and trouble - maker ; ( 2 ) doing well at school and [ not ] a trouble - maker ; and ( 3 ) sporty . unique relationships with variables representing more objective and/or self - report behavioural measures suggest these dimensions are markers of peer , scholastic and sports status . these analyses suggest multiple dimensions of adolescent social hierarchy can be very simply measured and contribute towards the development of more robust instruments within this area .

Introduction Methods Results Discussion

wherever we look , we have hierarchies , people varying in their social status ( marmot , 2004 , p. 89 ) , and , it has been suggested , attempting to achieve status in one s social group is both ubiquitous and important in terms of the emotional and resource - based benefits it brings ( anderson , john , keltner , & kring , 2001 ) . in this paper , we describe the development of ladders to measure a number of dimensions of subjective social status among 15-year old scottish school pupils , and the relationships between these dimensions and a range of individual characteristics . subjective status has been defined as a person s sense of place within a hierarchy , which may or may not agree with objective status ( adler & stewart , 2007 ; davis , 1956 ) . our measures were adapted from the macarthur scales of subjective social status , developed in the us and subsequently used in studies of health inequalities among both adults ( e.g. , 2001 ; we begin by describing the macarthur scales , note evidence of , and review the literature relating to , multiple dimensions of social status in adolescence , and , finally , discuss the meaning of subjective status measurements . capture individuals sense of their place in the social ladder which takes into account standing on multiple dimensions of socio - economic status and social position it is a simple visual scale ( a picture of a ladder ) which asks respondents how they measure up to a particular comparison group . similarly , on the basis that the most salient community is that of the school , the highest rung of the community ladder is described as representing the people in your school with the most respect , the highest grades , and the highest standing . however , the range of tasks facing school pupils include both school - defined and social goals ( gorman , kim , & schimmelbusch , 2002 ; wentzel , 1999 ) , suggesting that pupil standing within the school community is likely to be located on more than one dimension . the key school - defined goal is academic attainment , pupils being differentiated formally ( streams or sets ) or via the results of tests or exams . however , within the school context , pupils also need to maintain and establish interpersonal relationships , develop social identities and a sense of belongingness . consistent with this , pilot study feedback from finnish pupils on the school community ladder suggested that respect and highest standing represented a different dimension from the self - concept literature emphases its multidimensional structure and identifies academic and non - academic self - perceptions in childhood and adolescence ( byrne & shavelson , 1996 ; marsh , barnes , cairns , & tidman , 1984 ; song & hattie , 1985 ) . thus , harter s self - perception profile measures were based on the distinction between separate domains of competence ( academic , social , sports , appearance and behaviour ) and the possibility of varying self - assessments across domains ( harter , 1982 , 1985 , 1988 ) . evidence that this multidimensional self - concept structure becomes increasingly differentiated , stable and realistic with age ( byrne & shavelson , 1996 ; cole et al . a key question relates to the meaning of self - assessments and , specifically , subjective status measurements . the self - concept literature makes a strong case for suggesting that self - perceptions map well onto more objective measures and/or perceptions held by others . similarly , in a range of studies , dimensions of self- and teacher - report self - concept were found to be substantially correlated ( marsh et al . , 1984 ) , while grade points were found to have a much stronger relationship with academic than other dimensions of self - concept ( song & hattie , 1985 ) . set against this is evidence from studies of adolescent peer relations which suggests self - reports of status should be treated with caution ( mayeux , sandstrom , & cillessen , 2008 ) . there is evidence that self - reports may be more valid when acknowledging negative rather than positive attributes ( hymel & franke , 1985 ) ; further , certain children may be relatively insensitive to evidence of peer dislike and so unaware of their social status ( zakriski & coie , 1996 ) . other characteristics linked with adolescent social status include appearance , material possessions , spending power , athletic ability and academic performance , the significance of each differing according to gender ( meisinger , blake , lease , palardy , & olejnik , 2007 ) . a study of 13-year old scottish pupils found the popularity of top girls was attributed to their spending power and appearance ( mature and street - wise ) , and although top boys were also distinguished by their good looks and brand - label clothes , their main interest was sport ( michell , 1997 ) . several studies suggest that while cognitive ability has a positive effect on social status in childhood , the associations are less consistent in adolescence ( juvonen & murdock , 1995 ; lubbers , van der werf , kuyper , & offringa , 2006 ) , and , for many adolescent males , academic achievement may be a potentially degrading stigma ( adler et al . against this background , the overarching aims of this study are to describe the structure of adolescent school - based hierarchies and examine their associations with a range of individual characteristics . our questions are therefore : firstly , how do 15-year old pupils respond to a set of ladders representing different domains of subjective social status within the school community ; secondly , how are responses on the ladders inter - related ( what is the underlying factor structure ) ; and thirdly , how are the resulting factors associated with a range of variables representing other- and self report objective and behavioural characteristics ( ses and spending power ; physical appearance ; peer relationships including friendship nominations ; academic orientation and achievement ; sports participation and interest ) . subsequent analyses found that participating and non - participating schools did not differ significantly in respect of these dimensions , nor for total pupil roll or exam achievement by the end of statutory schooling ( sweeting , young , & west , 2008 ) . within selected schools , all pupils in ( scottish ) secondary 4 ( s4 ) , the final statutory school year , were invited to participate via letters sent to parents , including opt - out consent forms . during school - based sessions , pupils filled in a questionnaire in examination - type conditions , completed a brief interview and had their height and weight measured ( west , sweeting , young , & kelly , 2010 ) . ladders relating to domains identified within the self - concept literature ( doing well at school , popular , sporty , attractive and stylish , and rebellious ) and , because of our interest in status , two further areas ( powerful and , echoing the wording in the original school community ladder , respected ) , were tested in a pilot study in which 15-year old focus group participants completed the . top = most powerful people can get others to do what they want , in good or bad ways ; how much of a trouble - maker are you compared with the rest of your year group ? top = people who make the most trouble ; how attractive or stylish are you compared with the rest of your year group ? social class was coded using the uk standard occupational classification ( soc ; office of population census and surveys , 1990 ) and collapsed into three categories : non - manual ( comprising class i , professional ; class ii , managerial and technical ; and class iiinm , skilled non - manual occupations , n = 1663 ) ; skilled manual ( class iiim , n = 670 ) ; and semi or unskilled manual ( classes iv and v , n = 412 ) . this variable was collapsed into four categories , with mean values 0.36 ( n = 647 ) , 5.38 ( n = 577 ) , 10.08 ( n = 1089 ) and 21.86 ( n = 783 ) . body mass index ( kg / m ) was categorised as underweight ( n = 518 ) and obese ( n = 169 ) based on standard ( international obesity task force ) definitions for children and adolescents ( cole , bellizzi , flegal , & dietz , 2000 ; cole , flegal , nicholls , & jackson , 2007 ) . ( n = 1252 ) , average ( n = 1474 ) and ( very ) poor ( n = 294 ) . the resulting nominations received variable was collapsed into none ( n = 223 ) , one ( n = 349 ) , two or three ( n = 1009 ) , four or five ( n = 894 ) and six or more ( n = 719 ) . finally , each school sociogram was inspected manually in conjunction with each participant s description of their own friendship group , and any ( relatively rare ) obvious misclassifications corrected . group size was collapsed into isolated pupils ( no reciprocated links , n = 449 ) , dyad ( n = 156 ) , three to five members ( n = 527 ) , six to eleven ( n = 1231 ) and twelve or more ( n = 831 ) . since our study was based on data obtained from single school year groups , we recognise that isolates may have had friends outside the s4 year group , or within the year group who did not complete the questionnaire . however , for simplicity , and in line with others ( ennett & bauman , 1993 ) we use the term isolates . , with yes ( n = 2549 ) or no ( n = 603 ) response options . hit and kicked , no - one would talk to me ) , scored on a 4-point scale ( never = 1 to most days these were used to categorise pupils as having been victimised in any way weekly or more ( n = 398 ) , less often ( n = 1492 ) or never ( n = 1258 ) . questionnaire items about school included if i get the chance to skip school , i do ( n = 1392 ) , agree ( n = 534 ) and strongly agree ( n = 160 ) . scottish pupils take standard grade exams at the end of statutory education , and can be entered at different grade levels , credit being the highest . number of credit - level standard grades entered for , collapsed into none ( n = 648 ) , between one and four ( n = 1095 ) , five or six ( n = 601 ) and seven or eight ( n = 647 ) was used as a proxy for academic achievement . pupils were also asked about their plans on leaving school , responses collapsed into tertiary education ( higher and further , n = 2372 ) and apprenticeship , training or work ( n = 649 ) . pupils were asked how frequently they engaged in a range of leisure activities including exercise or do sports and go to watch sports matches , each coded never ( n = 281 for playing and n = 1179 for watching ) , weekly or less ( n = 1534 and n = 1660 ) and daily or most days ( n = 1329 and n = 310 ) . in general , means ranged from 5.0 to 7.0 , but were lower for trouble - maker . males rated themselves significantly higher on each ladder , the greatest gender difference occurring for sporty and the smallest for doing well at school . all were positive , apart from that between doing well at school and trouble - maker . , attractive or stylish and respected , the weakest between doing well at school and popular , attractive or stylish , respected and sporty , and between sporty and trouble - maker . despite no clear gender differences in the pattern of relationships , correlations were significantly higher among males for sporty with popular ( ( p = .009 ) , attractive or stylish ( p < .001 ) and respected ( p < .001 ) , and for respected with popular ( p < .001 ) . correlations were significantly higher among females for doing well at school with sporty ( p = .004 ) and for powerful with trouble - maker the first factor , explaining half the total variance , included popular , powerful , respected and attractive or stylish , each loading over .800 , and the item loading most strongly on the second factor , which explained almost one - fifth of the total variance , was doing well at school ; trouble - maker had a negative loading ( .672 ) ; this factor is titled scholastic status . finally , the third factor , titled sports status , and explaining one - tenth of the total variance , was represented by the single item analyses of variance showed higher scores ( all p < .001 ) for males on the peer ( f = 82.1 ) and sports ( f = 362.0 ) factors , and for females on the scholastic factor ( f = 13.2 ) , the latter reflecting the much lower scores of females on the trouble - maker ladder , which had a negative loading on the scholastic factor . having determined the underlying factor structure of our ladder measures , the next step was to establish the correlates of these dimensions . however , family affluence was positively related to each factor , and most strongly to peer . as shown in table 5 , interviewer - rated attractiveness and physical maturity ratings were positively related to peer , attractiveness also being positively , although much more weakly , associated with scholastic and sports . relationships between nominations and scholastic and sports were also positive , but considerably weaker . peer scores were higher among those who reported having a group or gang at school , and higher scores on all three factors were associated with less frequent victimisation . each academic - related variable ( school skipping , credit - level entries and post - school plans ) was very strongly associated with scholastic , relationships with the other two factors being weaker or non - existent . school skipping was positively related to peer and negatively to scholastic and sports , while number of exam entries was positively associated with the significant gender interaction between exam entries and scholastic occurred because the pattern for males was stronger and more clearly linear than that for females . scholastic and ( weakly ) inversely associated with both peer and sports . thus , exercising or playing sports was positively associated with peer only among males , with watching sports matches was associated with peer ( positively ) and scholastic ( negatively ) among males only , and the positive relationship between sports watching and sports was much stronger for males . this paper has shown that it is possible to identify different dimensions in respect of pupils subjective social status within the school community via a simple set of items which are understood by adolescents and which generate a very high response rate . the distinction between ladders representing popularity , power , receipt of respect , attractiveness or style and trouble - making , and those of academic achievement and conforming to behavioural standards , confirms that respect and highest standing the particular relationships with variables representing more objective measures suggest the dimensions identified are a reflection of peer , scholastic and sports status . the dominant factor ( peer ) included popular , powerful , respected , attractive or stylish and , although weighting less strongly , trouble - maker . but in addition , there was a clear linear relationship between scores on this factor and friendship nominations , a strong indication that those high on self - report peer status were also liked by others . the relative effect sizes suggest that of the three dimensions , self - report scholastic assessments relate most strongly to more objective measures , as might be expected given the significance and visibility of position in the academic hierarchy within the school system . contrasting with peer , scholastic was negatively associated with pocket money , corresponding with another study which found adolescents from higher ses backgrounds and those with less spending money were less likely to own consumer goods such as electrical items , but more likely to own books ( west , sweeting , young , & robins , 2006 ) . scholastic was positively related to friendship nominations , and , more clearly than the peer factor , to group size , but not with self - report group or gang membership , suggesting rather different types of friendship among middle groups by participants in another scottish study ; academically engaged and the kind of adolescents most adults would typically find pleasant and easy to get on with ( michell , 1997 , p. 8) . for example , males rated themselves higher on every ladder , including doing well at school and attractive and stylish , despite that fact that females were entered for significantly more credit - level standard grade exams and were more likely to receive however , the pattern of associations which each of the three factors identified in our analyses had with a range of characteristics previously demonstrated to relate to distinct and meaningful features of adolescent social status suggests these dimensions map well onto more objective measures . an additional perspective is taken by those who point towards reciprocal and interpretivist models , citing evidence that academic self - concept affects subsequent academic interest and achievement ( marsh et al . the existence of different dimensions in pupils subjective social status suggests that studies using multidimensional status measures might find each to be differentially associated with other constructs . in this same study peer , scholastic and sports status are related to the stress hormone , cortisol , in different ways ( west et al . thus , scholastic status might be more positively related to attributes such as leadership , and perhaps also associated with pupil well - being in schools which place a stronger emphasis on academic achievement . first is that while the ladders in our questionnaire largely represented domains identified in the self - concept literature , it is possible , as noted above , that had we included additional characteristics , alternative factors might have emerged . they did not include academic achievement in the form of test results or teacher ratings , nor the ratings of teachers or others of behaviour ; they relied on single measures ; we were unable to assess the reliability of the two interviewer ratings ; and the fact that the majority of variables were self - report introduces the problem of shared method variance . pupils within the same friendship network might have answered the group or gang question differently depending on their self - perceived peer or scholastic status . however , other - report variables were included and most self - report variables were objective ( e.g. the elaboration of multiple dimensions of social hierarchy among young people , and demonstration that they correspond with more objective and/or behavioural measures , represents a first step towards the development of more robust instruments within this area .

there is a shortage of dentists working in australian rural locations.1,2 this problem will increase in severity as the cohort of dentists who qualified between 19651975 begin to retire , especially as many of them work in rural areas . it is essential to encourage new dental graduates to consider working in country areas to alleviate this problem . research literature in other health disciplines shows that a rural placement can increase rural recruitment.3,4,5 in 2005 the james cook university school of medicine in northern australia provided eight week rural internships for all 6 year medical students . the internships were evaluated via questionnaires , site visits , interviews and follow up teleconferences , including a compulsory debrief session . the students reported that the internship was one of the most important clinical experiences of the final year course and they were able to meet their academic curriculum learning objectives.3 the study did not examine whether the students took up rural employment post - graduation and the researchers noted that the long term destination of the students would be an important aspect of further investigation.3 a longitudinal study of medical students in western australia identified rural background , self - reported value of the experience , placement duration and voluntary attendance at the rural placement as some of the factors which could persuade students to work in a rural practice.6 a study in 2007 also concluded rural background is a key predictor for working in a rural medical practice.7 other studies add to the substantial evidence that having a rural background is the single most significant predictor of a subsequent rural career for medical practitioners.8,9,10 the timing of a rural placement is important , with a study showing that offering it to 3 year undergraduates instead of 4 year students led to lower graduate recruitment into rural sectors.11 a rural placement program in tasmania reported that it provided a positive influence on the students intention to work rurally after their graduation.12 despite these positive reports a review published in 200713 claimed that there is still no definitive evidence of the effectiveness of rural intern - ships and recommends isolating the factors which positively influence graduates working in rural regions by producing methodological , structured , rigorous , longitudinal studies which follow up graduates and control for the independent predictors of rural practice . the evidence of the influence of rural placements on dental student s future plans is more limited . nunn and freeman14 highlighted that undergraduate teaching in many parts of the world is focused on techniques which are suited for developed populations and education is providing less encouragement for undergraduates to practice in remote areas . they felt a significant clinical outreach experience away from the large centralized teaching hospitals might encourage students to work in a rural location . one example at the university of western australia incorporated an optional rural , remote and indigenous placement ( rrip).15 the program lasted from 20022005 with 143 students graduating in this period , 55% had a rural placement . there was a gradual increase in the proportion of students who had participated in the placement who then went on to work in a rural area within 12 months of graduation ; 26% in 2002 , 38% in 2003 , 60% in 2004 and 48% in 2005 . the majority ( 95% ) were very positive in their feedback and found the placements to be a valuable learning experience.15 melbourne dental school16 provided a 4 week rural outplacement in 20062007 for 70 students in groups of 6 to 10 to an outreach clinic in the small town of shepparton and questionnaires provided feedback of their experience . the study found 19.5% of students were considering rural practice post placement with only one student uninterested and 79.1% expressed definite affirmation . all the students gained confidence in performing most procedures during their placement and it helped prepare them for dentistry post - graduation . mascarenhas et al17 stated that community based experiences are focussed on patient care and time management skills rather than learning specific clinical skills . a placement program in south australia showed that dental students can make a significant contribution to the provision of public dental services in rural communities , in terms of reducing patient waiting lists , providing patient care and giving students the opportunity to acquire an appreciation of the factors that influence rural dental healthcare practice.18 a community based dental education program set in the united states19 reported that students in community clinics see approximately 6 or more patients per day , whereas traditional dental school clinics which are focused on education see approximately 2 to 3 patients per day . the benefits for students from community programs were noted as the acquirement of knowledge , skills and values that are not readily available in dental school based clinical training . they concluded that students care for more patients , gain teamwork skills and have the opportunity to treat a more diverse range of patients.19 an outreach scheme developed in the uk involved fourth year dental students seconded to primary care centers in socially deprived areas with inadequate general dental services and poor oral health.20 the students completed questionnaires prior to and after the program . qualitative and quantitative research methodology was used in the program assessment using likert scales and semi structured open questions . post outreach the students reported an increase in confidence , improved time management skills and an enhanced ability to identify certain clinical problems . although not a rural experience it did show that working in a primary care location was popular and improved clinical skills.20,21 the faculty of dentistry , university of sydney decided to implement a rural placement program drawing on the positive experiences of the universities of western australia , melbourne and manchester as part of a strategy to promote rural training funded by the australian department of health and ageing . this paper discusses the effectiveness of rural placements in dental undergraduate teaching in terms of the suitability of the clinical training provided , its effect on the students clinical skills and confidence and whether the program influences students to consider rural work postgraduation . with the approval of the ethics review committee ( rpah zone ) data reported here were collected from fourth final year bachelor of dentistry ( bdent ) undergraduates . the rural placement program was offered as a voluntary option to all final year undergraduates ( n=80 ) and 40% ( n=32 ) volunteered . a placement lasted one month at one of three rural clinics , working under supervision four clinical days per week with one day allowed for study . there were four one month placements at bowral ( 6 students per placement ) and bathurst and orange ( taking 2 students each . ) each placement site had a registered dentist acting as a clinical supervisor for the students who received a training course to learn the faculty s teaching protocols . the students worked in pairs on the placement , one being the operator and the other the assistant , rotating these roles every half day . a faculty staff member was on hand to respond to student queries and issues throughout the placements . pre placement meetings were set up to brief the students on the type of clinical care they would practice and to answer any concerns . the students were asked to voluntarily complete a questionnaire which was designed to seek the students , thoughts and opinions on the rural placement program , obtaining feedback on all aspects from the supervisors , accommodation , clinical tasks , non - clinical tasks , the effect the program has had on them , likert scales scores on the students self - perceived clinical confidence and interest in rural practice and why . this paper focuses on the quantitative clinical likert data with some reference to the qualitative findings from the open questions relating to the students clinical work . three clinical supervisors , one from each rural clinic and three university of sydney faculty members were interviewed as part of the evaluation and responses included in this paper pertain to the students clinical work . the clinical supervisors were interviewed via phone and the faculty members were interviewed in person . student respondents were separated into those who volunteered to participate ( volunteers ) and those who did not ( non - volunteers ) . a self - administered pre and post placement questionnaire was distributed to volunteers and non - volunteers in person and via email , questions were a mixture of closed and semi - structured open questions . likert scales were used to measure the students self reported clinical confidence and ability in five key skills , namely , treatment planning , time management , communication skills , clinical ability and treatment skills . pre questionnaires were distributed to volunteers and non - volunteers two weeks prior to the first rural placement . a follow up questionnaire was given to the volunteers when they returned from their placement , and the non - volunteers follow up questionnaire was distributed at the end of the placement program . non respondents were resent questionnaires via email and a follow up telephone call was made one week later to students who had still not responded . with the approval of the ethics review committee ( rpah zone ) data reported here were collected from fourth final year bachelor of dentistry ( bdent ) undergraduates . the rural placement program was offered as a voluntary option to all final year undergraduates ( n=80 ) and 40% ( n=32 ) volunteered . a placement lasted one month at one of three rural clinics , working under supervision four clinical days per week with one day allowed for study . there were four one month placements at bowral ( 6 students per placement ) and bathurst and orange ( taking 2 students each . ) each placement site had a registered dentist acting as a clinical supervisor for the students who received a training course to learn the faculty s teaching protocols . the students worked in pairs on the placement , one being the operator and the other the assistant , rotating these roles every half day . a faculty staff member was on hand to respond to student queries and issues throughout the placements . pre placement meetings were set up to brief the students on the type of clinical care they would practice and to answer any concerns . the students were asked to voluntarily complete a questionnaire which was designed to seek the students , thoughts and opinions on the rural placement program , obtaining feedback on all aspects from the supervisors , accommodation , clinical tasks , non - clinical tasks , the effect the program has had on them , likert scales scores on the students self - perceived clinical confidence and interest in rural practice and why . this paper focuses on the quantitative clinical likert data with some reference to the qualitative findings from the open questions relating to the students clinical work . three clinical supervisors , one from each rural clinic and three university of sydney faculty members were interviewed as part of the evaluation and responses included in this paper pertain to the students clinical work . the clinical supervisors were interviewed via phone and the faculty members were interviewed in person . student respondents were separated into those who volunteered to participate ( volunteers ) and those who did not ( non - volunteers ) . a self - administered pre and post placement questionnaire was distributed to volunteers and non - volunteers in person and via email , questions were a mixture of closed and semi - structured open questions . likert scales were used to measure the students self reported clinical confidence and ability in five key skills , namely , treatment planning , time management , communication skills , clinical ability and treatment skills . pre questionnaires were distributed to volunteers and non - volunteers two weeks prior to the first rural placement . a follow up questionnaire was given to the volunteers when they returned from their placement , and the non - volunteers follow up questionnaire was distributed at the end of the placement program . non respondents were resent questionnaires via email and a follow up telephone call was made one week later to students who had still not responded . the first part of the analysis provides a profile of the students socio - demographic profile including their rural background . data were entered into sas 9.1 for frequency , mean , percentages and paired t - tests were used to compare the various likert scale group data . open ended responses were analysed using framework analysis to identify trends and themes in the group responses . all the volunteer students ( n=32 ) completed a pre - placement and post placement questionnaire whilst 67% of the non - volunteers ( n=32 ) completed a pre - questionnaire and 65% ( n=20 ) of this group completed a post placement questionnaire . a post placement questionnaire was only offered to non - volunteers who completed the pre - placement questionnaire for comparison reasons . all three clinical supervisors and three faculty members who were involved in the placement program were interviewed . the mean age of the volunteers and non - volunteers was both 27 years and this is displayed in table 1 along with the group s gender distribution . rural background has been broadly defined as any rural experience or rural exposure.12 students were asked what type of environment they had grown - up in and to comment on any rural experience they had encountered . there was little difference between the volunteers and non - volunteers , with 38.7% in both groups having lived in a city environment . there were more non - volunteers raised in small town areas ( 8.1% ) than the volunteers ( 3.2% ) . however there is not enough difference between the two groups to confound the results and no significance difference between the groups when a paired t - test was performed with both groups predominantly from a city environment . table 2 shows the mean scores of the five clinical aspects for pre and post volunteers and non - volunteers using the likert scales . based on a 5-point likert scale where 5 is the highest possible self - perceived score , it can be seen that the mean scores for these five areas of clinical practice increased in the volunteer group , but generally remained static for those students who did not complete the rural placement . for example the volunteers reported an increase in the important areas of treatment planning of 3.4 to 3.8 , communication skills 3.9 to 4.3 and treatment skills 3.3 to 3.8 while the non - volunteers increased less in their post placement scores with 3.6 to 3.8 for treatment planning , communication 4.3 to 4.4 and treatment skills 3.5 to 3.7 . the area the volunteer group perceived their greatest improvement was in treatment skills with a 0.5 increase . table 3 presents the combined scores for the five different likert skills , and shows that the post volunteers had almost the same mean score ( 19.3 ) as the post non - volunteers ( 19.2 ) despite the fact that the non - volunteers had a higher self - perceived score prior to the beginning of the rural placement program of 18.5 compared to a lower 17.3 from the volunteers . displayed as a percentage the volunteer group shows an 8% ( 69.2% to 77.2% ) increase in their combined self - reported skill level post placement , whereas the non - volunteers increase from pre to post placement was less than 3% ( 74% to 76.8% ) . a paired t - test showed a significantly higher mean skill score in the pre - placement non volunteers scores compared to the pre placement volunteers ( t=2.26 p=<.05 ) , with a mean higher score of 1.2 ( 95% ci 0.1 to 2.2 ) . the volunteer group reported a lower level of overall clinical ability compared with the pre - placement non - volunteers . the volunteers and non - volunteers changing scores from pre to post placement were compared to ascertain if there was a significant difference . table 4 shows the paired t - test results and identified that the rural placement volunteers had a significantly greater increase in their mean combined clinical scores of 2.0 compared to 0.5 of the non - volunteers ( t53 = 2.35 ; p<.02 ) . the clinical treatment episodes undertaken by the students were entered into the nsw dental public service data collection information system for oral health ( isoh ) and the main categories of care are shown in table 5 . the most common clinical procedure in bathurst was dental extractions , whilst at bowral and orange restorative care was the most common procedure , followed by acute care at bowral and dental extractions at orange . the system recorded across all the placements a total of 712 episodes of restorative care , 500 episodes of acute care , 420 dental extractions , 160 treatment plans and prevention , 143 of periodontal care and 45 episodes of denture care and fixed prosthodontics . the mean number of episodes of care for each student across the three placements per placement was 59 . this data was drawn from the semi - structured open questionnaire responses from students and interviews with the clinical supervisors and university faculty members . other procedures mentioned by several students were detecting caries , patient management , root canal therapy , acute care management , dealing with special care patients and cutting crown preparations . they reported that they were expected to complete patient treatment at a much faster pace than at the teaching metropolitan hospitals . according to the students they saw between 6 and 8 patients per day whereas at the university teaching hospitals non volunteers reported seeing approximately 3 to 4 patients per day . the non - volunteers reported completing more specialist dentistry such as prosthodontics and less basic clinical dentistry . non volunteers gave reasons for regretting not participating in the placement such as missing a valuable clinical experience , the opportunity to make new friends and making professional contacts . the minority of non - volunteers who had no regrets explained they felt being at the university teaching hospitals provided greater opportunity for more specialized clinical training such as fixed prosthodontics . the clinical supervisors reported the students increased in confidence , as well as improved clinical ability and clinical efficiency . supervisors also commented that students improved in their treatment planning , time managements and communication skills . they also confirmed the students saw between 68 patients per day as their clinical efficiency improved . they commented the pre - placement volunteers were as clinically competent as the non - volunteers and many of the more capable students volunteered for the program . the faculty members noted some of the returning students had stronger communication skills and confidence based on debriefing conversations . some students enquired about rural clinical employment at the rural clinics for post - graduation and almost all the students were very positive towards potentially working in a rural environment . table 6 displays the students reported interest in working in a rural location pre placement from two groups , 54.8% of the pre - placement volunteers were considering working in a rural placement after graduation compared to 35.5% of the non - volunteers . table 6 also shows that 96.9% of the volunteers post placement would consider working in a rural location after they graduate , a 42% increase in favor of working rurally . the non - volunteers were not asked this question in the follow up questionnaire as they had not had a rural experience to alter their perspective . the non - volunteer group was asked if they regretted not taking part in the rural placement after hearing reports from the returning volunteers and 57% of them regretted not participating , 30% were undecided and 13% had no regrets . the first part of the analysis provides a profile of the students socio - demographic profile including their rural background . data were entered into sas 9.1 for frequency , mean , percentages and paired t - tests were used to compare the various likert scale group data . open ended responses were analysed using framework analysis to identify trends and themes in the group responses . all the volunteer students ( n=32 ) completed a pre - placement and post placement questionnaire whilst 67% of the non - volunteers ( n=32 ) completed a pre - questionnaire and 65% ( n=20 ) of this group completed a post placement questionnaire . a post placement questionnaire was only offered to non - volunteers who completed the pre - placement questionnaire for comparison reasons . all three clinical supervisors and three faculty members who were involved in the placement program were interviewed . the mean age of the volunteers and non - volunteers was both 27 years and this is displayed in table 1 along with the group s gender distribution . rural background has been broadly defined as any rural experience or rural exposure.12 students were asked what type of environment they had grown - up in and to comment on any rural experience they had encountered . there was little difference between the volunteers and non - volunteers , with 38.7% in both groups having lived in a city environment . there were more non - volunteers raised in small town areas ( 8.1% ) than the volunteers ( 3.2% ) . however there is not enough difference between the two groups to confound the results and no significance difference between the groups when a paired t - test was performed with both groups predominantly from a city environment . table 2 shows the mean scores of the five clinical aspects for pre and post volunteers and non - volunteers using the likert scales . based on a 5-point likert scale where 5 is the highest possible self - perceived score , it can be seen that the mean scores for these five areas of clinical practice increased in the volunteer group , but generally remained static for those students who did not complete the rural placement . for example the volunteers reported an increase in the important areas of treatment planning of 3.4 to 3.8 , communication skills 3.9 to 4.3 and treatment skills 3.3 to 3.8 while the non - volunteers increased less in their post placement scores with 3.6 to 3.8 for treatment planning , communication 4.3 to 4.4 and treatment skills 3.5 to 3.7 . the area the volunteer group perceived their greatest improvement was in treatment skills with a 0.5 increase . table 3 presents the combined scores for the five different likert skills , and shows that the post volunteers had almost the same mean score ( 19.3 ) as the post non - volunteers ( 19.2 ) despite the fact that the non - volunteers had a higher self - perceived score prior to the beginning of the rural placement program of 18.5 compared to a lower 17.3 from the volunteers . displayed as a percentage the volunteer group shows an 8% ( 69.2% to 77.2% ) increase in their combined self - reported skill level post placement , whereas the non - volunteers increase from pre to post placement was less than 3% ( 74% to 76.8% ) . a paired t - test showed a significantly higher mean skill score in the pre - placement non volunteers scores compared to the pre placement volunteers ( t=2.26 p=<.05 ) , with a mean higher score of 1.2 ( 95% ci 0.1 to 2.2 ) . the volunteer group reported a lower level of overall clinical ability compared with the pre - placement non - volunteers . the volunteers and non - volunteers changing scores from pre to post placement were compared to ascertain if there was a significant difference . table 4 shows the paired t - test results and identified that the rural placement volunteers had a significantly greater increase in their mean combined clinical scores of 2.0 compared to 0.5 of the non - volunteers ( t53 = 2.35 ; p<.02 ) . the clinical treatment episodes undertaken by the students were entered into the nsw dental public service data collection information system for oral health ( isoh ) and the main categories of care are shown in table 5 . the most common clinical procedure in bathurst was dental extractions , whilst at bowral and orange restorative care was the most common procedure , followed by acute care at bowral and dental extractions at orange . the system recorded across all the placements a total of 712 episodes of restorative care , 500 episodes of acute care , 420 dental extractions , 160 treatment plans and prevention , 143 of periodontal care and 45 episodes of denture care and fixed prosthodontics . the mean number of episodes of care for each student across the three placements per placement was 59 . this data was drawn from the semi - structured open questionnaire responses from students and interviews with the clinical supervisors and university faculty members . other procedures mentioned by several students were detecting caries , patient management , root canal therapy , acute care management , dealing with special care patients and cutting crown preparations . they reported that they were expected to complete patient treatment at a much faster pace than at the teaching metropolitan hospitals . according to the students they saw between 6 and 8 patients per day whereas at the university teaching hospitals non volunteers reported seeing approximately 3 to 4 patients per day . the non - volunteers reported completing more specialist dentistry such as prosthodontics and less basic clinical dentistry . non volunteers gave reasons for regretting not participating in the placement such as missing a valuable clinical experience , the opportunity to make new friends and making professional contacts . the minority of non - volunteers who had no regrets explained they felt being at the university teaching hospitals provided greater opportunity for more specialized clinical training such as fixed prosthodontics . the clinical supervisors reported the students increased in confidence , as well as improved clinical ability and clinical efficiency . supervisors also commented that students improved in their treatment planning , time managements and communication skills . they also confirmed the students saw between 68 patients per day as their clinical efficiency improved . they commented the pre - placement volunteers were as clinically competent as the non - volunteers and many of the more capable students volunteered for the program . the faculty members noted some of the returning students had stronger communication skills and confidence based on debriefing conversations . some students enquired about rural clinical employment at the rural clinics for post - graduation and almost all the students were very positive towards potentially working in a rural environment . table 6 displays the students reported interest in working in a rural location pre placement from two groups , 54.8% of the pre - placement volunteers were considering working in a rural placement after graduation compared to 35.5% of the non - volunteers . table 6 also shows that 96.9% of the volunteers post placement would consider working in a rural location after they graduate , a 42% increase in favor of working rurally . the non - volunteers were not asked this question in the follow up questionnaire as they had not had a rural experience to alter their perspective . the non - volunteer group was asked if they regretted not taking part in the rural placement after hearing reports from the returning volunteers and 57% of them regretted not participating , 30% were undecided and 13% had no regrets . the overall response rate from the students was excellent , with all the volunteers and a high percentage of the non - volunteers ( pre questionnaire - 67% and post - 65% ) completing the pre and post placement questionnaires . non respondents were followed up using email and mobile phones , with a mixture of phoning and sms texting to encourage them to complete the questionnaire . the use of a control group increased the validity of the findings and by recording information pre and post completion of the rural placements . the program targeted 4 year students11 and each placement lasted one month because the research concluded that this timeframe encouraged students to consider rural work post - graduation.6 rural background was measured as the literature identified it as a potential confounder.6,7,8,9,10 the two student groups were found to be not significantly different in terms of their rural background allowing for comparisons to be made . likert scale methods and some open ended semi structured questions were used to provide both quantitative and qualitative data as the literature review showed these methods are a valuable research tool.3,4,15,20 also clinical supervisors via phone interviews were asked semi structured questions regarding the clinical work completed to verify the student responses and isoh data . the data collection system allowed a large amount of data to be acquired in a short time . the likert scales and students self - perceived levels of treatment planning , time management , communication skills , clinical ability and treatment skills were all positive for the rural sample . our results are similar to the manchester20 and leeds studies21 which found that students increased in their confidence , time management and diagnosis skills . we also found that their self - perceived clinical skills increased , the students saw more patients per day in the rural clinic than at the dental school clinics and this was confirmed by the rural clinical supervisors . this supports the evidence by baiit19 which also found students saw approximately 6 patients per day in a community clinic compared to 2 and 3 at the dental school teaching clinics . the non - volunteers had a significantly higher mean pre - placement score ( 18.5 ) than the volunteers ( 17.3 ) . an explanation as to why the pre volunteers had a lower likert score across the five skills could be due to pre placement anxiety . the finding that greater improvement occurs in the volunteers from pre to post placement than the non - volunteers is important as it demonstrates the significant increase in clinical confidence of the volunteer students post placement . the volunteer and non - volunteer group were directly compared and there was a significantly ( p<.05 ) larger increase in skill level from pre to post placement for the volunteers than the non - volunteers despite starting with a lower pre placement self - perceived score . this increase may be due to the volunteers considerable clinical work undertaken whilst on the placement in seeing more patients per day and completing more basic clinical dentistry than at the metropolitan teaching hospitals . the supervisors commented that the students helped reduce the patient waiting lists at the orange and bathurst clinics supporting richards et al18 who felt that dental students can make a significant contribution to the public dental services waiting lists . the clinical data entered into the nsw information system for oral health ( isoh ) supports the reported responses from the students about their level of clinical activity , and is an independent verification that considerable practical primary care dentistry was undertaken . in our study 97% of the volunteers post placement felt they were more likely to work in a rural setting after graduation , demonstrating a dramatic change in attitude which supports the findings of western australia15 and melbourne16 who found students were encouraged to work rurally post placement . this shows the positive influence the rural placements had on the students attitude towards considering working in a rural location upon graduation . longitudinal follow up is important for monitoring the long term value of rural placements ; therefore a follow up of these students will be undertaken in 18 months time to record their working location . after the students returned from the rural placement , the non - volunteers were asked if they had any regrets based on what they had heard from the volunteers , more than half of the non - volunteers felt they had missed out on a valuable teaching experience and the chance to make and build contacts and friendships . this demonstrates the positive feedback from the volunteers post placement regarding the placement when reintegrated with the non - volunteers . self - report was used so there is potential for recall bias , judgement error , memory loss and providing socially desirable answers . however with this type of study self - report is probably the most practical way to collect data given the intensity of the dental undergraduates curriculum . the weakness with semi - structured questions and likert scales is that the students were somewhat restricted as they were asked a set of specific questions . an interview for each participant would have provided more detailed in depth responses , however this would have been very time consuming and labour intensive for both the student and interviewer . likert scale scores are open to some interpretation and there was a higher response rate from the volunteer group and this may have had some impact . the timing of the completion of questionnaires varied and this is a potential weakness , pre volunteers completed their questionnaire one week pre placement and within 14 days post placement . non volunteers completed their pre placement questionnaires prior to the first placement and post placement they completed their questionnaires at the end of the teaching year . this had to be the case as the volunteers needed to be questioned soon after their placement to avoid memory loss and the non - volunteers responses were dependant on interactions with the returning volunteers , hence had to be undertaken after the final rural placement . by asking the non - volunteers at the end of the final year after all their clinical training , you would expect a positive shift in their perceived clinical scores and hence the groups final mean clinical score would be expected to be higher not lower than if taken earlier in the year . the questions were based on the questionnaire design and placement evaluation set up by the manchester20 study , however the questionnaire was not a validated instrument and had nt been used previously . the multiple sources of information gathering using student , faculty and clinician views on the students clinical work provided consistent data22 which increases the validity and reliability of the findings . this paper has focussed on the quantitative data from the students perspective of the rural placement program with relevant comments on the clinical training from faculty staff and clinical supervisors . self - report was used so there is potential for recall bias , judgement error , memory loss and providing socially desirable answers . however with this type of study self - report is probably the most practical way to collect data given the intensity of the dental undergraduates curriculum . the weakness with semi - structured questions and likert scales is that the students were somewhat restricted as they were asked a set of specific questions . an interview for each participant would have provided more detailed in depth responses , however this would have been very time consuming and labour intensive for both the student and interviewer . likert scale scores are open to some interpretation and there was a higher response rate from the volunteer group and this may have had some impact . the timing of the completion of questionnaires varied and this is a potential weakness , pre volunteers completed their questionnaire one week pre placement and within 14 days post placement . non volunteers completed their pre placement questionnaires prior to the first placement and post placement they completed their questionnaires at the end of the teaching year . this had to be the case as the volunteers needed to be questioned soon after their placement to avoid memory loss and the non - volunteers responses were dependant on interactions with the returning volunteers , hence had to be undertaken after the final rural placement . by asking the non - volunteers at the end of the final year after all their clinical training , you would expect a positive shift in their perceived clinical scores and hence the groups final mean clinical score would be expected to be higher not lower than if taken earlier in the year . the questions were based on the questionnaire design and placement evaluation set up by the manchester20 study , however the questionnaire was not a validated instrument and had nt been used previously . the multiple sources of information gathering using student , faculty and clinician views on the students clinical work provided consistent data22 which increases the validity and reliability of the findings . this paper has focussed on the quantitative data from the students perspective of the rural placement program with relevant comments on the clinical training from faculty staff and clinical supervisors . the rural placement program provided an excellent clinical experience as students reported that their clinical confidence and skills had improved . they also reported an increased likelihood to consider working in a rural location after graduation .

objective : to evaluate the effectiveness of a rural clinical placement on students self - perceived clinical skills and work location choice post-graduation.methods:a one month rural placement program was introduced in 20082009 for 4th year dental undergraduates . student s views on the rural exercise were collected by pre and post self - completion questionnaires , which were distributed to rural placement students and to the students who did not participate in the placement . information was collected on self - reported skill levels in various clinical techniques using likert scales and future rural work intentions . clinical supervisors and university faculty members opinions on the students clinical work were also collected via interviews.results:the mean age of the respondents was 27 years and the majority were female ( 57% ) . all the placement students ( volunteers ) completed pre and post placement questionnaires and ( 67% ) of the non - placement students completed the pre questionnaire and ( 65% ) a post questionnaire . when the two groups were compared in terms of their likert scores , there was a significantly ( p<.02 ) larger increase in skill level in the rural group . clinical supervisors confirmed volunteers increased in confidence and ability in clinical skills post placement . the majority ( 96% ) of students who completed a placement reported that they were more likely to work in a rural environment after graduation.conclusions:the rural placement improved the self - perceived clinical skills of the volunteers and enhanced positive attitudes towards working in a rural location .

INTRODUCTION MATERIALS AND METHODS Sample and procedures RESULTS Data Analysis Socio-demographic profile Self-Analysis of the Students Clinical Skills using Likert Scales Clinical ISOH Scores Qualitative data on Clinical care Student Open Question Responses on Rural Clinical Experience Clinical Supervisor Responses Faculty Members Responses Students Rural Intentions DISCUSSION Limitations CONCLUSIONS

the students reported that the internship was one of the most important clinical experiences of the final year course and they were able to meet their academic curriculum learning objectives.3 the study did not examine whether the students took up rural employment post - graduation and the researchers noted that the long term destination of the students would be an important aspect of further investigation.3 a longitudinal study of medical students in western australia identified rural background , self - reported value of the experience , placement duration and voluntary attendance at the rural placement as some of the factors which could persuade students to work in a rural practice.6 a study in 2007 also concluded rural background is a key predictor for working in a rural medical practice.7 other studies add to the substantial evidence that having a rural background is the single most significant predictor of a subsequent rural career for medical practitioners.8,9,10 the timing of a rural placement is important , with a study showing that offering it to 3 year undergraduates instead of 4 year students led to lower graduate recruitment into rural sectors.11 a rural placement program in tasmania reported that it provided a positive influence on the students intention to work rurally after their graduation.12 despite these positive reports a review published in 200713 claimed that there is still no definitive evidence of the effectiveness of rural intern - ships and recommends isolating the factors which positively influence graduates working in rural regions by producing methodological , structured , rigorous , longitudinal studies which follow up graduates and control for the independent predictors of rural practice . they felt a significant clinical outreach experience away from the large centralized teaching hospitals might encourage students to work in a rural location . there was a gradual increase in the proportion of students who had participated in the placement who then went on to work in a rural area within 12 months of graduation ; 26% in 2002 , 38% in 2003 , 60% in 2004 and 48% in 2005 . the majority ( 95% ) were very positive in their feedback and found the placements to be a valuable learning experience.15 melbourne dental school16 provided a 4 week rural outplacement in 20062007 for 70 students in groups of 6 to 10 to an outreach clinic in the small town of shepparton and questionnaires provided feedback of their experience . a placement program in south australia showed that dental students can make a significant contribution to the provision of public dental services in rural communities , in terms of reducing patient waiting lists , providing patient care and giving students the opportunity to acquire an appreciation of the factors that influence rural dental healthcare practice.18 a community based dental education program set in the united states19 reported that students in community clinics see approximately 6 or more patients per day , whereas traditional dental school clinics which are focused on education see approximately 2 to 3 patients per day . they concluded that students care for more patients , gain teamwork skills and have the opportunity to treat a more diverse range of patients.19 an outreach scheme developed in the uk involved fourth year dental students seconded to primary care centers in socially deprived areas with inadequate general dental services and poor oral health.20 the students completed questionnaires prior to and after the program . post outreach the students reported an increase in confidence , improved time management skills and an enhanced ability to identify certain clinical problems . although not a rural experience it did show that working in a primary care location was popular and improved clinical skills.20,21 the faculty of dentistry , university of sydney decided to implement a rural placement program drawing on the positive experiences of the universities of western australia , melbourne and manchester as part of a strategy to promote rural training funded by the australian department of health and ageing . this paper discusses the effectiveness of rural placements in dental undergraduate teaching in terms of the suitability of the clinical training provided , its effect on the students clinical skills and confidence and whether the program influences students to consider rural work postgraduation . the rural placement program was offered as a voluntary option to all final year undergraduates ( n=80 ) and 40% ( n=32 ) volunteered . the students worked in pairs on the placement , one being the operator and the other the assistant , rotating these roles every half day . the students were asked to voluntarily complete a questionnaire which was designed to seek the students , thoughts and opinions on the rural placement program , obtaining feedback on all aspects from the supervisors , accommodation , clinical tasks , non - clinical tasks , the effect the program has had on them , likert scales scores on the students self - perceived clinical confidence and interest in rural practice and why . this paper focuses on the quantitative clinical likert data with some reference to the qualitative findings from the open questions relating to the students clinical work . three clinical supervisors , one from each rural clinic and three university of sydney faculty members were interviewed as part of the evaluation and responses included in this paper pertain to the students clinical work . student respondents were separated into those who volunteered to participate ( volunteers ) and those who did not ( non - volunteers ) . a self - administered pre and post placement questionnaire was distributed to volunteers and non - volunteers in person and via email , questions were a mixture of closed and semi - structured open questions . likert scales were used to measure the students self reported clinical confidence and ability in five key skills , namely , treatment planning , time management , communication skills , clinical ability and treatment skills . pre questionnaires were distributed to volunteers and non - volunteers two weeks prior to the first rural placement . a follow up questionnaire was given to the volunteers when they returned from their placement , and the non - volunteers follow up questionnaire was distributed at the end of the placement program . the rural placement program was offered as a voluntary option to all final year undergraduates ( n=80 ) and 40% ( n=32 ) volunteered . the students worked in pairs on the placement , one being the operator and the other the assistant , rotating these roles every half day . the students were asked to voluntarily complete a questionnaire which was designed to seek the students , thoughts and opinions on the rural placement program , obtaining feedback on all aspects from the supervisors , accommodation , clinical tasks , non - clinical tasks , the effect the program has had on them , likert scales scores on the students self - perceived clinical confidence and interest in rural practice and why . this paper focuses on the quantitative clinical likert data with some reference to the qualitative findings from the open questions relating to the students clinical work . three clinical supervisors , one from each rural clinic and three university of sydney faculty members were interviewed as part of the evaluation and responses included in this paper pertain to the students clinical work . student respondents were separated into those who volunteered to participate ( volunteers ) and those who did not ( non - volunteers ) . a self - administered pre and post placement questionnaire was distributed to volunteers and non - volunteers in person and via email , questions were a mixture of closed and semi - structured open questions . likert scales were used to measure the students self reported clinical confidence and ability in five key skills , namely , treatment planning , time management , communication skills , clinical ability and treatment skills . pre questionnaires were distributed to volunteers and non - volunteers two weeks prior to the first rural placement . a follow up questionnaire was given to the volunteers when they returned from their placement , and the non - volunteers follow up questionnaire was distributed at the end of the placement program . all the volunteer students ( n=32 ) completed a pre - placement and post placement questionnaire whilst 67% of the non - volunteers ( n=32 ) completed a pre - questionnaire and 65% ( n=20 ) of this group completed a post placement questionnaire . a post placement questionnaire was only offered to non - volunteers who completed the pre - placement questionnaire for comparison reasons . all three clinical supervisors and three faculty members who were involved in the placement program were interviewed . the mean age of the volunteers and non - volunteers was both 27 years and this is displayed in table 1 along with the group s gender distribution . there was little difference between the volunteers and non - volunteers , with 38.7% in both groups having lived in a city environment . table 2 shows the mean scores of the five clinical aspects for pre and post volunteers and non - volunteers using the likert scales . based on a 5-point likert scale where 5 is the highest possible self - perceived score , it can be seen that the mean scores for these five areas of clinical practice increased in the volunteer group , but generally remained static for those students who did not complete the rural placement . for example the volunteers reported an increase in the important areas of treatment planning of 3.4 to 3.8 , communication skills 3.9 to 4.3 and treatment skills 3.3 to 3.8 while the non - volunteers increased less in their post placement scores with 3.6 to 3.8 for treatment planning , communication 4.3 to 4.4 and treatment skills 3.5 to 3.7 . table 3 presents the combined scores for the five different likert skills , and shows that the post volunteers had almost the same mean score ( 19.3 ) as the post non - volunteers ( 19.2 ) despite the fact that the non - volunteers had a higher self - perceived score prior to the beginning of the rural placement program of 18.5 compared to a lower 17.3 from the volunteers . displayed as a percentage the volunteer group shows an 8% ( 69.2% to 77.2% ) increase in their combined self - reported skill level post placement , whereas the non - volunteers increase from pre to post placement was less than 3% ( 74% to 76.8% ) . a paired t - test showed a significantly higher mean skill score in the pre - placement non volunteers scores compared to the pre placement volunteers ( t=2.26 p=<.05 ) , with a mean higher score of 1.2 ( 95% ci 0.1 to 2.2 ) . the volunteers and non - volunteers changing scores from pre to post placement were compared to ascertain if there was a significant difference . table 4 shows the paired t - test results and identified that the rural placement volunteers had a significantly greater increase in their mean combined clinical scores of 2.0 compared to 0.5 of the non - volunteers ( t53 = 2.35 ; p<.02 ) . this data was drawn from the semi - structured open questionnaire responses from students and interviews with the clinical supervisors and university faculty members . they commented the pre - placement volunteers were as clinically competent as the non - volunteers and many of the more capable students volunteered for the program . some students enquired about rural clinical employment at the rural clinics for post - graduation and almost all the students were very positive towards potentially working in a rural environment . table 6 displays the students reported interest in working in a rural location pre placement from two groups , 54.8% of the pre - placement volunteers were considering working in a rural placement after graduation compared to 35.5% of the non - volunteers . table 6 also shows that 96.9% of the volunteers post placement would consider working in a rural location after they graduate , a 42% increase in favor of working rurally . the non - volunteer group was asked if they regretted not taking part in the rural placement after hearing reports from the returning volunteers and 57% of them regretted not participating , 30% were undecided and 13% had no regrets . all the volunteer students ( n=32 ) completed a pre - placement and post placement questionnaire whilst 67% of the non - volunteers ( n=32 ) completed a pre - questionnaire and 65% ( n=20 ) of this group completed a post placement questionnaire . a post placement questionnaire was only offered to non - volunteers who completed the pre - placement questionnaire for comparison reasons . all three clinical supervisors and three faculty members who were involved in the placement program were interviewed . the mean age of the volunteers and non - volunteers was both 27 years and this is displayed in table 1 along with the group s gender distribution . there was little difference between the volunteers and non - volunteers , with 38.7% in both groups having lived in a city environment . table 2 shows the mean scores of the five clinical aspects for pre and post volunteers and non - volunteers using the likert scales . based on a 5-point likert scale where 5 is the highest possible self - perceived score , it can be seen that the mean scores for these five areas of clinical practice increased in the volunteer group , but generally remained static for those students who did not complete the rural placement . for example the volunteers reported an increase in the important areas of treatment planning of 3.4 to 3.8 , communication skills 3.9 to 4.3 and treatment skills 3.3 to 3.8 while the non - volunteers increased less in their post placement scores with 3.6 to 3.8 for treatment planning , communication 4.3 to 4.4 and treatment skills 3.5 to 3.7 . table 3 presents the combined scores for the five different likert skills , and shows that the post volunteers had almost the same mean score ( 19.3 ) as the post non - volunteers ( 19.2 ) despite the fact that the non - volunteers had a higher self - perceived score prior to the beginning of the rural placement program of 18.5 compared to a lower 17.3 from the volunteers . displayed as a percentage the volunteer group shows an 8% ( 69.2% to 77.2% ) increase in their combined self - reported skill level post placement , whereas the non - volunteers increase from pre to post placement was less than 3% ( 74% to 76.8% ) . a paired t - test showed a significantly higher mean skill score in the pre - placement non volunteers scores compared to the pre placement volunteers ( t=2.26 p=<.05 ) , with a mean higher score of 1.2 ( 95% ci 0.1 to 2.2 ) . the volunteers and non - volunteers changing scores from pre to post placement were compared to ascertain if there was a significant difference . table 4 shows the paired t - test results and identified that the rural placement volunteers had a significantly greater increase in their mean combined clinical scores of 2.0 compared to 0.5 of the non - volunteers ( t53 = 2.35 ; p<.02 ) . this data was drawn from the semi - structured open questionnaire responses from students and interviews with the clinical supervisors and university faculty members . the clinical supervisors reported the students increased in confidence , as well as improved clinical ability and clinical efficiency . they commented the pre - placement volunteers were as clinically competent as the non - volunteers and many of the more capable students volunteered for the program . some students enquired about rural clinical employment at the rural clinics for post - graduation and almost all the students were very positive towards potentially working in a rural environment . table 6 displays the students reported interest in working in a rural location pre placement from two groups , 54.8% of the pre - placement volunteers were considering working in a rural placement after graduation compared to 35.5% of the non - volunteers . table 6 also shows that 96.9% of the volunteers post placement would consider working in a rural location after they graduate , a 42% increase in favor of working rurally . the non - volunteer group was asked if they regretted not taking part in the rural placement after hearing reports from the returning volunteers and 57% of them regretted not participating , 30% were undecided and 13% had no regrets . the overall response rate from the students was excellent , with all the volunteers and a high percentage of the non - volunteers ( pre questionnaire - 67% and post - 65% ) completing the pre and post placement questionnaires . the use of a control group increased the validity of the findings and by recording information pre and post completion of the rural placements . the program targeted 4 year students11 and each placement lasted one month because the research concluded that this timeframe encouraged students to consider rural work post - graduation.6 rural background was measured as the literature identified it as a potential confounder.6,7,8,9,10 the two student groups were found to be not significantly different in terms of their rural background allowing for comparisons to be made . the likert scales and students self - perceived levels of treatment planning , time management , communication skills , clinical ability and treatment skills were all positive for the rural sample . we also found that their self - perceived clinical skills increased , the students saw more patients per day in the rural clinic than at the dental school clinics and this was confirmed by the rural clinical supervisors . the non - volunteers had a significantly higher mean pre - placement score ( 18.5 ) than the volunteers ( 17.3 ) . the finding that greater improvement occurs in the volunteers from pre to post placement than the non - volunteers is important as it demonstrates the significant increase in clinical confidence of the volunteer students post placement . the volunteer and non - volunteer group were directly compared and there was a significantly ( p<.05 ) larger increase in skill level from pre to post placement for the volunteers than the non - volunteers despite starting with a lower pre placement self - perceived score . this increase may be due to the volunteers considerable clinical work undertaken whilst on the placement in seeing more patients per day and completing more basic clinical dentistry than at the metropolitan teaching hospitals . in our study 97% of the volunteers post placement felt they were more likely to work in a rural setting after graduation , demonstrating a dramatic change in attitude which supports the findings of western australia15 and melbourne16 who found students were encouraged to work rurally post placement . this shows the positive influence the rural placements had on the students attitude towards considering working in a rural location upon graduation . after the students returned from the rural placement , the non - volunteers were asked if they had any regrets based on what they had heard from the volunteers , more than half of the non - volunteers felt they had missed out on a valuable teaching experience and the chance to make and build contacts and friendships . this demonstrates the positive feedback from the volunteers post placement regarding the placement when reintegrated with the non - volunteers . non volunteers completed their pre placement questionnaires prior to the first placement and post placement they completed their questionnaires at the end of the teaching year . this had to be the case as the volunteers needed to be questioned soon after their placement to avoid memory loss and the non - volunteers responses were dependant on interactions with the returning volunteers , hence had to be undertaken after the final rural placement . by asking the non - volunteers at the end of the final year after all their clinical training , you would expect a positive shift in their perceived clinical scores and hence the groups final mean clinical score would be expected to be higher not lower than if taken earlier in the year . the multiple sources of information gathering using student , faculty and clinician views on the students clinical work provided consistent data22 which increases the validity and reliability of the findings . this paper has focussed on the quantitative data from the students perspective of the rural placement program with relevant comments on the clinical training from faculty staff and clinical supervisors . non volunteers completed their pre placement questionnaires prior to the first placement and post placement they completed their questionnaires at the end of the teaching year . this had to be the case as the volunteers needed to be questioned soon after their placement to avoid memory loss and the non - volunteers responses were dependant on interactions with the returning volunteers , hence had to be undertaken after the final rural placement . by asking the non - volunteers at the end of the final year after all their clinical training , you would expect a positive shift in their perceived clinical scores and hence the groups final mean clinical score would be expected to be higher not lower than if taken earlier in the year . the multiple sources of information gathering using student , faculty and clinician views on the students clinical work provided consistent data22 which increases the validity and reliability of the findings . this paper has focussed on the quantitative data from the students perspective of the rural placement program with relevant comments on the clinical training from faculty staff and clinical supervisors . the rural placement program provided an excellent clinical experience as students reported that their clinical confidence and skills had improved . they also reported an increased likelihood to consider working in a rural location after graduation .

cardiac output ( co ) , expressed in liters / minute , is the amount of blood pumped by heart in one minute . mathematically , co is the product of heart rate and the stroke volume [ 1 - 8 ] . the heart rate , in beats / min , is the number of beats per minute and the stoke volume is the volume of blood , in milliliters ( ml ) , pumped out of the heart on each beat . cardiac output depends on cardiovascular system parameters and cardiac or even extra - cardiac factors . also , co is a fundamental determinant of oxygen delivered to tissues and is an essential indicator of how well the heart can meet the demands of the body . therefore , co is one of the most important parameters for monitoring cardiac function , estimating global oxygen delivery and for understanding the causes of high blood pressure . in other words , co is a critical factor for monitoring hemodynamic status of patients [ 1 - 11 ] . ultimately , measuring cardiac output has always been a matter of interest to researchers and clinicians . the methods developed so far for measuring co can be categorized as : flowmetry , fick [ 13 - 16 ] , the relative exhaled carbon dioxide , thermo dilution , esophageal doppler ultrasound , esophageal doppler [ 17,18,19,27 - 31 ] , bio - electrical impedance [ 32 - 34 ] , plethysmography impedance and arterial analysis method [ 12,13,14,35 - 43 ] . most of these methods are either invasive or too expensive and need special expertise or experience ; hence , they are not risk free . for example , fick and thermo dilution methods are both clinically possible , but they are invasive methods and could only be conducted in an equipped environment such as intensive care units ( icus ) and the cardiac catheterization laboratories . in addition , these methods require the injection of cold saline or dye into a big vessel in which the whole cardiac output is flowing in . in this method , swan - ganz catheter is used which enters the right side of heart through the inferior vena cava and then the pulmonary artery , but it is both invasive and needs special expertise and experience . in fick method , the measurement of mixed venous oxygen needs blood sample from the pulmonary artery which is an invasive method . doppler ultrasound method is non - invasive and accurate , but needs expensive equipment and the operator must be an expert . therefore , to facilitate the conduction and to prevent the side effects of the invasive methods for measuring the cardiac output , a system was designed to measure the cardiac output in a non - invasive way ( with the least invasion ) through the arterial pulse wave . the developed co estimation system is generally based on the theory that blood pressure in an artery is related to the stroke volume ( sv ) . the relation between sv , blood pressure and vascular resistance ( z ) is as follows [ 12,13,14,35 - 43 ] : sv=ejection[pa(t)-pd]dtz ( 1 ) where pa(t ) is the arterial pressure at time t and ptd is the arterial pressure at the end of diastole . in fact , this integral calculates the area under systolic portion of the arterial pressure waveform , from the end - diastole to the end of the ejection phase ; this corresponds to stroke volume . figure 1shows an example of the area under the curve calculated by the integral used in equation 1 . the area under the wave of the arterial pressure curve for estimating the stroke volume pressure changes in the artery are obtained via analyzing arterial pulse ; details will be explained below . through beat - to - beat analysis of the arterial pressure waveform , the cardiac output can be measured continuously . an important advantage of this approach is that the patient does not require endotracheal intubation or sedation to be monitored . most steps involved in the developed system are processing and analyzing the arterial pressure waveform . figure 2 illustrates the main steps of the developed system . in short , from a complex arterial pulse , we find the starting point and the dicrotic notch corresponding to the systolic portion of the arterial pressures , then , the area under the curve between these two points is calculated . the area under the curve is proportional to the stroke volume ; if it is multiplied by the heart rate , then we can calculate the cardiac output . steps of the developed cardiac output estimating system the signal is recorded using a blood pressure transducer which is attached to the arterial lane ( radial artery , brachial or femoral ) . in this method , at first an arterial route is created by a catheter 22 ( arterial line ) , and then the arterial lane is connected to the transducer . then , mechanical pulses of the heart beat change into electrical signals through the diaphragm and the life signals are transmitted to the monitor . finally , by connecting the monitor to a computer , the signals are stored . although estimating the area under curve seems easy , the accurate estimation of co is difficult due to many artifacts that can affect the signal . arterial waveform of the signal often interferes with different noises such as the power line noise , base line wandering noise and emg signals . these noises are often created as a result of electrical signal fluctuations , motion or breathing artifacts . extracting information from the arterial waveform relies on a clean and noise - free signal ; therefore , before using any algorithms to extract clinical information from the arterial waveform , it is necessary to remove the unusual waveforms and noise signals . the purpose of this step is to remove noise from an acquired arterial pressure signal . baseline wondering noise is removed from a given signal by polynomial fitting technique ; first the noise in the signal is estimated and then the estimated noise is subtracted from the signal . to estimate the noise level , the polynomial estimating the baseline was fitted by requiring it to pass through the sample of the signal . ultimately , the baseline wondering noise was removed from the signal by simply subtracting the obtained polynomial curve from the signal . ( the second row ) signal after removing noise using savitzky - golay filter , ( the thrid row ) signals after base line correction . other noises and artifacts in the signal are removed by filtering ; various low pass filters including median filtering , the kalman filter and savitzky - golay filter were studied . frequency limit of the arterial waveform is up to 100 hz range ; thus , the filter cutoff frequency was also adjusted in this range . the objective in this step was to extract important features for each individual beat of arterial pulse waveform for next steps . the following three features were employed ( figure 4 ) : total pulse duration ( t ) , systolic amplitude ( as ) and diastolic amplitude ( ad ) of the pressure waveform . the characteristics of the arterial pulse wave used in this study modified from total pulse duration ( t ) is the distance between the beginning and the end of pulse waveform . onset of the pulse was determined using the first and second derivatives of the signal as it was discussed in ; the first derivative of the pulse was scanned and the onset of the signal was considered as the first zero crossing point from negative to positive that led to the maximum point on the first derivative curve ( see systolic amplitude ( as ) is the peak pressure of the waveform which is simply determined by scanning the pulse to find its maximum value . diastolic amplitude ( ad ) is the trough of pressure waveform that is determined by finding the minimum value of the pulse the objective in this step is to flag inconsistent beats and remove them from further analyses . the inconsistent pulses are pulses that are not consistent with physiological characteristics of the cardiovascular system . for instance , the systolic domain could never be three times more than the average systolic domain [ 1 - 3 ] . the criteria used in this study for identifying inconsistent beats are listed in table 1 . this step is completed using the morphological features calculated for each pulse in the previous step . criteria for identifying abnormal pulses ( unacceptable ) . as is the systolic domain ( peak of the pulse wave form ) , am is the average systolic domain , ad is the diastolic adomain ( least diastolic pulse ) , t is the width of the pulse and tm is the mean pulse width . in this step , the area under the systolic portion of the arterial pressure waveform is calculated . the systolic portion is from the end - diastole to the end of the ejection phase ; therefore , for estimating this area , the location of ventricular ejection and the closure of the aortic valve on each pulse should be identified . the location of ventricular ejection corresponds to the onset of signal that was specified in sub - section 2 - 3 . the location of the closure of the aortic valve on arterial pressure waveform corresponds to a point called dicrotic notch . dicrotic notch point in each pulse was determined by using the algorithm discussed in . it is considered to be at the first point after the position of the peak at which the sign of the 3rd derivative of the pulse changes . finally , after identifying the onset point and the dicrotic notch point , the area under the curve was calculated using the trapezoidal method . to decrease the effect of variation in the arterial pressure waveform on estimating co , area under the curve the pulses were detected every 10-sec of the signal and it was segmented , aligned based on their peaks and then averaged to estimate a representative of these pulses . as it was mentioned in equation ( 1 ) , the stroke volume has an inverse relation with the coronary vascular resistance . therefore , in addition to the area under the systolic portion of the arterial pressure waveform indicated in figure 1 , the vascular resistance is required for calculating the stroke volume and the cardiac output . this parameter varies from patient to patient , thus it should be set for each patient individually . , this parameter was taken into account as a calibration coefficient ( k ) , as follows : co = k hr sv ( 2 ) where hr is the number of heart rate per minute and sv is the stroke volume which is calculated by using equation 1 , and the methods explained in sub - sections 1 to 6 . based on equation 2 , the developed system must be calibrated for each patient . in other words , parameter k has to be estimated for each patient , individually . in this study , this parameter was estimated in the first few seconds of recording the signal using co value estimated via thermodilution technique . our goal in this work was to present an interactive system that works as an interactive environment for continuously measuring co through developing a matlab interactive software package . the purpose of interaction in the developed system is to facilitate the use of a computer for estimating , analyzing and storing co and several parameters that may be valuable in monitoring hemodynamic status of high - risk surgical patients and critically ill patients in intensive care units ( icu ) . the developed software package provides graphical user interfaces ( guis ) to filter an acquired arterial pressures signal , extract relevant features for each beat , estimate important parameters , calibrate the system , store data and reload and reanalyze a signal . using this system , arterial pressure signals are acquired from a patient monitor used to screen patient parameters and then several parameters such as the cardiac output , systolic blood pressure , diastolic blood pressure , mean blood pressure , heart rate and the stroke volume are continuously estimated and displayed as a numerical value . in addition , this software provides a time series graph for each of these parameters that assist in tracking changes of each parameter and ultimately better management of patient ( see result section for example outputs of the software ) . in this software , a color alarm system is provided so that the background color of the monitor changes to red when the cardiac output is less than 2 liters /minute , to yellow when the cardiac output is between 2 and 5 liters , and it changes to green if the cardiac output is more than 5 liters . thus , this option enables the operator to use his / her vision in addition to the sense of hearing in noisy environment . the signal is recorded using a blood pressure transducer which is attached to the arterial lane ( radial artery , brachial or femoral ) . in this method , at first an arterial route is created by a catheter 22 ( arterial line ) , and then the arterial lane is connected to the transducer . then , mechanical pulses of the heart beat change into electrical signals through the diaphragm and the life signals are transmitted to the monitor . finally , by connecting the monitor to a computer , the signals are stored . although estimating the area under curve seems easy , the accurate estimation of co is difficult due to many artifacts that can affect the signal . arterial waveform of the signal often interferes with different noises such as the power line noise , base line wandering noise and emg signals . these noises are often created as a result of electrical signal fluctuations , motion or breathing artifacts . extracting information from the arterial waveform relies on a clean and noise - free signal ; otherwise , it can lead to inaccurate results and may mislead physicians . therefore , before using any algorithms to extract clinical information from the arterial waveform , it is necessary to remove the unusual waveforms and noise signals . the purpose of this step is to remove noise from an acquired arterial pressure signal . baseline wondering noise is removed from a given signal by polynomial fitting technique ; first the noise in the signal is estimated and then the estimated noise is subtracted from the signal . to estimate the noise level , the polynomial estimating the baseline was fitted by requiring it to pass through the sample of the signal . ultimately , the baseline wondering noise was removed from the signal by simply subtracting the obtained polynomial curve from the signal . ( the second row ) signal after removing noise using savitzky - golay filter , ( the thrid row ) signals after base line correction . other noises and artifacts in the signal are removed by filtering ; various low pass filters including median filtering , the kalman filter and savitzky - golay filter were studied . frequency limit of the arterial waveform is up to 100 hz range ; thus , the filter cutoff frequency was also adjusted in this range . the objective in this step was to extract important features for each individual beat of arterial pulse waveform for next steps . the following three features were employed ( figure 4 ) : total pulse duration ( t ) , systolic amplitude ( as ) and diastolic amplitude ( ad ) of the pressure waveform . the characteristics of the arterial pulse wave used in this study modified from total pulse duration ( t ) is the distance between the beginning and the end of pulse waveform . onset of the pulse was determined using the first and second derivatives of the signal as it was discussed in ; the first derivative of the pulse was scanned and the onset of the signal was considered as the first zero crossing point from negative to positive that led to the maximum point on the first derivative curve ( see systolic amplitude ( as ) is the peak pressure of the waveform which is simply determined by scanning the pulse to find its maximum value . diastolic amplitude ( ad ) is the trough of pressure waveform that is determined by finding the minimum value of the pulse the objective in this step is to flag inconsistent beats and remove them from further analyses . the inconsistent pulses are pulses that are not consistent with physiological characteristics of the cardiovascular system . for instance , the systolic domain could never be three times more than the average systolic domain [ 1 - 3 ] . the criteria used in this study for identifying inconsistent beats are listed in table 1 . this step is completed using the morphological features calculated for each pulse in the previous step . criteria for identifying abnormal pulses ( unacceptable ) . as is the systolic domain ( peak of the pulse wave form ) , am is the average systolic domain , ad is the diastolic adomain ( least diastolic pulse ) , t is the width of the pulse and tm is the mean pulse width . in this step , the area under the systolic portion of the arterial pressure waveform is calculated . the systolic portion is from the end - diastole to the end of the ejection phase ; therefore , for estimating this area , the location of ventricular ejection and the closure of the aortic valve on each pulse should be identified . the location of ventricular ejection corresponds to the onset of signal that was specified in sub - section 2 - 3 . the location of the closure of the aortic valve on arterial pressure waveform corresponds to a point called dicrotic notch . dicrotic notch point in each pulse was determined by using the algorithm discussed in . it is considered to be at the first point after the position of the peak at which the sign of the 3rd derivative of the pulse changes . finally , after identifying the onset point and the dicrotic notch point , the area under the curve was calculated using the trapezoidal method . to decrease the effect of variation in the arterial pressure waveform on estimating co , area under the curve the pulses were detected every 10-sec of the signal and it was segmented , aligned based on their peaks and then averaged to estimate a representative of these pulses . as it was mentioned in equation ( 1 ) , the stroke volume has an inverse relation with the coronary vascular resistance . therefore , in addition to the area under the systolic portion of the arterial pressure waveform indicated in figure 1 , the vascular resistance is required for calculating the stroke volume and the cardiac output . this parameter varies from patient to patient , thus it should be set for each patient individually . , this parameter was taken into account as a calibration coefficient ( k ) , as follows : co = k hr sv ( 2 ) where hr is the number of heart rate per minute and sv is the stroke volume which is calculated by using equation 1 , and the methods explained in sub - sections 1 to 6 . based on equation 2 , the developed system must be calibrated for each patient . in other words , parameter k has to be estimated for each patient , individually . in this study , this parameter was estimated in the first few seconds of recording the signal using co value estimated via thermodilution technique . our goal in this work was to present an interactive system that works as an interactive environment for continuously measuring co through developing a matlab interactive software package . the purpose of interaction in the developed system is to facilitate the use of a computer for estimating , analyzing and storing co and several parameters that may be valuable in monitoring hemodynamic status of high - risk surgical patients and critically ill patients in intensive care units ( icu ) . the developed software package provides graphical user interfaces ( guis ) to filter an acquired arterial pressures signal , extract relevant features for each beat , estimate important parameters , calibrate the system , store data and reload and reanalyze a signal . using this system , arterial pressure signals are acquired from a patient monitor used to screen patient parameters and then several parameters such as the cardiac output , systolic blood pressure , diastolic blood pressure , mean blood pressure , heart rate and the stroke volume are continuously estimated and displayed as a numerical value . in addition , this software provides a time series graph for each of these parameters that assist in tracking changes of each parameter and ultimately better management of patient ( see result section for example outputs of the software ) . in this software , a color alarm system is provided so that the background color of the monitor changes to red when the cardiac output is less than 2 liters /minute , to yellow when the cardiac output is between 2 and 5 liters , and it changes to green if the cardiac output is more than 5 liters . thus , this option enables the operator to use his / her vision in addition to the sense of hearing in noisy environment . the performance of the designed co estimation system was evaluated using mimic data available at mibh website . the measured co values and co values estimated using developed system are summarized in table 2 . in this table only the results for the signals , that the co values estimated by using thermodilution method were reported . as it can be seen , this system with an average error of 6.5% has an acceptable efficacy in estimating cardiac output . statistical analysis using student t - test ( =0.05 ) also proved that there was no significant difference between co values estimated using the developed system and those measured using thermodilution method . cardiac output values estimated using the developed system ( estimated co ) compared with those measured using the thermodilution method ( co ) . the average estimation error of the developed system is negative which shows that the system underestimates co ; nevertheless , overall there is no significant difference between the estimated co values and gold standard values . the estimation error for signal # 4 is relatively large , this may be due to the fact that this signal is very noisy and the system could not detect dicrotic notch correctly . as it was discussed earlier , by using this system , we can track the changes in cardiac output , systolic blood pressure , diastolic blood pressure , average blood pressure , heart beat and the stroke volume of a patient via time series graphs provided for these parameters . an example output of the developed system that can be useful for tracking a patient important parameters . systolic pressure ( a ) , mean arterial pressure ( map ) ( b ) , heart rate ( c ) , and cardiac output ( d ) versus time . cardiac output is a critical factor for monitoring hemodynamic status of high - risk surgical patients and critically ill patients in intensive care units ( icu ) , because it can be used for monitoring cardiac function , estimating global oxygen delivery and understanding the causes of high blood pressure . in this paper , the system consists of five main steps : signal acquisition , signal preprocessing , feature extraction , morphological analysis and inconsistent pulse removal , area under the systolic portion calculation and calibration . performance analysis using 7 real signals demonstrated that co values provided by the system are not statistically different from the values obtained using thermodilution method . the system has several advantages including being minimally invasive , easy to use , not expensive and finally providing continuous recording of co and several important parameters for tracking these parameters and ultimately patient s conditions . consequently , the developed system could be a suitable system for monitoring hemodynamic status of high - risk surgical patients and critically ill patients in intensive care units ( icu ) .

background : cardiac output ( co ) is the total volume of blood pumped by the heart per minute and is a function of heart rate and stroke volume . co is one of the most important parameters for monitoring cardiac function , estimating global oxygen delivery and understanding the causes of high blood pressure . hence , measuring co has always been a matter of interest to researchers and clinicians . several methods have been developed for this purpose , but a majority of them are either invasive , too expensive or need special expertise and experience . besides , they are not usually risk free and have consequences . objective : here , a semi - invasive system was designed and developed for continuous co measurement via analyzing and processing arterial pulse waves . results : quantitative evaluation of developed co estimation system was performed using 7 signals . it showed that it has an acceptable average error of ( 6.5% ) in estimating co. in addition , this system has the ability to consistently estimate this parameter and to provide a co versus time curve that assists in tracking changes of co. moreover , the system provides such curve for systolic blood pressure , diastolic blood pressure , average blood pressure , heart rate and stroke volume . conclusion : evaluation of the results showed that the developed system is capable of accurately estimating co. the curves which the system provides for important parameters may be valuable in monitoring hemodynamic status of high - risk surgical patients and critically ill patients in intensive care units ( icu ) . therefore , it could be a suitable system for monitoring hemodynamic status of critically ill patients .

Introduction Methods Signal Acquisition Signal Pre-processing Feature Extraction Morphological Analysis and Inconsistent Pulse Removal Calculating the Curve of Systolic Phase of Arterial Waveform Calibration Software Development Results and Discussions Conclusion

cardiac output ( co ) , expressed in liters / minute , is the amount of blood pumped by heart in one minute . mathematically , co is the product of heart rate and the stroke volume [ 1 - 8 ] . the heart rate , in beats / min , is the number of beats per minute and the stoke volume is the volume of blood , in milliliters ( ml ) , pumped out of the heart on each beat . cardiac output depends on cardiovascular system parameters and cardiac or even extra - cardiac factors . also , co is a fundamental determinant of oxygen delivered to tissues and is an essential indicator of how well the heart can meet the demands of the body . therefore , co is one of the most important parameters for monitoring cardiac function , estimating global oxygen delivery and for understanding the causes of high blood pressure . in other words , co is a critical factor for monitoring hemodynamic status of patients [ 1 - 11 ] . ultimately , measuring cardiac output has always been a matter of interest to researchers and clinicians . the methods developed so far for measuring co can be categorized as : flowmetry , fick [ 13 - 16 ] , the relative exhaled carbon dioxide , thermo dilution , esophageal doppler ultrasound , esophageal doppler [ 17,18,19,27 - 31 ] , bio - electrical impedance [ 32 - 34 ] , plethysmography impedance and arterial analysis method [ 12,13,14,35 - 43 ] . most of these methods are either invasive or too expensive and need special expertise or experience ; hence , they are not risk free . for example , fick and thermo dilution methods are both clinically possible , but they are invasive methods and could only be conducted in an equipped environment such as intensive care units ( icus ) and the cardiac catheterization laboratories . in addition , these methods require the injection of cold saline or dye into a big vessel in which the whole cardiac output is flowing in . in this method , swan - ganz catheter is used which enters the right side of heart through the inferior vena cava and then the pulmonary artery , but it is both invasive and needs special expertise and experience . doppler ultrasound method is non - invasive and accurate , but needs expensive equipment and the operator must be an expert . therefore , to facilitate the conduction and to prevent the side effects of the invasive methods for measuring the cardiac output , a system was designed to measure the cardiac output in a non - invasive way ( with the least invasion ) through the arterial pulse wave . the developed co estimation system is generally based on the theory that blood pressure in an artery is related to the stroke volume ( sv ) . the relation between sv , blood pressure and vascular resistance ( z ) is as follows [ 12,13,14,35 - 43 ] : sv=ejection[pa(t)-pd]dtz ( 1 ) where pa(t ) is the arterial pressure at time t and ptd is the arterial pressure at the end of diastole . in fact , this integral calculates the area under systolic portion of the arterial pressure waveform , from the end - diastole to the end of the ejection phase ; this corresponds to stroke volume . figure 1shows an example of the area under the curve calculated by the integral used in equation 1 . the area under the wave of the arterial pressure curve for estimating the stroke volume pressure changes in the artery are obtained via analyzing arterial pulse ; details will be explained below . through beat - to - beat analysis of the arterial pressure waveform , the cardiac output can be measured continuously . most steps involved in the developed system are processing and analyzing the arterial pressure waveform . figure 2 illustrates the main steps of the developed system . in short , from a complex arterial pulse , we find the starting point and the dicrotic notch corresponding to the systolic portion of the arterial pressures , then , the area under the curve between these two points is calculated . the area under the curve is proportional to the stroke volume ; if it is multiplied by the heart rate , then we can calculate the cardiac output . steps of the developed cardiac output estimating system the signal is recorded using a blood pressure transducer which is attached to the arterial lane ( radial artery , brachial or femoral ) . then , mechanical pulses of the heart beat change into electrical signals through the diaphragm and the life signals are transmitted to the monitor . finally , by connecting the monitor to a computer , the signals are stored . although estimating the area under curve seems easy , the accurate estimation of co is difficult due to many artifacts that can affect the signal . arterial waveform of the signal often interferes with different noises such as the power line noise , base line wandering noise and emg signals . extracting information from the arterial waveform relies on a clean and noise - free signal ; therefore , before using any algorithms to extract clinical information from the arterial waveform , it is necessary to remove the unusual waveforms and noise signals . to estimate the noise level , the polynomial estimating the baseline was fitted by requiring it to pass through the sample of the signal . ultimately , the baseline wondering noise was removed from the signal by simply subtracting the obtained polynomial curve from the signal . frequency limit of the arterial waveform is up to 100 hz range ; thus , the filter cutoff frequency was also adjusted in this range . the following three features were employed ( figure 4 ) : total pulse duration ( t ) , systolic amplitude ( as ) and diastolic amplitude ( ad ) of the pressure waveform . the characteristics of the arterial pulse wave used in this study modified from total pulse duration ( t ) is the distance between the beginning and the end of pulse waveform . onset of the pulse was determined using the first and second derivatives of the signal as it was discussed in ; the first derivative of the pulse was scanned and the onset of the signal was considered as the first zero crossing point from negative to positive that led to the maximum point on the first derivative curve ( see systolic amplitude ( as ) is the peak pressure of the waveform which is simply determined by scanning the pulse to find its maximum value . diastolic amplitude ( ad ) is the trough of pressure waveform that is determined by finding the minimum value of the pulse the objective in this step is to flag inconsistent beats and remove them from further analyses . the inconsistent pulses are pulses that are not consistent with physiological characteristics of the cardiovascular system . as is the systolic domain ( peak of the pulse wave form ) , am is the average systolic domain , ad is the diastolic adomain ( least diastolic pulse ) , t is the width of the pulse and tm is the mean pulse width . in this step , the area under the systolic portion of the arterial pressure waveform is calculated . the systolic portion is from the end - diastole to the end of the ejection phase ; therefore , for estimating this area , the location of ventricular ejection and the closure of the aortic valve on each pulse should be identified . the location of the closure of the aortic valve on arterial pressure waveform corresponds to a point called dicrotic notch . it is considered to be at the first point after the position of the peak at which the sign of the 3rd derivative of the pulse changes . finally , after identifying the onset point and the dicrotic notch point , the area under the curve was calculated using the trapezoidal method . to decrease the effect of variation in the arterial pressure waveform on estimating co , area under the curve the pulses were detected every 10-sec of the signal and it was segmented , aligned based on their peaks and then averaged to estimate a representative of these pulses . as it was mentioned in equation ( 1 ) , the stroke volume has an inverse relation with the coronary vascular resistance . therefore , in addition to the area under the systolic portion of the arterial pressure waveform indicated in figure 1 , the vascular resistance is required for calculating the stroke volume and the cardiac output . this parameter varies from patient to patient , thus it should be set for each patient individually . , this parameter was taken into account as a calibration coefficient ( k ) , as follows : co = k hr sv ( 2 ) where hr is the number of heart rate per minute and sv is the stroke volume which is calculated by using equation 1 , and the methods explained in sub - sections 1 to 6 . based on equation 2 , the developed system must be calibrated for each patient . in this study , this parameter was estimated in the first few seconds of recording the signal using co value estimated via thermodilution technique . the purpose of interaction in the developed system is to facilitate the use of a computer for estimating , analyzing and storing co and several parameters that may be valuable in monitoring hemodynamic status of high - risk surgical patients and critically ill patients in intensive care units ( icu ) . the developed software package provides graphical user interfaces ( guis ) to filter an acquired arterial pressures signal , extract relevant features for each beat , estimate important parameters , calibrate the system , store data and reload and reanalyze a signal . using this system , arterial pressure signals are acquired from a patient monitor used to screen patient parameters and then several parameters such as the cardiac output , systolic blood pressure , diastolic blood pressure , mean blood pressure , heart rate and the stroke volume are continuously estimated and displayed as a numerical value . in addition , this software provides a time series graph for each of these parameters that assist in tracking changes of each parameter and ultimately better management of patient ( see result section for example outputs of the software ) . in this software , a color alarm system is provided so that the background color of the monitor changes to red when the cardiac output is less than 2 liters /minute , to yellow when the cardiac output is between 2 and 5 liters , and it changes to green if the cardiac output is more than 5 liters . thus , this option enables the operator to use his / her vision in addition to the sense of hearing in noisy environment . the signal is recorded using a blood pressure transducer which is attached to the arterial lane ( radial artery , brachial or femoral ) . then , mechanical pulses of the heart beat change into electrical signals through the diaphragm and the life signals are transmitted to the monitor . finally , by connecting the monitor to a computer , the signals are stored . although estimating the area under curve seems easy , the accurate estimation of co is difficult due to many artifacts that can affect the signal . extracting information from the arterial waveform relies on a clean and noise - free signal ; otherwise , it can lead to inaccurate results and may mislead physicians . therefore , before using any algorithms to extract clinical information from the arterial waveform , it is necessary to remove the unusual waveforms and noise signals . to estimate the noise level , the polynomial estimating the baseline was fitted by requiring it to pass through the sample of the signal . other noises and artifacts in the signal are removed by filtering ; various low pass filters including median filtering , the kalman filter and savitzky - golay filter were studied . frequency limit of the arterial waveform is up to 100 hz range ; thus , the filter cutoff frequency was also adjusted in this range . the objective in this step was to extract important features for each individual beat of arterial pulse waveform for next steps . the following three features were employed ( figure 4 ) : total pulse duration ( t ) , systolic amplitude ( as ) and diastolic amplitude ( ad ) of the pressure waveform . the characteristics of the arterial pulse wave used in this study modified from total pulse duration ( t ) is the distance between the beginning and the end of pulse waveform . onset of the pulse was determined using the first and second derivatives of the signal as it was discussed in ; the first derivative of the pulse was scanned and the onset of the signal was considered as the first zero crossing point from negative to positive that led to the maximum point on the first derivative curve ( see systolic amplitude ( as ) is the peak pressure of the waveform which is simply determined by scanning the pulse to find its maximum value . diastolic amplitude ( ad ) is the trough of pressure waveform that is determined by finding the minimum value of the pulse the objective in this step is to flag inconsistent beats and remove them from further analyses . the inconsistent pulses are pulses that are not consistent with physiological characteristics of the cardiovascular system . as is the systolic domain ( peak of the pulse wave form ) , am is the average systolic domain , ad is the diastolic adomain ( least diastolic pulse ) , t is the width of the pulse and tm is the mean pulse width . in this step , the area under the systolic portion of the arterial pressure waveform is calculated . the systolic portion is from the end - diastole to the end of the ejection phase ; therefore , for estimating this area , the location of ventricular ejection and the closure of the aortic valve on each pulse should be identified . the location of the closure of the aortic valve on arterial pressure waveform corresponds to a point called dicrotic notch . it is considered to be at the first point after the position of the peak at which the sign of the 3rd derivative of the pulse changes . finally , after identifying the onset point and the dicrotic notch point , the area under the curve was calculated using the trapezoidal method . to decrease the effect of variation in the arterial pressure waveform on estimating co , area under the curve the pulses were detected every 10-sec of the signal and it was segmented , aligned based on their peaks and then averaged to estimate a representative of these pulses . as it was mentioned in equation ( 1 ) , the stroke volume has an inverse relation with the coronary vascular resistance . therefore , in addition to the area under the systolic portion of the arterial pressure waveform indicated in figure 1 , the vascular resistance is required for calculating the stroke volume and the cardiac output . this parameter varies from patient to patient , thus it should be set for each patient individually . , this parameter was taken into account as a calibration coefficient ( k ) , as follows : co = k hr sv ( 2 ) where hr is the number of heart rate per minute and sv is the stroke volume which is calculated by using equation 1 , and the methods explained in sub - sections 1 to 6 . based on equation 2 , the developed system must be calibrated for each patient . in this study , this parameter was estimated in the first few seconds of recording the signal using co value estimated via thermodilution technique . our goal in this work was to present an interactive system that works as an interactive environment for continuously measuring co through developing a matlab interactive software package . the purpose of interaction in the developed system is to facilitate the use of a computer for estimating , analyzing and storing co and several parameters that may be valuable in monitoring hemodynamic status of high - risk surgical patients and critically ill patients in intensive care units ( icu ) . the developed software package provides graphical user interfaces ( guis ) to filter an acquired arterial pressures signal , extract relevant features for each beat , estimate important parameters , calibrate the system , store data and reload and reanalyze a signal . using this system , arterial pressure signals are acquired from a patient monitor used to screen patient parameters and then several parameters such as the cardiac output , systolic blood pressure , diastolic blood pressure , mean blood pressure , heart rate and the stroke volume are continuously estimated and displayed as a numerical value . in addition , this software provides a time series graph for each of these parameters that assist in tracking changes of each parameter and ultimately better management of patient ( see result section for example outputs of the software ) . in this software , a color alarm system is provided so that the background color of the monitor changes to red when the cardiac output is less than 2 liters /minute , to yellow when the cardiac output is between 2 and 5 liters , and it changes to green if the cardiac output is more than 5 liters . thus , this option enables the operator to use his / her vision in addition to the sense of hearing in noisy environment . the performance of the designed co estimation system was evaluated using mimic data available at mibh website . the measured co values and co values estimated using developed system are summarized in table 2 . in this table only the results for the signals , that the co values estimated by using thermodilution method were reported . as it can be seen , this system with an average error of 6.5% has an acceptable efficacy in estimating cardiac output . statistical analysis using student t - test ( =0.05 ) also proved that there was no significant difference between co values estimated using the developed system and those measured using thermodilution method . cardiac output values estimated using the developed system ( estimated co ) compared with those measured using the thermodilution method ( co ) . the average estimation error of the developed system is negative which shows that the system underestimates co ; nevertheless , overall there is no significant difference between the estimated co values and gold standard values . the estimation error for signal # 4 is relatively large , this may be due to the fact that this signal is very noisy and the system could not detect dicrotic notch correctly . as it was discussed earlier , by using this system , we can track the changes in cardiac output , systolic blood pressure , diastolic blood pressure , average blood pressure , heart beat and the stroke volume of a patient via time series graphs provided for these parameters . an example output of the developed system that can be useful for tracking a patient important parameters . systolic pressure ( a ) , mean arterial pressure ( map ) ( b ) , heart rate ( c ) , and cardiac output ( d ) versus time . cardiac output is a critical factor for monitoring hemodynamic status of high - risk surgical patients and critically ill patients in intensive care units ( icu ) , because it can be used for monitoring cardiac function , estimating global oxygen delivery and understanding the causes of high blood pressure . in this paper , the system consists of five main steps : signal acquisition , signal preprocessing , feature extraction , morphological analysis and inconsistent pulse removal , area under the systolic portion calculation and calibration . performance analysis using 7 real signals demonstrated that co values provided by the system are not statistically different from the values obtained using thermodilution method . the system has several advantages including being minimally invasive , easy to use , not expensive and finally providing continuous recording of co and several important parameters for tracking these parameters and ultimately patient s conditions . consequently , the developed system could be a suitable system for monitoring hemodynamic status of high - risk surgical patients and critically ill patients in intensive care units ( icu ) .

risk evaluation and mitigation strategies ( remss ) , particularly ones that require elements to assure the safe use of drugs , are operationally complex and burdensome for physicians , pharmacists , drug distributors , regulators , and manufacturers.a demonstration of the impact of remss on improving safety was a key request from the legislator but , based on our review of public information , this impact does not appear to have been evaluated or made public.we recommend that rems programs should be evaluated for their effectiveness to improve drug safety . approximately 10 years ago , the us food and drug administration ( fda ) implemented a safety risk monitoring system for new marketed drugs and for drugs already on the market and for which a new risk was identified . the risk evaluation and mitigation strategy , or rems , requires multiple and complex processes , particularly remss that include elements to assure safe use ( etasu ) . these etasu involve the participation of physician prescribers and drug companies but can also involve patients , pharmacists , and drug distributors [ 2 , 3 ] . pre - existing drug safety monitoring systems , such as the black box warning , have been frequently criticized [ 46 ] and , in particular , concerns were raised because their utility is difficult to evaluate . consequently , a key provision in the fda amendments act of 2007 was the mandate to study the effectiveness of remss to evaluate their impact on improving drug safety . logically , the addition of a new and more complex system , such as remss with etasu , could only be justified if evidence - based results demonstrate that such a process improves drug safety . in 2013 , the inspector general of the department of health and human services conducted an extensive evaluation of remss launched between 2007 and 2011 . the report concluded that the fda could not determine whether remss were improving drug safety . this report highlighted that the federal agency did not have the capacity to fulfill one of the most important aspects of the amendments act of 2007 , which was to verify that the rems system was efficient . since this report , new remss have been implemented , and few have been discontinued . as remss accumulate , so does the administrative burden and workload on the health system . this study evaluates remss issued , or modified , from december 2011 ( right after the period covered by the inspector general report ) until august 2015 . the study evaluates rems rationale , characteristics , and consistencies and evaluates whether the impact of remss to improve drug safety could be documented . the focus was on remss with etasu , as these plans concern drugs that pose the most significant safety risks . remss requiring etasu are requested for drugs that represent the highest known , and clinically relevant , safety concerns . the main rationale for etasu is to provide patients with safe access to a drug that would otherwise be unavailable to them . we evaluated the individual characteristics of remss with etasu that were issued from december 2011 to september 2015 . we searched the fda website ( http://www.accessdata.fda.gov/scripts/cder/rems/index.cfm ) to determine which of the following elements were incorporated into remss with etasu ( points 36 being specific for etasu):a medication guide . this could , for instance , takes the form of a patients package insert.a communication plan to healthcare providers . this plan may include letters or other means of communication ( e - mail , website , professional societies).an implementation system . this process is specific for etasus and is intended for the sponsor of the drug to monitor , evaluate , and eventually correct the level of compliance by healthcare providers and pharmacists . this system may also include wholesalers , distributors , or other parties.the need for certification . for drug prescribers and drug dispensers ( e.g. , pharmacists ) , a certification may be required to demonstrate their ability to diagnose the condition , understand the risk / benefit of the drug , read the educational material , and , eventually , treat potential adverse drug reactions . pharmacists certifications might require an agreement to fill a prescription and dispense the drug only after receiving prior authorization , checking laboratory values , or checking for the presence of stickers . stickers indicate that the patient has met all criteria for receiving the product ( qualification stickers ) to fill a prescription . pharmacists might also be required to dispense a drug only within a specified period , as well as to fill prescriptions only from enrolled prescribers.the need to enroll patients . the registry can be used to document that the drug is dispensed to patients with documentation of safe - use conditions , or to document that the patient is enrolled in a mandatory monitoring system . the registry can collect clinical outcomes , including safety information , compliance with prescribing protocols , and assessment regarding the impact of actions taken to ensure compliance.the need to train prescribers . a prescriber s training generally requires the review of clinical documents , successfully answering questionnaires , and the documentation of these activities . a medication guide . this could , for instance , takes the form of a patients package insert . a communication plan to healthcare providers . this plan may include letters or other means of communication ( e - mail , website , professional societies ) . an implementation system . this process is specific for etasus and is intended for the sponsor of the drug to monitor , evaluate , and eventually correct the level of compliance by healthcare providers and pharmacists . the need for certification . for drug prescribers and drug dispensers ( e.g. , pharmacists ) , a certification may be required to demonstrate their ability to diagnose the condition , understand the risk / benefit of the drug , read the educational material , and , eventually , treat potential adverse drug reactions . pharmacists certifications might require an agreement to fill a prescription and dispense the drug only after receiving prior authorization , checking laboratory values , or checking for the presence of stickers . stickers indicate that the patient has met all criteria for receiving the product ( qualification stickers ) to fill a prescription . pharmacists might also be required to dispense a drug only within a specified period , as well as to fill prescriptions only from enrolled prescribers . the registry can be used to document that the drug is dispensed to patients with documentation of safe - use conditions , or to document that the patient is enrolled in a mandatory monitoring system . the registry can collect clinical outcomes , including safety information , compliance with prescribing protocols , and assessment regarding the impact of actions taken to ensure compliance . a prescriber s training generally requires the review of clinical documents , successfully answering questionnaires , and the documentation of these activities . for each rems with etasu , the number of versions was documented , including the number of revisions following the first issue . for each drug subjected to a rems with etasu , the dedicated rems website was accessed and the information available on the fda s website was eventually completed . the following information was compiled : the drug name ( usan / trade name ) , the labeled indication(s ) , whether the indication was covering an orphan disease or not , and the clinical description of the safety risk(s ) . discontinuations were first searched on the fda website , verified on the drug s sponsor website by consulting the press release section , and confirmed on each rems website address . last , for each drug subjected to a rems with etasu , the drug package insert was reviewed for the presence of a black box warning and to check whether the black box warning risks matched the risks mentioned in the rems . a pubmed search was performed ( http://www.ncbi.nlm.nih.gov/pubmed ) from 2007 to 2015 for publications concerning remss . rems with etasu. a similar search was conducted on the fda website , with no time limits , with the additional term rems working group ( www.fda.gov ) . this last search was done because the inspector general and the fda had agreed that a working group would report on an evidence - based approach that would evaluate the efficiency of remss by march 2015 . forty - two remss with etasus were issued , or modified , during the study period . we could not find data on the fda s website or from fda publications demonstrating the effectiveness of remss to improve drug safety . from the general literature it was a survey on the effectiveness of the rems medication guide for conveying information on the risks of varenicline . a second study evaluated the bosentan rems program for its compliance to a required monthly testing of liver function . the study was conducted by the same consulting group in collaboration with the maker of the drug . a third study evaluating retigabine / ezogabine rems was conducted by the maker of the drug and surveyed pharmacists and physicians for their understanding of the risk of urinary retention associated with the drug . of the 1028 individuals the study demonstrated an insufficient level of understanding of the risk , especially among pharmacists . the initial risk minimization program was later continued as part of a rems with etasu . the study was conducted by the maker of the drug and concluded that , in the absence of pregnancy report in female patients or female partners of male patients , the program was effective in preventing fetal exposure to the drug . a recent study reported on an education program for prescribers of extended - release / long - acting opioid analgesics . this continuing education program , mandated by the fda , was funded by the manufacturers of these products . in the immediate period following the implementation of the program , the 2850 participants surveyed demonstrated a significant improvement in the number of correct responses to knowledge questions ( from 60 to 84% ) and 82% of participants declared they were planning to change their practice . after 2 months , however , when a subset of 476 participants were tested , the results were an improvement from 60 to 69 , and 67% of participants planned to change their practice , indicating that the positive effect was waning . of the 42 remss with etasu , 35 were designed for a single drug ( table 1 ) , and seven were shared programs that included multiple drugs of the same class ( table 2 ) . these shared programs were implemented across multiple manufacturers.table 1single remss with etasu : drugs , sponsors , indications , nature of risk(s ) and presence of a black box warningdrugssponsor(s)indicationrisk(s)black boxloxapinetevaacute agitation with schizophrenia or bipolar type ibronchospasmyesvandetanibastra zenecamedullary thyroid klong qt syndromeyesalvimopancubist / merckshorten gi recovery following bowel surgeryincrease incidence of miyesicodextrinbaxterperitoneal dialysis solutionincorrect blood glucose resultsyesteduglutideshireshort bowel syndromepossible acceleration of neoplastic growth and enhancement of colon polyp growth , gi obstruction , biliary and pancreatic disordersnolomitapideaegerionhomozygous familial hypercholesterolemiahepatotoxicityyesmipomersensanofihomozygous familial hypercholesterolemiahepatotoxicityyesambrisentangileadpulmonary arterial hypertensionserious birth defectsyesalosetronprometheusdiarrhea predominant ibsischemic colitisyesalglucosidase alphasanofipompe diseaseanaphylaxisyesmifepristonedancomedical termination of pregnancylife - threatening bleeding , infections , or other problemsyesmycophenolatemultipleprophylaxis of organ transplant rejectionpregnancy loss and congenital malformations , other serious risksyesromiplostimamgenchronic immune thrombocytopeniaprogression of myelodysplasia and acute myeloid leukemia , thromboembolism , marrow fibrosisnopomalidomidecelgenemultiple myelomarisk of embryo fetal exposure and other risksyeseltrombopaggskchronic immune thrombocytopeniahepatotoxicity , bone marrow fibrosis , thromboembolismyesphentermine / topiramatevivuschronic weight managementbirth defect ( cleft lift / cleft palate)nolenalidomidecelgenemyelodysplastic syndrome , mantle cell lymphomaembryo - fetal toxicityyesvigabatrinlundbeckrefractory complex partial seizurevision lossyeseculizumabalexionparoxysmal nocturnal hemoglobinuriaatypical husmeningococcal infectionyesthalidomidecelgenemultiple myelomaerythema nodosumembryo - fetal toxicityyesdofetilidepfizeratrial fibrillationflutterarrhythmiayesbosentanactelionpulmonary arterial hypertensionbirth defects , hepatotoxicityyesemtricitabine / tenofovir / disoproxilgileadpre - exposure prophylaxis of hiv-1adherence to regimen , control of hiv-1 status , counselingyesnatalizumabbiogenmultiple sclerosisprogressive multifocal leukoencephalopathyyesclozapinejazzschizophreniasevere neutropeniayescollagenase clostridium histolyticumendodupuytren diseaselapeyronie diseasetendon rupture , anaphylaxis , corporal ruptureyesolanzapinelillyschizophreniamitigate risk of post - injection delirium / sedation syndromeyesmacitentanactelionpulmonary arterial hypertensionserious birth defectyesriociguatbayerpulmonary arterial hypertensionteratogenicityyessacrosidaseqol medicalsucrase - isomaltase deficiencysevere allergic reactionnotestosterone undecanoateendoprimary hypogonadismhypogonadotropic hypogonadismpulmonary oil micro - emboli , anaphylaxisyesmetreleptinaegeriongeneralized lipodystrophyneutralizing antibodies , risk of lymphomayesalemtuzimabsanofirelapsing multiple sclerosisauto - immune conditions , infusion reactions , malignanciesyessodium oxybatejazznarcolepsycns and respiratory depression , potential abuse / misuse , ci with hypnotics and alcohol , handling and storageyesalosetronboehringer ingelheimchronic ibsischemic colitis , serious complication of constipationyes ci contra - indicated , cns central nervous system , etasu elements to assure safe use , gi gastro - intestinal , hus hemolytic uremic syndrome , ibs irritable bowel syndrome , mi myocardial infarction , rems risk evaluation and mitigation strategy table 2common remss with etasu : drugs , sponsors , indications , nature of risk(s ) and presence of a black box warningdrugssponsor(s)indicationrisk(s)black boxepoetin alphaamgen , janssenanemia of chronic kidney disease and chemotherapyshortened survival , increased risk of tumor progression / recurrenceyesdarbepoetin alphaamgenyesbuprenorphinemultipleopioid dependenceaccidental overdose , misuse or abusenobuprenorphine and naloxonemultipleopioid dependenceaccidental overdose , misuse or abusenoextended - release and long - acting opioid analgesicsmultipleanalgesiaaddiction , abuse , and misuseyesisotretinoinmultiplesevere recalcitrant nodular acnesevere birth defectsyesrosiglitazone and its combinationsgsktype 2 diabetesischemic cardiovascular riskyesfentanylmultipleanalgesia in cancer patientsmitigate the risk of misuse , abuse , addiction , overdose and serious complications due to medication errorsyes etasu elements to assure safe use , rems risk evaluation and mitigation strategy single remss with etasu : drugs , sponsors , indications , nature of risk(s ) and presence of a black box warning ci contra - indicated , cns central nervous system , etasu elements to assure safe use , gi gastro - intestinal , hus hemolytic uremic syndrome , ibs irritable bowel syndrome , mi myocardial infarction , rems risk evaluation and mitigation strategy common remss with etasu : drugs , sponsors , indications , nature of risk(s ) and presence of a black box warning etasu elements to assure safe use , rems risk evaluation and mitigation strategy the nature of the risks involved could be common to more than one rems ( table 3 ) . the risk of birth defect(s ) or embryo - fetal toxicity and the risk of potential abuse or misuse were the most frequent risks addressed in remss with etasu ( 8/42 [ 19% ] and 6/42 [ 14% ] , respectively ) . some risks were mentioned in only one rems . these were biliary / pancreatic disorders ; ischemic colitis and complication of constipation ; delirium , sedation , and vision loss ; severe neutropenia ; and congestive heart failure . these were the adherence to the prescribed regimen and counseling , mistake in blood glucose reading , and tendon or cavernous body rupture following a local drug injection.table 3risks addressed in remss with etasurisksnumber of drugsbirth defect(s ) or embryo - fetal toxicity8potential abuse or misuse6allergic reaction5tumor or neoplastic progression4hepatotoxicity4infection3thromboembolism3cardiac arrhythmia2myocardial infarction2bone marrow fibrosis2biliary / pancreatic disorders1ischemic colitis and complication of constipation1delirium , sedation , and vision loss1severe neutropenia1congestive heart failure1adherence to regimen and counseling1incorrect blood glucose reading1tendon or cavernous body rupture after a local injection1 etasu elements to assure safe use , rems risk evaluation and mitigation strategy risks addressed in remss with etasu etasu elements to assure safe use , rems risk evaluation and mitigation strategy the risk(s ) mentioned in a rems with etasu could be single or multiple . for 24 remss , only a single risk was identified ( e.g. , hepatotoxicity for mipomersen or bronchospasm for adasuve ) . for 17 remss , multiple risks were mentioned ( e.g. , progression of myelodysplastic syndrome and acute myeloid leukemia , thromboembolism , and bone marrow fibrosis for romiplostim ) . some , however , were not documented with clinical data and remained hypothetical ( e.g. , ischemic cardiac disease and possible acceleration of neoplastic growth for teduglutide ) . in that case , the risks could be inferred from the drug s mechanism of action or hypothesized based on non - clinical data . some risks were difficult for individual prescribers to evaluate as they were clinical events that could have multiple etiologies ( e.g. , myocardial infarction with alvimopan ) or were too rare to be encountered in a practice with a limited number of patients . for such rare events , the collection of data from a large population and/or multiple prescribers could only document an increased risk of the 42 remss with etasu , 36 drugs ( including 31 individual drugs and five classes of drugs ) had a label that contained a black box warning . these remss concerned four individual drugs ( teduglutide , romiplostim , phentermine / topiramate , and sacrosidase ) and two classes of drugs ( buprenorphine - containing products including transmucosal formulations and buprenorphine / naloxone product combinations ) . there was no apparent difference in the type of risks for drugs with a black box warning and drugs without such a warning . for instance , the same risks ( misuse or abuse , thromboembolism , birth defect , or acceleration of tumor growth ) were mentioned for drugs with or without black box warnings . apart from information contained in the drug label , the severity or the frequency of the risk was generally not quantified in rems documents . when a drug had a black box warning , the risks mentioned in the rems with etasu were similar to risks mentioned in the black box for 33 drugs ( 23 individual drugs and two grouped remss ) . risks were dissimilar for 11 drugs ( eight individual remss and three common remss ) . in eight instances , fewer risks were mentioned in the rems than in the black box ( six single remss and two common remss ) . in three instances , more risks were mentioned in the rems than in the black box ( two single remss and one common rems ) . no rationale could be identified to explain these discrepancies . for extended - release and long - acting opioid analgesics , a drug approved for multiple indications could be submitted for a rems with etasu for one indication but not for another indication . for instance , topiramate was subjected to a rems when used in combination with phentermine , indicated for weight management , but was not submitted for a rems when indicated for epilepsy and migraine . similarly , mifepristone was subjected to a rems with etasu for the medical termination of intra - uterine pregnancy ( one 600-mg dose ) but not for the treatment of cushing s syndrome ( 300 mg daily , continuously ) . some medical specialties , such as neuropsychiatry , cardiovascular medicine , endocrinology and metabolic diseases , and oncology , were more frequently concerned by remss with etasu than other specialties ( table 4).table 4medical specialty concerned with rems with etasuspecialtynumber of remss with etasuneurology / psychiatry7cardiology / vascular7endocrinology / metabolic disease6oncology / hematology5hepato - gastro - intestinal4 etasu elements to assure safe use , rems risk evaluation and mitigation strategy medical specialty concerned with rems with etasu etasu elements to assure safe use , rems risk evaluation and mitigation strategy most drugs ( 24/42 , or 57% ) that were subjected to remss with etasu were indicated for an orphan disease . the most frequent indication was pulmonary arterial hypertension for which four drugs shared a risk of birth defects . the processes that were included in remss with etasu were , in order of decreasing frequency , an implementation system ( 81% of remss ) , prescriber training ( 71% ) , prescriber certification ( 69% ) , a medication guide ( 67% ) , dispenser certification ( 48% ) , patient enrolment ( 43% ) , and a communication plan ( 26% ) . three of the 42 remss with etasu ( 7% ) were discontinued ( table 5).table 5discontinuation of rems with etasudrugindicationriskreason for discontinuationeltrombopagthrombocytopenia in chronic immune thrombocytopeniaprogression of mds and aml , thromboembolism , marrow fibrosisnot availableromiplostimthrombocytopenia in chronic immune thrombocytopeniahepatotoxicity , bone marrow fibrosis , thromboembolismnot availablelumizymepompe diseaseanaphylactic reactionsdrug considered similar to myozymerosiglitazone and its combinationstype 2 diabetes mellitusischemic cardiovascular riskrisk not documented ( replaced by risk of congestive heart failure ) aml acute myeloid leukemia , etasu elements to assure safe use , mds myelodysplastic syndromes , rems risk evaluation and mitigation strategy discontinuation of rems with etasu aml acute myeloid leukemia , etasu elements to assure safe use , mds myelodysplastic syndromes , rems risk evaluation and mitigation strategy a rems is mandated by law and includes specific measures to ensure that the benefits of a drug outweigh its risks . a rems may be required by the fda as part of a new drug approval process , or for an approved product when new safety information emerges . one of the key provisions in establishing remss was to ensure that the fda can evaluate the impact of such programs and thus demonstrate their relevance in addressing and preventing safety risks [ 1 , 7 ] . in february 2013 , the office of the inspector general issued a comprehensive report covering remss issued since their implementation in 2007 until the end of 2011 . this report concluded that the fda did not have relevant data to determine whether remss improve drug safety and , despite the significant burden and cost associated with the rems system , the relevance of these programs could not be established . our study s first objective was to establish whether the situation regarding the relevance of remss has changed since the inspector general s report and whether the impact of remss to improve drug safety could now be established . our second objective was to establish the characteristics of remss with etasu , as these remss address drugs with the most significant safety risk . since the report from the office of the inspector general , 42 new remss with etasu have been issued ( 35 programs for individual drugs , and seven programs incorporating more than one drug ) . our study confirms that there is still only very limited information publicly available to demonstrate that remss with etasu address and/or prevent safety risks . while data on rems effectiveness might have been collected , they have not generally been made public by regulators , sponsors , or scientists . this is contrary to the public commitment made by the fda in its response to the inspector general s report . despite the burden that remss represent , remss continue to be requested and continue to be implemented despite the fact that the original intent of the law that established the rems system has not been fulfilled . the two most frequent risks addressed in the remss with etasu we reviewed were the risk of embryo - fetal toxicities and the risk for drug abuse . since the thalidomide tragedy , the risk of embryo - fetal toxicity has been a priority for regulators ; it is thus not a surprise to see this risk prominently addressed in remss . more surprising , however , is the great variability of the level of embryo - fetal risk associated with drugs submitted to a rems . the very high risk of embryo - fetal toxicity of oral retinoids used to treat severe and refractory acne is well known . this risk is further amplified as patients affected by this type of acne are mostly young females of reproductive age . over the years , multiple initiatives it is thus logical to incorporate the prescription of oral retinoids into a rems with etasu . oral retinoids are included in a common rems that is shared between manufacturers . to our knowledge , it is not known whether this program adequately addresses and prevents the risk of embryo - fetal toxicity . for other drugs , the risk of embryo - fetal toxicity is variable . for thalidomide itself and lenalidomide , a derivative of thalidomide , the potential of these drugs to induce embryo - fetal toxicity is evidently high . these drugs , however , are indicated for the treatment of multiple myeloma and the population affected by this disease is generally not at risk of pregnancy . indeed , a multiple myeloma is very rarely diagnosed during a woman s reproductive years [ 13 , 19 ] . additionally , the treatment of multiple myeloma incorporates multiple chemotherapeutic drugs that , while they are themselves embryo - toxic , are not submitted to a rems with etasu . while the situation is somewhat illogical , regulators probably could not avoid including thalidomide and its derivative into a rems . in these cases , the logic could be to prevent reproductive - aged females in contact with patients to have access to the drugs or could be more an issue of public perception in the context of the tragedy mentioned . the case of topiramate is also interesting . when indicated for the management of obesity , topiramate topiramate is known to be associated with a risk of birth defect , however the risk appears relatively low . at the same time , when used to treat migraine ( a disease that mostly affects women in their reproductive age ) , topiramate is not subject to a rems . it could be speculated that the true intent of the rems was to limit prescriptions for the management of obesity , as the risk of birth defect would have increased with a large number of prescriptions , or to indirectly address other potential risks associated with the drug , such as the cardiovascular risks . four drugs indicated for chronic pulmonary hypertension , a rare orphan disease , share a risk of embryo - fetal toxicity . each drug is subjected to an individual rems with etasu . the patient population is not only at high risk for pregnancy , but is also one for whom a pregnancy could have severe consequences for the health of the mother . in this case unfortunately , each individual rems has its own specific processes , and the burden for patients and prescribers would certainly be minimized with the implementation of a shared rems . the risk of drug abuse , particularly opioid abuse , with pain medications has been a longstanding and vexing problem . despite multiple actions taken over the years , a definitive solution has not been found . while there are no data to indicate that this shared rems is more effective than previous attempts , the recent admission by the fda that a complete overhaul was necessary indicates that the current system is no better than its predecessors . beyond embryo - fetal toxicity and opioid abuse , the other risks identified in remss , such as allergic reaction , hepatotoxicity , or thromboembolism , were of varying severity . for instance , allergic reactions with commonly prescribed drugs not subjected to a rems can nevertheless be life - threatening or fatal and continue to affect many patients . the same is true for the risk of thromboembolism , which can be fatal and is associated with widely prescribed drugs that are not subjected to remss [ 25 , 26 ] . overall , the rationale that supports the incorporation of a drug into a rems is certainly difficult to establish for regulators and this issue was not foreseen by the legislator . the risks addressed in a rems with etasu were generally documented during the drug development program of a candidate drug and were identified before the drug was approved . some risks , however , such as an increased incidence of tumor progression or an increase in cardiovascular events , could not be documented with the available data and remained speculative . such risks , because of their lack of specificity and their rarity , can not be identified at the level of individual prescribers . most drugs that were included in a rems with etasu also had a black box warning in their label . the content of black box warnings and the content of remss were frequently divergent without apparent reasons . the black box warning system has been previously criticized and is generally considered inefficient [ 46 , 27 , 28 ] . in this context , regulators might be tempted to supplement a black box warning with a rems with etasu . remss , however , are immensely more complex and logistically challenging that black box warnings . there is thus a legitimate concern that the legislator added an unproven , cumbersome system on top of a potentially ineffective but simple system . additionally , a significant number of remss with etasu were requested for drugs that did not have a black box warning in their label . most remss with etasu were implemented for drugs that were indicated for a rare orphan disease . in this case , regulators might be requesting remss with the objective to continue to collect safety information while favoring early patient access to life - saving medication . however , in the absence of data to evaluate the efficiency of rems , this remains speculative . in the context of conditional approval for orphan drugs and for oncology drugs , post - marketing data collection is necessary to get a final approval [ 29 , 30 ] and can be more informative that rems . also , patient registries that become the norm for rare orphan diseases provide useful clinical information , including safety information . for many orphan drugs , registries are an efficient way to collect safety information that could be evaluated against the rems system . finally , most patients affected by an orphan disease are under the care of highly specialized physicians who are likely to manage any risks in a prudent and efficient way . with these considerations , the benefits of orphan diseases remss remain unproven . having multiple remss for the same orphan indication renders medical practice more cumbersome and , ultimately , could result in limiting patient access to lifesaving medications , which would be the exact opposite of the legislator s intent . the processes requested as part of a rems with etasu highlight their complexity and administrative burden . rems revisions , mostly dedicated to procedural details , are frequent and slow to implement . very few remss with etasu were discontinued and when they were , no rationale was made publicly available , or the rationale was difficult to understand . while remss can be negotiated between the fda and a drug manufacturer , the fda has sole decision power . in this context , the negotiation , implementation , or potential discontinuation , of a rems is particularly challenging for small drug manufacturers who do not have the necessary staff and must contract these functions out at a significant additional cost . it also appears challenging for regulators to implement rems programs across multiple review divisions in a consistent and logical manner . the current rems system does not appear to meet the intent of the law which requested a mandatory evaluation of these programs to demonstrate that they were improving drug safety . the decision - making process to require a rems is not transparent and results in programs that contain inconsistent processes and unclear objectives . each year , new rems are issued , and very few are discontinued , but today , it is still unknown whether the rems system is useful or not . while risk minimization strategies are always difficult to implement [ 33 , 34 ] , we have identified multiple challenges that need to be addressed .

risk evaluation and mitigation strategies ( remss ) with elements to assure safe use ( etasu ) are requested for drugs with significant safety risks . we reviewed rems programs issued since 2011 to evaluate their rationales , characteristics , and consistencies , and evaluated their impact on improving drug safety . we conducted a literature search and a survey of relevant websites ( fda , manufacturers , and remss ) . etasu characteristics were summarized . rems risks were compared with labeled risks , including black box warnings . forty - two programs were analyzed . seven incorporated drugs of the same class . most drugs ( 57% ) were indicated for an orphan disease . a single risk was mentioned in 24 remss , and multiple risks in 18 . embryo - fetal toxicity and abuse or misuse were the most frequent risks . all risks were identified during clinical development but some were hypothetical . thirty - six drugs had a black box warning . rems risks and black box risks differed for 11 drugs . a drug with multiple indications could have a rems for one of them but not for another . most remss required prescriber training and certification , half required dispenser certification and patient enrolment . remss were revised multiple times and only three ( 7% ) were discontinued . no data were available to establish whether remss were effective in improving drug safety . some remss were deemed inefficient . remss with etasu continue to be implemented but their impact on improving drug safety is still not documented . hence , one of the main requirements of the fda amendments act of 2007 is not being addressed . in addition , remss are complex and their logic is inconsistent ; we recommend a thorough re - evaluation of the rems program .

Key Points Introduction Methods Results Discussion Conclusion

risk evaluation and mitigation strategies ( remss ) , particularly ones that require elements to assure the safe use of drugs , are operationally complex and burdensome for physicians , pharmacists , drug distributors , regulators , and manufacturers.a demonstration of the impact of remss on improving safety was a key request from the legislator but , based on our review of public information , this impact does not appear to have been evaluated or made public.we recommend that rems programs should be evaluated for their effectiveness to improve drug safety . approximately 10 years ago , the us food and drug administration ( fda ) implemented a safety risk monitoring system for new marketed drugs and for drugs already on the market and for which a new risk was identified . the risk evaluation and mitigation strategy , or rems , requires multiple and complex processes , particularly remss that include elements to assure safe use ( etasu ) . pre - existing drug safety monitoring systems , such as the black box warning , have been frequently criticized [ 46 ] and , in particular , concerns were raised because their utility is difficult to evaluate . consequently , a key provision in the fda amendments act of 2007 was the mandate to study the effectiveness of remss to evaluate their impact on improving drug safety . logically , the addition of a new and more complex system , such as remss with etasu , could only be justified if evidence - based results demonstrate that such a process improves drug safety . the report concluded that the fda could not determine whether remss were improving drug safety . this report highlighted that the federal agency did not have the capacity to fulfill one of the most important aspects of the amendments act of 2007 , which was to verify that the rems system was efficient . the study evaluates rems rationale , characteristics , and consistencies and evaluates whether the impact of remss to improve drug safety could be documented . the focus was on remss with etasu , as these plans concern drugs that pose the most significant safety risks . remss requiring etasu are requested for drugs that represent the highest known , and clinically relevant , safety concerns . we searched the fda website ( http://www.accessdata.fda.gov/scripts/cder/rems/index.cfm ) to determine which of the following elements were incorporated into remss with etasu ( points 36 being specific for etasu):a medication guide . for each drug subjected to a rems with etasu , the dedicated rems website was accessed and the information available on the fda s website was eventually completed . the following information was compiled : the drug name ( usan / trade name ) , the labeled indication(s ) , whether the indication was covering an orphan disease or not , and the clinical description of the safety risk(s ) . last , for each drug subjected to a rems with etasu , the drug package insert was reviewed for the presence of a black box warning and to check whether the black box warning risks matched the risks mentioned in the rems . forty - two remss with etasus were issued , or modified , during the study period . from the general literature it was a survey on the effectiveness of the rems medication guide for conveying information on the risks of varenicline . of the 42 remss with etasu , 35 were designed for a single drug ( table 1 ) , and seven were shared programs that included multiple drugs of the same class ( table 2 ) . these shared programs were implemented across multiple manufacturers.table 1single remss with etasu : drugs , sponsors , indications , nature of risk(s ) and presence of a black box warningdrugssponsor(s)indicationrisk(s)black boxloxapinetevaacute agitation with schizophrenia or bipolar type ibronchospasmyesvandetanibastra zenecamedullary thyroid klong qt syndromeyesalvimopancubist / merckshorten gi recovery following bowel surgeryincrease incidence of miyesicodextrinbaxterperitoneal dialysis solutionincorrect blood glucose resultsyesteduglutideshireshort bowel syndromepossible acceleration of neoplastic growth and enhancement of colon polyp growth , gi obstruction , biliary and pancreatic disordersnolomitapideaegerionhomozygous familial hypercholesterolemiahepatotoxicityyesmipomersensanofihomozygous familial hypercholesterolemiahepatotoxicityyesambrisentangileadpulmonary arterial hypertensionserious birth defectsyesalosetronprometheusdiarrhea predominant ibsischemic colitisyesalglucosidase alphasanofipompe diseaseanaphylaxisyesmifepristonedancomedical termination of pregnancylife - threatening bleeding , infections , or other problemsyesmycophenolatemultipleprophylaxis of organ transplant rejectionpregnancy loss and congenital malformations , other serious risksyesromiplostimamgenchronic immune thrombocytopeniaprogression of myelodysplasia and acute myeloid leukemia , thromboembolism , marrow fibrosisnopomalidomidecelgenemultiple myelomarisk of embryo fetal exposure and other risksyeseltrombopaggskchronic immune thrombocytopeniahepatotoxicity , bone marrow fibrosis , thromboembolismyesphentermine / topiramatevivuschronic weight managementbirth defect ( cleft lift / cleft palate)nolenalidomidecelgenemyelodysplastic syndrome , mantle cell lymphomaembryo - fetal toxicityyesvigabatrinlundbeckrefractory complex partial seizurevision lossyeseculizumabalexionparoxysmal nocturnal hemoglobinuriaatypical husmeningococcal infectionyesthalidomidecelgenemultiple myelomaerythema nodosumembryo - fetal toxicityyesdofetilidepfizeratrial fibrillationflutterarrhythmiayesbosentanactelionpulmonary arterial hypertensionbirth defects , hepatotoxicityyesemtricitabine / tenofovir / disoproxilgileadpre - exposure prophylaxis of hiv-1adherence to regimen , control of hiv-1 status , counselingyesnatalizumabbiogenmultiple sclerosisprogressive multifocal leukoencephalopathyyesclozapinejazzschizophreniasevere neutropeniayescollagenase clostridium histolyticumendodupuytren diseaselapeyronie diseasetendon rupture , anaphylaxis , corporal ruptureyesolanzapinelillyschizophreniamitigate risk of post - injection delirium / sedation syndromeyesmacitentanactelionpulmonary arterial hypertensionserious birth defectyesriociguatbayerpulmonary arterial hypertensionteratogenicityyessacrosidaseqol medicalsucrase - isomaltase deficiencysevere allergic reactionnotestosterone undecanoateendoprimary hypogonadismhypogonadotropic hypogonadismpulmonary oil micro - emboli , anaphylaxisyesmetreleptinaegeriongeneralized lipodystrophyneutralizing antibodies , risk of lymphomayesalemtuzimabsanofirelapsing multiple sclerosisauto - immune conditions , infusion reactions , malignanciesyessodium oxybatejazznarcolepsycns and respiratory depression , potential abuse / misuse , ci with hypnotics and alcohol , handling and storageyesalosetronboehringer ingelheimchronic ibsischemic colitis , serious complication of constipationyes ci contra - indicated , cns central nervous system , etasu elements to assure safe use , gi gastro - intestinal , hus hemolytic uremic syndrome , ibs irritable bowel syndrome , mi myocardial infarction , rems risk evaluation and mitigation strategy table 2common remss with etasu : drugs , sponsors , indications , nature of risk(s ) and presence of a black box warningdrugssponsor(s)indicationrisk(s)black boxepoetin alphaamgen , janssenanemia of chronic kidney disease and chemotherapyshortened survival , increased risk of tumor progression / recurrenceyesdarbepoetin alphaamgenyesbuprenorphinemultipleopioid dependenceaccidental overdose , misuse or abusenobuprenorphine and naloxonemultipleopioid dependenceaccidental overdose , misuse or abusenoextended - release and long - acting opioid analgesicsmultipleanalgesiaaddiction , abuse , and misuseyesisotretinoinmultiplesevere recalcitrant nodular acnesevere birth defectsyesrosiglitazone and its combinationsgsktype 2 diabetesischemic cardiovascular riskyesfentanylmultipleanalgesia in cancer patientsmitigate the risk of misuse , abuse , addiction , overdose and serious complications due to medication errorsyes etasu elements to assure safe use , rems risk evaluation and mitigation strategy single remss with etasu : drugs , sponsors , indications , nature of risk(s ) and presence of a black box warning ci contra - indicated , cns central nervous system , etasu elements to assure safe use , gi gastro - intestinal , hus hemolytic uremic syndrome , ibs irritable bowel syndrome , mi myocardial infarction , rems risk evaluation and mitigation strategy common remss with etasu : drugs , sponsors , indications , nature of risk(s ) and presence of a black box warning etasu elements to assure safe use , rems risk evaluation and mitigation strategy the nature of the risks involved could be common to more than one rems ( table 3 ) . the risk of birth defect(s ) or embryo - fetal toxicity and the risk of potential abuse or misuse were the most frequent risks addressed in remss with etasu ( 8/42 [ 19% ] and 6/42 [ 14% ] , respectively ) . these were the adherence to the prescribed regimen and counseling , mistake in blood glucose reading , and tendon or cavernous body rupture following a local drug injection.table 3risks addressed in remss with etasurisksnumber of drugsbirth defect(s ) or embryo - fetal toxicity8potential abuse or misuse6allergic reaction5tumor or neoplastic progression4hepatotoxicity4infection3thromboembolism3cardiac arrhythmia2myocardial infarction2bone marrow fibrosis2biliary / pancreatic disorders1ischemic colitis and complication of constipation1delirium , sedation , and vision loss1severe neutropenia1congestive heart failure1adherence to regimen and counseling1incorrect blood glucose reading1tendon or cavernous body rupture after a local injection1 etasu elements to assure safe use , rems risk evaluation and mitigation strategy risks addressed in remss with etasu etasu elements to assure safe use , rems risk evaluation and mitigation strategy the risk(s ) mentioned in a rems with etasu could be single or multiple . for 24 remss , only a single risk was identified ( e.g. for 17 remss , multiple risks were mentioned ( e.g. some risks were difficult for individual prescribers to evaluate as they were clinical events that could have multiple etiologies ( e.g. for such rare events , the collection of data from a large population and/or multiple prescribers could only document an increased risk of the 42 remss with etasu , 36 drugs ( including 31 individual drugs and five classes of drugs ) had a label that contained a black box warning . there was no apparent difference in the type of risks for drugs with a black box warning and drugs without such a warning . for instance , the same risks ( misuse or abuse , thromboembolism , birth defect , or acceleration of tumor growth ) were mentioned for drugs with or without black box warnings . when a drug had a black box warning , the risks mentioned in the rems with etasu were similar to risks mentioned in the black box for 33 drugs ( 23 individual drugs and two grouped remss ) . risks were dissimilar for 11 drugs ( eight individual remss and three common remss ) . in eight instances , fewer risks were mentioned in the rems than in the black box ( six single remss and two common remss ) . in three instances , more risks were mentioned in the rems than in the black box ( two single remss and one common rems ) . for extended - release and long - acting opioid analgesics , a drug approved for multiple indications could be submitted for a rems with etasu for one indication but not for another indication . similarly , mifepristone was subjected to a rems with etasu for the medical termination of intra - uterine pregnancy ( one 600-mg dose ) but not for the treatment of cushing s syndrome ( 300 mg daily , continuously ) . some medical specialties , such as neuropsychiatry , cardiovascular medicine , endocrinology and metabolic diseases , and oncology , were more frequently concerned by remss with etasu than other specialties ( table 4).table 4medical specialty concerned with rems with etasuspecialtynumber of remss with etasuneurology / psychiatry7cardiology / vascular7endocrinology / metabolic disease6oncology / hematology5hepato - gastro - intestinal4 etasu elements to assure safe use , rems risk evaluation and mitigation strategy medical specialty concerned with rems with etasu etasu elements to assure safe use , rems risk evaluation and mitigation strategy most drugs ( 24/42 , or 57% ) that were subjected to remss with etasu were indicated for an orphan disease . the processes that were included in remss with etasu were , in order of decreasing frequency , an implementation system ( 81% of remss ) , prescriber training ( 71% ) , prescriber certification ( 69% ) , a medication guide ( 67% ) , dispenser certification ( 48% ) , patient enrolment ( 43% ) , and a communication plan ( 26% ) . three of the 42 remss with etasu ( 7% ) were discontinued ( table 5).table 5discontinuation of rems with etasudrugindicationriskreason for discontinuationeltrombopagthrombocytopenia in chronic immune thrombocytopeniaprogression of mds and aml , thromboembolism , marrow fibrosisnot availableromiplostimthrombocytopenia in chronic immune thrombocytopeniahepatotoxicity , bone marrow fibrosis , thromboembolismnot availablelumizymepompe diseaseanaphylactic reactionsdrug considered similar to myozymerosiglitazone and its combinationstype 2 diabetes mellitusischemic cardiovascular riskrisk not documented ( replaced by risk of congestive heart failure ) aml acute myeloid leukemia , etasu elements to assure safe use , mds myelodysplastic syndromes , rems risk evaluation and mitigation strategy discontinuation of rems with etasu aml acute myeloid leukemia , etasu elements to assure safe use , mds myelodysplastic syndromes , rems risk evaluation and mitigation strategy a rems is mandated by law and includes specific measures to ensure that the benefits of a drug outweigh its risks . one of the key provisions in establishing remss was to ensure that the fda can evaluate the impact of such programs and thus demonstrate their relevance in addressing and preventing safety risks [ 1 , 7 ] . this report concluded that the fda did not have relevant data to determine whether remss improve drug safety and , despite the significant burden and cost associated with the rems system , the relevance of these programs could not be established . our study s first objective was to establish whether the situation regarding the relevance of remss has changed since the inspector general s report and whether the impact of remss to improve drug safety could now be established . our second objective was to establish the characteristics of remss with etasu , as these remss address drugs with the most significant safety risk . since the report from the office of the inspector general , 42 new remss with etasu have been issued ( 35 programs for individual drugs , and seven programs incorporating more than one drug ) . our study confirms that there is still only very limited information publicly available to demonstrate that remss with etasu address and/or prevent safety risks . despite the burden that remss represent , remss continue to be requested and continue to be implemented despite the fact that the original intent of the law that established the rems system has not been fulfilled . the two most frequent risks addressed in the remss with etasu we reviewed were the risk of embryo - fetal toxicities and the risk for drug abuse . since the thalidomide tragedy , the risk of embryo - fetal toxicity has been a priority for regulators ; it is thus not a surprise to see this risk prominently addressed in remss . more surprising , however , is the great variability of the level of embryo - fetal risk associated with drugs submitted to a rems . to our knowledge , it is not known whether this program adequately addresses and prevents the risk of embryo - fetal toxicity . additionally , the treatment of multiple myeloma incorporates multiple chemotherapeutic drugs that , while they are themselves embryo - toxic , are not submitted to a rems with etasu . four drugs indicated for chronic pulmonary hypertension , a rare orphan disease , share a risk of embryo - fetal toxicity . the patient population is not only at high risk for pregnancy , but is also one for whom a pregnancy could have severe consequences for the health of the mother . beyond embryo - fetal toxicity and opioid abuse , the other risks identified in remss , such as allergic reaction , hepatotoxicity , or thromboembolism , were of varying severity . the risks addressed in a rems with etasu were generally documented during the drug development program of a candidate drug and were identified before the drug was approved . most drugs that were included in a rems with etasu also had a black box warning in their label . the content of black box warnings and the content of remss were frequently divergent without apparent reasons . in this context , regulators might be tempted to supplement a black box warning with a rems with etasu . remss , however , are immensely more complex and logistically challenging that black box warnings . additionally , a significant number of remss with etasu were requested for drugs that did not have a black box warning in their label . most remss with etasu were implemented for drugs that were indicated for a rare orphan disease . very few remss with etasu were discontinued and when they were , no rationale was made publicly available , or the rationale was difficult to understand . while remss can be negotiated between the fda and a drug manufacturer , the fda has sole decision power . the current rems system does not appear to meet the intent of the law which requested a mandatory evaluation of these programs to demonstrate that they were improving drug safety . each year , new rems are issued , and very few are discontinued , but today , it is still unknown whether the rems system is useful or not .

g protein coupled receptors ( gpcrs ) are nodal regulators of mammalian cell physiology because they transduce cell signals from diverse ligands such as neurohormones , sensory stimuli , and ions through heterotrimeric g proteins . in the heart , they represent the major modulators of both function and morphology with -adrenergic receptors ( ars ) representing the heads of the line , and for this reason they are considered the most important molecular targets in the cardiovascular system . currently , 3 ar subtypes ( 1ar , 2ar , and 3ar ) have been identified in the myocardium , with 1 and 2ars the most expressed and studied . however , since its discovery in 1989 , it soon seemed clear that 3ars , the isoform with minor expression , can influence cardiovascular physiology . in particular , 3ars seem to have multiple roles that go from regulation of metabolism , vasodilation , and relaxation to cardiac contractility . thus , this receptor is of high interest especially for new potential therapeutic approaches for heart disease . in this review , we will discuss what is known about the cardiac role of 3ars and how not only their activation but also the blockade could be beneficial or not in cardiac physiology and in disease . the mammalian 3ar sequence consists of about 400408 amino acids in a protein that has the typical structure of all gpcrs . the 3ar has 7 transmembrane domains ( 7-tmds ) with an extracellular n - terminal that is glycosylated , and an intracellular c - terminal domain . further , the cys361 residue in the fourth intracellular domain is palmitoylated , a feature that has been shown to be associated with g protein - coupling and adenylyl cyclase stimulation following agonist stimulation of the receptor ( fig . as shown in figure 2 , the protein sequence alignment between different mammalian species demonstrates that most of the homology between the 3ar amino acid sequences is concentrated in the 7-tmds and in the membrane - proximal regions of the intracellular loops . interestingly , when the 3ar protein sequence is compared with other ar ( 1 and 2ar ) isoforms , it is still possible to observe a high level of homology in the 7-tmd sequence , but a significant divergence is present both in the third intracellular loop and in the c - terminal domain ( fig . this difference probably represents the major factor affecting the pharmacologic regulation of the receptors and their response to a ligand . in this regard , the c - terminus of both 1 and 2ars is rich in serine and threonine residues , and is subjected to gpcr kinase ( grk)-mediated regulation through phosphorylation . further , these receptors also harbor a consensus sequence for protein kinase a ( pka ) . of note , the 3ar lacks all of these sites , and is more resistant to agonist - induced desensitization / downregulation . finally , these sequence divergences also support differential and intracellular signaling ( including g protein - coupling ) between the 3 ar isoforms , which may determine their relative roles in physiology and in the disease . the receptor is a gpcr with 7-tmds , an extracellular n - terminal domain ( exd1 ) , and an intracellular c - terminal domain ( ind4 ) . the receptor presents also 6 loops , 3 are intracellular ( ind1 , ind2 , and ind3 ) , and 3 are extracellular ( exd2 , exd3 , and exd4 ) . indicated with arrows are the asparagine ( n ) residues , in the exd1 , that are sites of n - glycosylation ; tryptophan ( w ) in position 64 that is the location of 3ar - polymorphism ( trp64arg ) and the cysteine ( c ) in position 361 that is a site subjected to palmitoylation . protein sequence alignment of mouse , dog , pig , human , and macaca mulatta 3ar . the tmd is highlighted in yellow ; the tryptophan ( w ) in position 64 is in red ; the cysteine ( c ) in position 361 is in green . the human 3ar was cloned in 1989 , and the studies demonstrated that this new ar isoform was mainly implicated in lipolysis and thermogenesis regulation in adipose tissues . however , over the last 2 decades , different reports have also clearly shown that 3ars are present in the cardiovascular system , mainly in myocardium and endothelium , where they have a prominent role in modulating cardiac function and angiogenesis , respectively . in this context , determining the specific pathways associated with these effects represents a tough challenge and remains a largely unresolved question regarding 3ar function . this is due , at least in part , to the fact that the role of the cardiac 3ar has not been studied with the same intensity as the 1- and 2ars . moreover , all the studies concerning 3ar function have not been focused on similar cell types , and the agonist and the doses used are significantly different between most studies . in fact , it is known that in the mouse , the gene encoding for the 3ar undergoes alternative splicing and gives rise to 3aar and 3bar variants . these 2 3ar isoforms are coupled to adenylyl cyclase stimulatory g ( gs ) or to the inhibitory g ( gi ) protein subunits ( 3bar ) , or exclusively to the gs protein ( 3aar ) . by contrast , in humans , although some reports have proposed that 3ar can activate gs signaling in cho / k1 cells and in adipocytes , it is a general assumption that , at least in ventricular myocardium , 3ars are mainly coupled with gi proteins . for these reasons , the 3ar leads to effects that are either comparable or opposite to those elicited by 1- and 2ar stimulation . in fact , stimulation of 3ar , through gs activation , increases the generation of cyclic amp ( camp ) and the activation of the pka , similar to 1- and 2ars . in the myocardium , after catecholamines stimulation of ars , pka phosphorylates many ca handling proteins and some myofilament components leading to positive inotropic , lusitropic , and chronotropic effects ( fig . 4 ) . however , because 3ars are also coupled with gi , they can act as a brake to prevent 1 and 2ars overactivation , and this has been proposed as a mechanism in the heart ( fig . moreover , in the heart , the stimulation of 3ars leads to increased endothelial nitric oxide ( no ) synthase ( enos ) or neuronal ( nnos ) activation , giving rise to no generation and activation of soluble guanylate cyclase to produce cgmp and cgmp - dependent protein kinase g ( pkg ) activation ( fig . pkg is a serine / threonine kinase that mediates many of the biological effects of no / cgmp . in particular , pkg downstream of 3ars can enhance myocytes relaxation but cause negative inotropy , possibly through the phosphorylation of troponin i and l - type ca channel ( fig . importantly , the 3ar / no - cgmp / pkg signaling axis seems to be a robust cardioprotective mechanism that can be beneficial in failing myocardium . in fact , pkg activation downstream of cgmp has been proven to reduce ca oscillations which can cause ventricular arrhythmias , hypercontracture and sarcolemmal rupture as well as mitochondrial permeability transition pore that is a cause of cell death . of note , both these phenomena occur during reperfusion of ischemic myocardium and can lead to progression toward heart failure ( hf ) . schematic representation of 3ar signaling activation in cardiomyocytes . 3ars are coupled to both stimulatory g proteins ( gs ) and inhibitory g proteins ( gi ) . although the gs pathway induces the generation of camp and cgmp which , in turn , activates the pka and pkg , respectively , the activation of gi signaling pathway is able to stimulate only the generation of cgmp and the activation of pkg . pka has multiple roles in cardiomyocytes and is able to induce the phosphorylation of several key factors involved in the regulation of contractility , such as cardiac troponin i ( ctni ) and phospholamban ( pln ) . furthermore , pka can phosphorylate the l - type ca channel ( ltcc ) increasing the influx of extracellular ca . importantly , pka can also activate protein kinase b ( akt ) with the subsequent activation of the endothelial no synthase ( enos ) . of note , enos activation increases the generation of no which , in turn , activates the soluble guanylate cyclase to produce cgmp and pkg activation . however , pkg can induce the inactivation of ltcc , thus reducing the extracellular ca influx . moreover , cgmp can stimulate phosphodiesterase 2 ( pde2 ) , reducing the camp levels and the activation of pka . of note , after the gi signaling pathway activation , the 3ar is able to give rise to no through both enos and neuronal nos ( nnos ) located on the sarcoplasmic reticulum ( sr ) . in fact , there is a nonsynonymous polymorphism at amino acid 64 where a tryptophan can exist instead of arginine ( figs . 1 , 2 ) . this mutation makes the 3ar less responsive to catecholamine stimulation , which has been associated with some pathophysiological conditions ( ie , obesity ) ( box 1 ) . this important difference suggests that nonhuman 3ar and its signaling may not fully mirror the human system . obesity , susceptibility todiabetesinsulin resistancehyperuricemia obesity , susceptibility to accumulating evidence has revealed that the 3ar , present in endothelium and myocardium , may have specific beneficial effects in the cardiovascular system including cardioprotection . this becomes critically crucial in cardiac diseases such as hf , a syndrome characterized by decreased cardiac output , caused by deficits in contractility and/or relaxation . importantly , after an injury such as ischemia , to preserve cardiac output , there is an increase in sympathetic activity and in catecholamine release to stimulate ar - mediated inotropic capacity . however , chronic exposure of the heart to high levels of catecholamines can lead to further pathologic changes in the heart that can induce a progressive deterioration of cardiac function and structure . of note , catecholamines directly stimulate ars , and the sustained activation of these receptors correlate with left ventricular ( lv ) dysfunction and mortality . in this regard , grk2 , the principal grk involved in ar regulation within the cardiomyocytes , phosphorylates the receptors attenuating their increased responsiveness . this process , called desensitization , at early stage represents a protective mechanism , but in chronic stage can cause 1 and 2ars dysregulation and signaling abnormalities ( eg , downregulation and overdesensitization ) and promote the progression of the disease . importantly , as discussed above , 3ars lack grk recognition sites and are not subject to desensitization and downregulation , and in fact , their levels within human failing myocardium remain unchanged or become upregulated . enhancement of 3ars could represent either a protective mechanism against the detrimental effects of chronic ar stimulation or a detrimental mechanism that may lead to further deterioration of hf . in this regard , the role of this receptor in the heart has been debated for years , and some reports have suggested that due to its cardiodepressant effect , sustained activation of 3ars in hf could contribute to impaired cardiac function . consistently , the antagonism of this receptor has been proposed as a potential strategy against hf development . however , by contrast with this hypothesis , it has been demonstrated that in the failing myocardium , 3ars are able to inhibit , through activation of na - k pump , the deleterious accumulation of na in cardiac myocytes thus blocking camp generation , and consequently , reducing the activation of the camp - downstream oxidative pathways . mechanistically , this effect decreases the glutathionylation of the 1 na - k pump subunit and enhances the na - k pump activity in presence of 3ar agonists . in line with the notion that 3ar activation in failing myocardium is not detrimental , studies strongly support the idea that overexpression or persistent activation of 3ar is cardioprotective and can attenuate pathological lv hypertrophy induced by continuous infusion of isoproterenol and angiotensin ii , or by transaortic constriction , in mice . importantly , as shown in these studies , the activation of nos and subsequent no generation represent the main mechanism responsible for 3ar - induced cardioprotection . in agreement with this mechanism of action , another study , using small ( mice ) and large ( pigs ) animal models of ischemia / reperfusion ( i / r ) injury , demonstrated that administration of selective 3ar agonist brl 37344 positively affected infarct size ( acutely ) and lv function ( chronically ) . further , this study showed that 3ar / no signaling decreased opening of the mitochondrial permeability transition pore , thus conferring protection to the cardiac cells against cell death . finally , it is well established that the role of exercise in cardioprotection , and 3ars , has been also shown to be a mediator of this effect , especially in a setting of cardiac i / r injury . of note , during exercise , it seems that gs is the key mediator of 3ar - induced protection that leads to the pka / akt / enos activation , thus suggesting that , in some conditions , the 3ar , similarly to the cardioprotective 2ar , is coupled with both gs and gi in cardiomyocytes ( fig . regard , this signaling pathway activation has a multiple protective role in the injured myocardium like the promotion of revascularization of the ischemic tissue . in fact , activation of akt and enos , and the consequent secretion of no , has been proven to directly stimulate endothelial cell function and promote the neoangiogenesis . further , as discussed above , the enos - mediated generation of no is also responsible for cgmp and pkg activation , thus directly conferring beneficial effects on the myocardium . as described above , 3ars have emerged as novel potential targets for the treatment of certain cardiovascular diseases including hf . the clinical relevance of this is further supported by the successful effects obtained with -blocker treatment in patients with hf as they can block the noxious effects of catecholamines and prevent further 1 and 2ars downregulation . importantly , as proposed by us and others , the use of -blockers may influence the expression / activity of 3ars . however , because there are some specific differences in -blockers ( 1ar selective or nonselective ) , the full extent of whether any molecular changes in 3ars are significant contributors to the therapeutic mechanisms of ar antagonism in hf still remains to be elucidated . accordingly , answering these mechanistic questions is important and may lead to novel therapeutic advances in hf . in 2007 in particular , in an hf rat model induced by transaortic constriction , it has been shown that , although metoprolol ( a selective 1-blocker ) treatment did not affect the expression levels of 3ar which was increased after hf , the use of carvedilol ( a nonselective -blocker ) resulted in a robust 3ar downregulation . nevertheless , soon after this report , a number of studies proposed that 1-blockers and their subsequent beneficial effects in hf could induce enhancement of the 3ar expression and activity . for example , sharma et al showed that metoprolol treatment was able to improve cardiac function in diabetic rats mainly through 3ar upregulation and no generation . analogous , in a canine model of mitral regurgitation , we recently found that metoprolol can promote 3ar upregulation and enhance its protective signaling ( ie , nnos / no / cgmp ) . moreover , correlative data have been recently shown for the potential of 3ar agonistic activity of nebivolol , a highly selective 1-blocker . in particular , in a model of i / r injury , nebivolol administration activated cardiac 3ars leading to a significant reduction of infarct size . similarly , zhang et al demonstrated , in a setting of hf in mice , induced by left anterior descending artery ligation , that nebivolol was able to reduce cardiac fibrosis and apoptosis and improved cardiac function . interestingly , this report showed also that after 4 weeks postmyocardial infarction , there was a significant reduction of cardiac 3ar levels , and that nebivolol was able to restore the expression of this receptor . importantly , further to the direct effect in cardiomyocytes , 1-blockers , such as nebivolol have been also reported to act on endothelium . of note , enhancement of neoangiogenesis in the failing heart is considered one of the mechanisms of protection activated by this class of drugs . in this context , nebivolol through 3ar activation increases the generation of no , a key mediator of endothelial function , enhancing endothelial proliferation and increasing vasodilation . as described above , the 3ar represents an emerging attractive target for pharmacological modulation in the injured heart that , for its compensatory effects , prevents the effects of excessive catecholamines stimulation on the heart . in fact , selective 3ar agonism has been proven to confer protection in the failing heart through a specific cgmp / no signaling pathway . a particular role for no has been associated with the enhancement of endothelial cell proliferation , and within failing myocardium it can lead to a beneficial effect on cardiac function and remodeling . as described above , 3ars are expressed not only in cardiomyocytes but also in endothelial cells , thus supporting the idea that this receptor can also promote proangiogenic mechanisms . moreover , in adipocytes , the 3ar is implicated in metabolic regulation [ fatty acid ( fa ) oxidation , lipolysis , and thermogenesis ] , that can also be a crucial mechanism to rescue the heart from failure . importantly , this mechanism is particularly important in several pathological conditions that affect cardiac function , such as ischemia and pressure overload . in fact , in these conditions , the heart , in the presence of limited oxygen supply , suppresses glucose and fa oxidation with a shift in cardiac substrate metabolism from fa oxidation to glycolysis . glycolysis without a concomitant increase in glucose oxidation results in an accumulation of different harmful catabolites such as lactate and protons that are the cause of intracellular acidosis and na and ca overload . these effects strongly reduce the capacity of the heart to provide sufficient energy for contractile work because more energy is spent to restore ion homeostasis and lead to an increase in lipid accumulation within the cardiac cells with consequent lipotoxicity . in this context , as recently demonstrated in adipocytes , nebivolol , through 3ar activation , is able to improve adipocyte metabolism . therefore , it is plausible that long - term 3ar stimulation , through selective agonists and/or -blockers ( ie , nebivolol ) , can be used as a novel therapeutical approach also to improve metabolism within the failing myocardium . currently , 2 clinical trials , the beta 3 agonist treatment in heart failure ( beat - hf ; clinicaltrials.gov : nct01876433 ) , or in the prevention of hf development , the assessment of efficacy of mirabegron , a new beta 3-adrenergic receptor in the prevention of heart failure ( beta3_lvh ; clinicaltrials.gov : nct02599480 ) , are trying to evaluate the effects of 3ar agonism on hf progression and development . in this context , patients will be treated with the selective 3ar agonist , mirabegron ( also known as ym-178 ) , a drug already approved in the united states , japan , and europe , for the treatment of overactive bladder . this drug , through the activation of 3ar , exerts a myorelaxant effect in the detrusor muscle thus improving the bladder filling . anyway , in the meantime that these trials will give us the proof - of - concept of the beneficial role of 3ars in human hf , the most important clinical evidence that we currently have regarding the role of this receptor remains the proved efficacy of -blockers . the cardioprotective effect of ar - blockade has been largely attributed , for decades , to antagonism of cardiac 1- and 2ars and a resulting heart rate reduction . however , as discussed above in this review , there is emerging evidence that this class of drugs may also act through 3ar signaling pathway activation which is not blocked by the drugs currently used in clinical hf - therapy ( box 2 ) . however , the main question that still remains unanswered is why not all patients respond favorably to these agents . for these reasons , further elucidation on specific mechanisms of action of these drugs is extremely interesting . in particular , it will be important to evaluate how different -blockers can impact , in a positive or a negative manner , 3ar activity , and if this effect is a significant contributor of therapeutic mechanisms in hf . the mammalian 3ar sequence consists of about 400408 amino acids in a protein that has the typical structure of all gpcrs . the 3ar has 7 transmembrane domains ( 7-tmds ) with an extracellular n - terminal that is glycosylated , and an intracellular c - terminal domain . further , the cys361 residue in the fourth intracellular domain is palmitoylated , a feature that has been shown to be associated with g protein - coupling and adenylyl cyclase stimulation following agonist stimulation of the receptor ( fig . as shown in figure 2 , the protein sequence alignment between different mammalian species demonstrates that most of the homology between the 3ar amino acid sequences is concentrated in the 7-tmds and in the membrane - proximal regions of the intracellular loops . interestingly , when the 3ar protein sequence is compared with other ar ( 1 and 2ar ) isoforms , it is still possible to observe a high level of homology in the 7-tmd sequence , but a significant divergence is present both in the third intracellular loop and in the c - terminal domain ( fig . this difference probably represents the major factor affecting the pharmacologic regulation of the receptors and their response to a ligand . in this regard , the c - terminus of both 1 and 2ars is rich in serine and threonine residues , and is subjected to gpcr kinase ( grk)-mediated regulation through phosphorylation . further , these receptors also harbor a consensus sequence for protein kinase a ( pka ) . of note , the 3ar lacks all of these sites , and is more resistant to agonist - induced desensitization / downregulation . finally , these sequence divergences also support differential and intracellular signaling ( including g protein - coupling ) between the 3 ar isoforms , which may determine their relative roles in physiology and in the disease . human 3ar structure . the receptor is a gpcr with 7-tmds , an extracellular n - terminal domain ( exd1 ) , and an intracellular c - terminal domain ( ind4 ) . the receptor presents also 6 loops , 3 are intracellular ( ind1 , ind2 , and ind3 ) , and 3 are extracellular ( exd2 , exd3 , and exd4 ) . indicated with arrows are the asparagine ( n ) residues , in the exd1 , that are sites of n - glycosylation ; tryptophan ( w ) in position 64 that is the location of 3ar - polymorphism ( trp64arg ) and the cysteine ( c ) in position 361 that is a site subjected to palmitoylation . protein sequence alignment of mouse , dog , pig , human , and macaca mulatta 3ar . the tmd is highlighted in yellow ; the tryptophan ( w ) in position 64 is in red ; the cysteine ( c ) in position 361 is in green . the human 3ar was cloned in 1989 , and the studies demonstrated that this new ar isoform was mainly implicated in lipolysis and thermogenesis regulation in adipose tissues . however , over the last 2 decades , different reports have also clearly shown that 3ars are present in the cardiovascular system , mainly in myocardium and endothelium , where they have a prominent role in modulating cardiac function and angiogenesis , respectively . in this context , determining the specific pathways associated with these effects represents a tough challenge and remains a largely unresolved question regarding 3ar function . this is due , at least in part , to the fact that the role of the cardiac 3ar has not been studied with the same intensity as the 1- and 2ars . moreover , all the studies concerning 3ar function have not been focused on similar cell types , and the agonist and the doses used are significantly different between most studies . in fact , it is known that in the mouse , the gene encoding for the 3ar undergoes alternative splicing and gives rise to 3aar and 3bar variants . these 2 3ar isoforms are coupled to adenylyl cyclase stimulatory g ( gs ) or to the inhibitory g ( gi ) protein subunits ( 3bar ) , or exclusively to the gs protein ( 3aar ) . by contrast , in humans , although some reports have proposed that 3ar can activate gs signaling in cho / k1 cells and in adipocytes , it is a general assumption that , at least in ventricular myocardium , 3ars are mainly coupled with gi proteins . for these reasons , the 3ar leads to effects that are either comparable or opposite to those elicited by 1- and 2ar stimulation . in fact , stimulation of 3ar , through gs activation , increases the generation of cyclic amp ( camp ) and the activation of the pka , similar to 1- and 2ars . in the myocardium , after catecholamines stimulation of ars , pka phosphorylates many ca handling proteins and some myofilament components leading to positive inotropic , lusitropic , and chronotropic effects ( fig . 4 ) . however , because 3ars are also coupled with gi , they can act as a brake to prevent 1 and 2ars overactivation , and this has been proposed as a mechanism in the heart ( fig . moreover , in the heart , the stimulation of 3ars leads to increased endothelial nitric oxide ( no ) synthase ( enos ) or neuronal ( nnos ) activation , giving rise to no generation and activation of soluble guanylate cyclase to produce cgmp and cgmp - dependent protein kinase g ( pkg ) activation ( fig . pkg is a serine / threonine kinase that mediates many of the biological effects of no / cgmp . in particular , pkg downstream of 3ars can enhance myocytes relaxation but cause negative inotropy , possibly through the phosphorylation of troponin i and l - type ca channel ( fig . importantly , the 3ar / no - cgmp / pkg signaling axis seems to be a robust cardioprotective mechanism that can be beneficial in failing myocardium . in fact , pkg activation downstream of cgmp has been proven to reduce ca oscillations which can cause ventricular arrhythmias , hypercontracture and sarcolemmal rupture as well as mitochondrial permeability transition pore that is a cause of cell death . of note , both these phenomena occur during reperfusion of ischemic myocardium and can lead to progression toward heart failure ( hf ) 3ars are coupled to both stimulatory g proteins ( gs ) and inhibitory g proteins ( gi ) . although the gs pathway induces the generation of camp and cgmp which , in turn , activates the pka and pkg , respectively , the activation of gi signaling pathway is able to stimulate only the generation of cgmp and the activation of pkg . pka has multiple roles in cardiomyocytes and is able to induce the phosphorylation of several key factors involved in the regulation of contractility , such as cardiac troponin i ( ctni ) and phospholamban ( pln ) . furthermore , pka can phosphorylate the l - type ca channel ( ltcc ) increasing the influx of extracellular ca . importantly , pka can also activate protein kinase b ( akt ) with the subsequent activation of the endothelial no synthase ( enos ) . of note , enos activation increases the generation of no which , in turn , activates the soluble guanylate cyclase to produce cgmp and pkg activation . however , pkg can induce the inactivation of ltcc , thus reducing the extracellular ca influx . moreover , cgmp can stimulate phosphodiesterase 2 ( pde2 ) , reducing the camp levels and the activation of pka . of note , after the gi signaling pathway activation , the 3ar is able to give rise to no through both enos and neuronal nos ( nnos ) located on the sarcoplasmic reticulum ( sr ) . in fact , there is a nonsynonymous polymorphism at amino acid 64 where a tryptophan can exist instead of arginine ( figs . 1 , 2 ) . this mutation makes the 3ar less responsive to catecholamine stimulation , which has been associated with some pathophysiological conditions ( ie , obesity ) ( box 1 ) . this important difference suggests that nonhuman 3ar and its signaling may not fully mirror the human system . accumulating evidence has revealed that the 3ar , present in endothelium and myocardium , may have specific beneficial effects in the cardiovascular system including cardioprotection . this becomes critically crucial in cardiac diseases such as hf , a syndrome characterized by decreased cardiac output , caused by deficits in contractility and/or relaxation . importantly , after an injury such as ischemia , to preserve cardiac output , there is an increase in sympathetic activity and in catecholamine release to stimulate ar - mediated inotropic capacity . however , chronic exposure of the heart to high levels of catecholamines can lead to further pathologic changes in the heart that can induce a progressive deterioration of cardiac function and structure . of note , catecholamines directly stimulate ars , and the sustained activation of these receptors correlate with left ventricular ( lv ) dysfunction and mortality . in this regard , grk2 , the principal grk involved in ar regulation within the cardiomyocytes , phosphorylates the receptors attenuating their increased responsiveness . this process , called desensitization , at early stage represents a protective mechanism , but in chronic stage can cause 1 and 2ars dysregulation and signaling abnormalities ( eg , downregulation and overdesensitization ) and promote the progression of the disease . importantly , as discussed above , 3ars lack grk recognition sites and are not subject to desensitization and downregulation , and in fact , their levels within human failing myocardium remain unchanged or become upregulated . enhancement of 3ars could represent either a protective mechanism against the detrimental effects of chronic ar stimulation or a detrimental mechanism that may lead to further deterioration of hf . in this regard , the role of this receptor in the heart has been debated for years , and some reports have suggested that due to its cardiodepressant effect , sustained activation of 3ars in hf could contribute to impaired cardiac function . consistently , the antagonism of this receptor has been proposed as a potential strategy against hf development . however , by contrast with this hypothesis , it has been demonstrated that in the failing myocardium , 3ars are able to inhibit , through activation of na - k pump , the deleterious accumulation of na in cardiac myocytes thus blocking camp generation , and consequently , reducing the activation of the camp - downstream oxidative pathways . mechanistically , this effect decreases the glutathionylation of the 1 na - k pump subunit and enhances the na - k pump activity in presence of 3ar agonists . in line with the notion that 3ar activation in failing myocardium is not detrimental , studies strongly support the idea that overexpression or persistent activation of 3ar is cardioprotective and can attenuate pathological lv hypertrophy induced by continuous infusion of isoproterenol and angiotensin ii , or by transaortic constriction , in mice . importantly , as shown in these studies , the activation of nos and subsequent no generation represent the main mechanism responsible for 3ar - induced cardioprotection . in agreement with this mechanism of action , another study , using small ( mice ) and large ( pigs ) animal models of ischemia / reperfusion ( i / r ) injury , demonstrated that administration of selective 3ar agonist brl 37344 positively affected infarct size ( acutely ) and lv function ( chronically ) . further , this study showed that 3ar / no signaling decreased opening of the mitochondrial permeability transition pore , thus conferring protection to the cardiac cells against cell death . finally , it is well established that the role of exercise in cardioprotection , and 3ars , has been also shown to be a mediator of this effect , especially in a setting of cardiac i / r injury . of note , during exercise , it seems that gs is the key mediator of 3ar - induced protection that leads to the pka / akt / enos activation , thus suggesting that , in some conditions , the 3ar , similarly to the cardioprotective 2ar , is coupled with both gs and gi in cardiomyocytes ( fig . regard , this signaling pathway activation has a multiple protective role in the injured myocardium like the promotion of revascularization of the ischemic tissue . in fact , activation of akt and enos , and the consequent secretion of no , has been proven to directly stimulate endothelial cell function and promote the neoangiogenesis . further , as discussed above , the enos - mediated generation of no is also responsible for cgmp and pkg activation , thus directly conferring beneficial effects on the myocardium . as described above , 3ars have emerged as novel potential targets for the treatment of certain cardiovascular diseases including hf . the clinical relevance of this is further supported by the successful effects obtained with -blocker treatment in patients with hf as they can block the noxious effects of catecholamines and prevent further 1 and 2ars downregulation . importantly , as proposed by us and others , the use of -blockers may influence the expression / activity of 3ars . however , because there are some specific differences in -blockers ( 1ar selective or nonselective ) , the full extent of whether any molecular changes in 3ars are significant contributors to the therapeutic mechanisms of ar antagonism in hf still remains to be elucidated . accordingly , answering these mechanistic questions is important and may lead to novel therapeutic advances in hf . in 2007 , it was first reported that a relationship between -blockers and 3ar may exist . in particular , in an hf rat model induced by transaortic constriction , it has been shown that , although metoprolol ( a selective 1-blocker ) treatment did not affect the expression levels of 3ar which was increased after hf , the use of carvedilol ( a nonselective -blocker ) resulted in a robust 3ar downregulation . nevertheless , soon after this report , a number of studies proposed that 1-blockers and their subsequent beneficial effects in hf could induce enhancement of the 3ar expression and activity . for example , sharma et al showed that metoprolol treatment was able to improve cardiac function in diabetic rats mainly through 3ar upregulation and no generation . analogous , in a canine model of mitral regurgitation , we recently found that metoprolol can promote 3ar upregulation and enhance its protective signaling ( ie , nnos / no / cgmp ) . moreover , correlative data have been recently shown for the potential of 3ar agonistic activity of nebivolol , a highly selective 1-blocker . in particular , in a model of i / r injury , nebivolol administration activated cardiac 3ars leading to a significant reduction of infarct size . similarly , zhang et al demonstrated , in a setting of hf in mice , induced by left anterior descending artery ligation , that nebivolol was able to reduce cardiac fibrosis and apoptosis and improved cardiac function . interestingly , this report showed also that after 4 weeks postmyocardial infarction , there was a significant reduction of cardiac 3ar levels , and that nebivolol was able to restore the expression of this receptor . importantly , further to the direct effect in cardiomyocytes , 1-blockers , such as nebivolol have been also reported to act on endothelium . of note , enhancement of neoangiogenesis in the failing heart is considered one of the mechanisms of protection activated by this class of drugs . in this context , nebivolol through 3ar activation increases the generation of no , a key mediator of endothelial function , enhancing endothelial proliferation and increasing vasodilation . as described above , the 3ar represents an emerging attractive target for pharmacological modulation in the injured heart that , for its compensatory effects , prevents the effects of excessive catecholamines stimulation on the heart . in fact , selective 3ar agonism has been proven to confer protection in the failing heart through a specific cgmp / no signaling pathway . a particular role for no has been associated with the enhancement of endothelial cell proliferation , and within failing myocardium it can lead to a beneficial effect on cardiac function and remodeling . as described above , 3ars are expressed not only in cardiomyocytes but also in endothelial cells , thus supporting the idea that this receptor can also promote proangiogenic mechanisms . moreover , in adipocytes , the 3ar is implicated in metabolic regulation [ fatty acid ( fa ) oxidation , lipolysis , and thermogenesis ] , that can also be a crucial mechanism to rescue the heart from failure . importantly , this mechanism is particularly important in several pathological conditions that affect cardiac function , such as ischemia and pressure overload . in fact , in these conditions , the heart , in the presence of limited oxygen supply , suppresses glucose and fa oxidation with a shift in cardiac substrate metabolism from fa oxidation to glycolysis . glycolysis without a concomitant increase in glucose oxidation results in an accumulation of different harmful catabolites such as lactate and protons that are the cause of intracellular acidosis and na and ca overload . these effects strongly reduce the capacity of the heart to provide sufficient energy for contractile work because more energy is spent to restore ion homeostasis and lead to an increase in lipid accumulation within the cardiac cells with consequent lipotoxicity . in this context , as recently demonstrated in adipocytes , nebivolol , through 3ar activation , is able to improve adipocyte metabolism . therefore , it is plausible that long - term 3ar stimulation , through selective agonists and/or -blockers ( ie , nebivolol ) , can be used as a novel therapeutical approach also to improve metabolism within the failing myocardium . currently , 2 clinical trials , the beta 3 agonist treatment in heart failure ( beat - hf ; clinicaltrials.gov : nct01876433 ) , or in the prevention of hf development , the assessment of efficacy of mirabegron , a new beta 3-adrenergic receptor in the prevention of heart failure ( beta3_lvh ; clinicaltrials.gov : nct02599480 ) , are trying to evaluate the effects of 3ar agonism on hf progression and development . in this context , patients will be treated with the selective 3ar agonist , mirabegron ( also known as ym-178 ) , a drug already approved in the united states , japan , and europe , for the treatment of overactive bladder . this drug , through the activation of 3ar , exerts a myorelaxant effect in the detrusor muscle thus improving the bladder filling . anyway , in the meantime that these trials will give us the proof - of - concept of the beneficial role of 3ars in human hf , the most important clinical evidence that we currently have regarding the role of this receptor remains the proved efficacy of -blockers . the cardioprotective effect of ar - blockade has been largely attributed , for decades , to antagonism of cardiac 1- and 2ars and a resulting heart rate reduction . however , as discussed above in this review , there is emerging evidence that this class of drugs may also act through 3ar signaling pathway activation which is not blocked by the drugs currently used in clinical hf - therapy ( box 2 ) . however , the main question that still remains unanswered is why not all patients respond favorably to these agents . for these reasons , further elucidation on specific mechanisms of action of these drugs is extremely interesting . in particular , it will be important to evaluate how different -blockers can impact , in a positive or a negative manner , 3ar activity , and if this effect is a significant contributor of therapeutic mechanisms in hf . the 3ar is a novel and intriguing receptor , with multiple functions within the cardiovascular system . in this review , we have discussed how many disparities have been generated around the signaling and the function of this receptor . however , the emerging concept in the literature is that 3ar is mostly protective for the cardiovascular system and its agonism with selective ligands or activation during 1- and 2ar blockade could represent a future therapeutic strategy to prevent development of hf . anyway , as discussed above , the 3ar function in the heart is still poorly investigated , and for this reason , further investigations are required to clarify the causal mechanistic relationship between 3ar expression and cardiac dysfunction and protection . more importantly , since presently , only 1ar - blockers have been associated with improvement of the 3ar signaling pathway , it will be crucial to define the specific signaling pathways associated with -blocker dependent activation / inhibition of 3ars because it can help in personalizing anti - hf therapy .

abstract : cardiac diseases , such as heart failure , remain leading causes of morbidity and mortality worldwide , with myocardial infarction as the most common etiology . hf is characterized by -adrenergic receptor ( ar ) dysregulation that is primarily due to the upregulation of g protein coupled receptor kinases that leads to overdesensitization of 1 and 2ars , and this clinically manifests as a loss of inotropic reserve . interestingly , the minor ar isoform , the 3ar , found in the heart , lacks g protein coupled receptor kinases recognition sites , and is not subject to desensitization , and as a consequence of this , in human failing myocardium , the levels of this receptor remain unchanged or are even increased . in different preclinical studies , it has been shown that 3ars can activate different signaling pathways that can protect the heart . the clinical relevance of this is also supported by the effects of -blockers which are well known for their proangiogenic and cardioprotective effects , and data are emerging showing that these are mediated , at least in part , by enhancement of 3ar activity . in this regard , targeting of 3ars could represent a novel potential strategy to improve cardiac metabolism , function , and remodeling .

INTRODUCTION The -Blockers and Clinical Perspectives of CONCLUSIONS

g protein coupled receptors ( gpcrs ) are nodal regulators of mammalian cell physiology because they transduce cell signals from diverse ligands such as neurohormones , sensory stimuli , and ions through heterotrimeric g proteins . in the heart , they represent the major modulators of both function and morphology with -adrenergic receptors ( ars ) representing the heads of the line , and for this reason they are considered the most important molecular targets in the cardiovascular system . currently , 3 ar subtypes ( 1ar , 2ar , and 3ar ) have been identified in the myocardium , with 1 and 2ars the most expressed and studied . however , since its discovery in 1989 , it soon seemed clear that 3ars , the isoform with minor expression , can influence cardiovascular physiology . the 3ar has 7 transmembrane domains ( 7-tmds ) with an extracellular n - terminal that is glycosylated , and an intracellular c - terminal domain . further , the cys361 residue in the fourth intracellular domain is palmitoylated , a feature that has been shown to be associated with g protein - coupling and adenylyl cyclase stimulation following agonist stimulation of the receptor ( fig . interestingly , when the 3ar protein sequence is compared with other ar ( 1 and 2ar ) isoforms , it is still possible to observe a high level of homology in the 7-tmd sequence , but a significant divergence is present both in the third intracellular loop and in the c - terminal domain ( fig . in this regard , the c - terminus of both 1 and 2ars is rich in serine and threonine residues , and is subjected to gpcr kinase ( grk)-mediated regulation through phosphorylation . of note , the 3ar lacks all of these sites , and is more resistant to agonist - induced desensitization / downregulation . indicated with arrows are the asparagine ( n ) residues , in the exd1 , that are sites of n - glycosylation ; tryptophan ( w ) in position 64 that is the location of 3ar - polymorphism ( trp64arg ) and the cysteine ( c ) in position 361 that is a site subjected to palmitoylation . this is due , at least in part , to the fact that the role of the cardiac 3ar has not been studied with the same intensity as the 1- and 2ars . in fact , it is known that in the mouse , the gene encoding for the 3ar undergoes alternative splicing and gives rise to 3aar and 3bar variants . by contrast , in humans , although some reports have proposed that 3ar can activate gs signaling in cho / k1 cells and in adipocytes , it is a general assumption that , at least in ventricular myocardium , 3ars are mainly coupled with gi proteins . however , because 3ars are also coupled with gi , they can act as a brake to prevent 1 and 2ars overactivation , and this has been proposed as a mechanism in the heart ( fig . moreover , in the heart , the stimulation of 3ars leads to increased endothelial nitric oxide ( no ) synthase ( enos ) or neuronal ( nnos ) activation , giving rise to no generation and activation of soluble guanylate cyclase to produce cgmp and cgmp - dependent protein kinase g ( pkg ) activation ( fig . importantly , the 3ar / no - cgmp / pkg signaling axis seems to be a robust cardioprotective mechanism that can be beneficial in failing myocardium . pka has multiple roles in cardiomyocytes and is able to induce the phosphorylation of several key factors involved in the regulation of contractility , such as cardiac troponin i ( ctni ) and phospholamban ( pln ) . obesity , susceptibility todiabetesinsulin resistancehyperuricemia obesity , susceptibility to accumulating evidence has revealed that the 3ar , present in endothelium and myocardium , may have specific beneficial effects in the cardiovascular system including cardioprotection . this becomes critically crucial in cardiac diseases such as hf , a syndrome characterized by decreased cardiac output , caused by deficits in contractility and/or relaxation . however , chronic exposure of the heart to high levels of catecholamines can lead to further pathologic changes in the heart that can induce a progressive deterioration of cardiac function and structure . in this regard , grk2 , the principal grk involved in ar regulation within the cardiomyocytes , phosphorylates the receptors attenuating their increased responsiveness . this process , called desensitization , at early stage represents a protective mechanism , but in chronic stage can cause 1 and 2ars dysregulation and signaling abnormalities ( eg , downregulation and overdesensitization ) and promote the progression of the disease . importantly , as discussed above , 3ars lack grk recognition sites and are not subject to desensitization and downregulation , and in fact , their levels within human failing myocardium remain unchanged or become upregulated . enhancement of 3ars could represent either a protective mechanism against the detrimental effects of chronic ar stimulation or a detrimental mechanism that may lead to further deterioration of hf . in this regard , the role of this receptor in the heart has been debated for years , and some reports have suggested that due to its cardiodepressant effect , sustained activation of 3ars in hf could contribute to impaired cardiac function . consistently , the antagonism of this receptor has been proposed as a potential strategy against hf development . however , by contrast with this hypothesis , it has been demonstrated that in the failing myocardium , 3ars are able to inhibit , through activation of na - k pump , the deleterious accumulation of na in cardiac myocytes thus blocking camp generation , and consequently , reducing the activation of the camp - downstream oxidative pathways . in line with the notion that 3ar activation in failing myocardium is not detrimental , studies strongly support the idea that overexpression or persistent activation of 3ar is cardioprotective and can attenuate pathological lv hypertrophy induced by continuous infusion of isoproterenol and angiotensin ii , or by transaortic constriction , in mice . finally , it is well established that the role of exercise in cardioprotection , and 3ars , has been also shown to be a mediator of this effect , especially in a setting of cardiac i / r injury . of note , during exercise , it seems that gs is the key mediator of 3ar - induced protection that leads to the pka / akt / enos activation , thus suggesting that , in some conditions , the 3ar , similarly to the cardioprotective 2ar , is coupled with both gs and gi in cardiomyocytes ( fig . the clinical relevance of this is further supported by the successful effects obtained with -blocker treatment in patients with hf as they can block the noxious effects of catecholamines and prevent further 1 and 2ars downregulation . in 2007 in particular , in an hf rat model induced by transaortic constriction , it has been shown that , although metoprolol ( a selective 1-blocker ) treatment did not affect the expression levels of 3ar which was increased after hf , the use of carvedilol ( a nonselective -blocker ) resulted in a robust 3ar downregulation . interestingly , this report showed also that after 4 weeks postmyocardial infarction , there was a significant reduction of cardiac 3ar levels , and that nebivolol was able to restore the expression of this receptor . as described above , the 3ar represents an emerging attractive target for pharmacological modulation in the injured heart that , for its compensatory effects , prevents the effects of excessive catecholamines stimulation on the heart . a particular role for no has been associated with the enhancement of endothelial cell proliferation , and within failing myocardium it can lead to a beneficial effect on cardiac function and remodeling . moreover , in adipocytes , the 3ar is implicated in metabolic regulation [ fatty acid ( fa ) oxidation , lipolysis , and thermogenesis ] , that can also be a crucial mechanism to rescue the heart from failure . in fact , in these conditions , the heart , in the presence of limited oxygen supply , suppresses glucose and fa oxidation with a shift in cardiac substrate metabolism from fa oxidation to glycolysis . therefore , it is plausible that long - term 3ar stimulation , through selective agonists and/or -blockers ( ie , nebivolol ) , can be used as a novel therapeutical approach also to improve metabolism within the failing myocardium . currently , 2 clinical trials , the beta 3 agonist treatment in heart failure ( beat - hf ; clinicaltrials.gov : nct01876433 ) , or in the prevention of hf development , the assessment of efficacy of mirabegron , a new beta 3-adrenergic receptor in the prevention of heart failure ( beta3_lvh ; clinicaltrials.gov : nct02599480 ) , are trying to evaluate the effects of 3ar agonism on hf progression and development . anyway , in the meantime that these trials will give us the proof - of - concept of the beneficial role of 3ars in human hf , the most important clinical evidence that we currently have regarding the role of this receptor remains the proved efficacy of -blockers . however , as discussed above in this review , there is emerging evidence that this class of drugs may also act through 3ar signaling pathway activation which is not blocked by the drugs currently used in clinical hf - therapy ( box 2 ) . in particular , it will be important to evaluate how different -blockers can impact , in a positive or a negative manner , 3ar activity , and if this effect is a significant contributor of therapeutic mechanisms in hf . further , the cys361 residue in the fourth intracellular domain is palmitoylated , a feature that has been shown to be associated with g protein - coupling and adenylyl cyclase stimulation following agonist stimulation of the receptor ( fig . interestingly , when the 3ar protein sequence is compared with other ar ( 1 and 2ar ) isoforms , it is still possible to observe a high level of homology in the 7-tmd sequence , but a significant divergence is present both in the third intracellular loop and in the c - terminal domain ( fig . in this regard , the c - terminus of both 1 and 2ars is rich in serine and threonine residues , and is subjected to gpcr kinase ( grk)-mediated regulation through phosphorylation . of note , the 3ar lacks all of these sites , and is more resistant to agonist - induced desensitization / downregulation . indicated with arrows are the asparagine ( n ) residues , in the exd1 , that are sites of n - glycosylation ; tryptophan ( w ) in position 64 that is the location of 3ar - polymorphism ( trp64arg ) and the cysteine ( c ) in position 361 that is a site subjected to palmitoylation . this is due , at least in part , to the fact that the role of the cardiac 3ar has not been studied with the same intensity as the 1- and 2ars . in fact , it is known that in the mouse , the gene encoding for the 3ar undergoes alternative splicing and gives rise to 3aar and 3bar variants . by contrast , in humans , although some reports have proposed that 3ar can activate gs signaling in cho / k1 cells and in adipocytes , it is a general assumption that , at least in ventricular myocardium , 3ars are mainly coupled with gi proteins . however , because 3ars are also coupled with gi , they can act as a brake to prevent 1 and 2ars overactivation , and this has been proposed as a mechanism in the heart ( fig . moreover , in the heart , the stimulation of 3ars leads to increased endothelial nitric oxide ( no ) synthase ( enos ) or neuronal ( nnos ) activation , giving rise to no generation and activation of soluble guanylate cyclase to produce cgmp and cgmp - dependent protein kinase g ( pkg ) activation ( fig . importantly , the 3ar / no - cgmp / pkg signaling axis seems to be a robust cardioprotective mechanism that can be beneficial in failing myocardium . pka has multiple roles in cardiomyocytes and is able to induce the phosphorylation of several key factors involved in the regulation of contractility , such as cardiac troponin i ( ctni ) and phospholamban ( pln ) . accumulating evidence has revealed that the 3ar , present in endothelium and myocardium , may have specific beneficial effects in the cardiovascular system including cardioprotection . however , chronic exposure of the heart to high levels of catecholamines can lead to further pathologic changes in the heart that can induce a progressive deterioration of cardiac function and structure . in this regard , grk2 , the principal grk involved in ar regulation within the cardiomyocytes , phosphorylates the receptors attenuating their increased responsiveness . this process , called desensitization , at early stage represents a protective mechanism , but in chronic stage can cause 1 and 2ars dysregulation and signaling abnormalities ( eg , downregulation and overdesensitization ) and promote the progression of the disease . importantly , as discussed above , 3ars lack grk recognition sites and are not subject to desensitization and downregulation , and in fact , their levels within human failing myocardium remain unchanged or become upregulated . enhancement of 3ars could represent either a protective mechanism against the detrimental effects of chronic ar stimulation or a detrimental mechanism that may lead to further deterioration of hf . in this regard , the role of this receptor in the heart has been debated for years , and some reports have suggested that due to its cardiodepressant effect , sustained activation of 3ars in hf could contribute to impaired cardiac function . consistently , the antagonism of this receptor has been proposed as a potential strategy against hf development . however , by contrast with this hypothesis , it has been demonstrated that in the failing myocardium , 3ars are able to inhibit , through activation of na - k pump , the deleterious accumulation of na in cardiac myocytes thus blocking camp generation , and consequently , reducing the activation of the camp - downstream oxidative pathways . in line with the notion that 3ar activation in failing myocardium is not detrimental , studies strongly support the idea that overexpression or persistent activation of 3ar is cardioprotective and can attenuate pathological lv hypertrophy induced by continuous infusion of isoproterenol and angiotensin ii , or by transaortic constriction , in mice . finally , it is well established that the role of exercise in cardioprotection , and 3ars , has been also shown to be a mediator of this effect , especially in a setting of cardiac i / r injury . of note , during exercise , it seems that gs is the key mediator of 3ar - induced protection that leads to the pka / akt / enos activation , thus suggesting that , in some conditions , the 3ar , similarly to the cardioprotective 2ar , is coupled with both gs and gi in cardiomyocytes ( fig . the clinical relevance of this is further supported by the successful effects obtained with -blocker treatment in patients with hf as they can block the noxious effects of catecholamines and prevent further 1 and 2ars downregulation . in particular , in an hf rat model induced by transaortic constriction , it has been shown that , although metoprolol ( a selective 1-blocker ) treatment did not affect the expression levels of 3ar which was increased after hf , the use of carvedilol ( a nonselective -blocker ) resulted in a robust 3ar downregulation . interestingly , this report showed also that after 4 weeks postmyocardial infarction , there was a significant reduction of cardiac 3ar levels , and that nebivolol was able to restore the expression of this receptor . as described above , the 3ar represents an emerging attractive target for pharmacological modulation in the injured heart that , for its compensatory effects , prevents the effects of excessive catecholamines stimulation on the heart . a particular role for no has been associated with the enhancement of endothelial cell proliferation , and within failing myocardium it can lead to a beneficial effect on cardiac function and remodeling . moreover , in adipocytes , the 3ar is implicated in metabolic regulation [ fatty acid ( fa ) oxidation , lipolysis , and thermogenesis ] , that can also be a crucial mechanism to rescue the heart from failure . in fact , in these conditions , the heart , in the presence of limited oxygen supply , suppresses glucose and fa oxidation with a shift in cardiac substrate metabolism from fa oxidation to glycolysis . therefore , it is plausible that long - term 3ar stimulation , through selective agonists and/or -blockers ( ie , nebivolol ) , can be used as a novel therapeutical approach also to improve metabolism within the failing myocardium . currently , 2 clinical trials , the beta 3 agonist treatment in heart failure ( beat - hf ; clinicaltrials.gov : nct01876433 ) , or in the prevention of hf development , the assessment of efficacy of mirabegron , a new beta 3-adrenergic receptor in the prevention of heart failure ( beta3_lvh ; clinicaltrials.gov : nct02599480 ) , are trying to evaluate the effects of 3ar agonism on hf progression and development . in this context , patients will be treated with the selective 3ar agonist , mirabegron ( also known as ym-178 ) , a drug already approved in the united states , japan , and europe , for the treatment of overactive bladder . anyway , in the meantime that these trials will give us the proof - of - concept of the beneficial role of 3ars in human hf , the most important clinical evidence that we currently have regarding the role of this receptor remains the proved efficacy of -blockers . however , as discussed above in this review , there is emerging evidence that this class of drugs may also act through 3ar signaling pathway activation which is not blocked by the drugs currently used in clinical hf - therapy ( box 2 ) . in particular , it will be important to evaluate how different -blockers can impact , in a positive or a negative manner , 3ar activity , and if this effect is a significant contributor of therapeutic mechanisms in hf . however , the emerging concept in the literature is that 3ar is mostly protective for the cardiovascular system and its agonism with selective ligands or activation during 1- and 2ar blockade could represent a future therapeutic strategy to prevent development of hf . anyway , as discussed above , the 3ar function in the heart is still poorly investigated , and for this reason , further investigations are required to clarify the causal mechanistic relationship between 3ar expression and cardiac dysfunction and protection . more importantly , since presently , only 1ar - blockers have been associated with improvement of the 3ar signaling pathway , it will be crucial to define the specific signaling pathways associated with -blocker dependent activation / inhibition of 3ars because it can help in personalizing anti - hf therapy .

we quantified the copy - number statistics of endogenous mrna using single - molecule fluorescence in situ hybridization ( smfish ) , following the method of , which we adapted for counting mrna in e. coli at single - transcript resolution ( see online methods ) . briefly , a set of ~5070 fluorescently labeled oligonucleotide probes , each 20 bases in length , was designed against the transcript - of - interest . probes were hybridized to fixed cells and imaged using epifluorescence microscopy . to estimate the number of mrna molecules from the gene - of - interest in a given cell , the total intensity of fluorescent foci in the cell was measured , yielding an estimate of the number of bound probes , in turn indicating the number of target mrna molecules . this approach follows the one previously used in live - cell studies of mrna kinetics using the ms2 system . figure 2 demonstrates the dynamic range and accuracy of measuring mrna copy - numbers using smfish , for the case of the plac promoter . mrna levels covering ~3 orders of magnitude ( ~0.160 molecules / cell ) could be measured . the smfish - based estimation of mrna numbers was in excellent agreement with measurements using quantitative pcr ( qpcr ) as well as with data from the literature . a similar comparison made in four other promoters yielded good agreement between smfish and other assays ( figures s2s5 ) . the smfish - based measurements allowed us to obtain the copy - number statistics of mrna transcripts from a gene - of - interest under a given growth condition . the mrna histograms were well described by a negative binomial distribution ( figure 2c ) , consistent with the prediction of the two - state model . in particular , the smfish data allowed us to accurately measure both the mean ( n ) and variance ( ) of mrna copy - number , and therefore calculate the burstiness parameter b = /n characterizing the transcriptional time - series . we used smfish to quantify mrna statistics from 20 promoters : plac , pgaletkm , pmarii , rrnbp1 , pbiobfcd , bacteriophage promoter pr , and 13 variants of the bacteriophage promoter prm ( see tables s1s3 ) . in cases where promoter activity is regulated by growth conditions ( e.g. the presence of a specific sugar or amino acid ) , a range of growth conditions was used so that the full range of mrna levels could be achieved ( see supplementary note ) . this ensemble of promoters allowed us to scan a range of expression levels ( ~0.0160 mrna / cell ) , different molecular mechanisms of transcription regulation ( activation , repression and combinations thereof ) and topologies of gene networks controlling gene activity ( such as the presence or absence of feedback ) ; see table s3 . all of these factors can conceivably affect the observed fluctuations in gene activity . in total , > 150 independent experiments were made , each one yielding the distribution of mrna copy - number from a given gene at a given stimulus level . to characterize the transcriptional time - series in the complete data set , we plot ( figures 3a and b ) the burstiness b and the noise from each experiment , as a function of the mean expression level n at that condition . the expression levels were corrected for the differences in gene copy number ( see figures s6 and s7 and supplementary note ) and mrna lifetime ( see table s4 , figures s8 and s9 and supplementary note ) , so that the characteristics of mrna production from a single - copy promoter can be examined . we first note that the cell - to - cell variability in mrna numbers is dominated by the inherent fluctuations of the two - state process ( intrinsic noise ) rather than by cell - to - cell difference in parameter values ( extrinsic noise ) . this is suggested by the following observations : ( i ) the noise decreases monotonically with n ( figure 3b ) , which is the typical behavior of intrinsic , but not of extrinsic , noise . ( ii ) in the limit of low n , 2n1 ( figure 3a ) , as expected for the intrinsic noise of a poisson process . that transcription is poissonian at very low expression level has been shown previously . ( iii ) in the limit of high n , decreases sharply rather than approach a plateau ( figure 3b ) . the observed dominance of intrinsic noise in mrna number fluctuations is consistent with previous observations , that extrinsic noise is an important factor at the level of the protein species , but not mrna . the most striking feature in figures 3a and 3b is that b and exhibit gene - independent behavior ; that is , the values from different genes and growth conditions show a clear trend , with a dependence on the expression level n alone . thus , the properties of the time - series seem to depend primarily on the mean mrna level , not on the specific gene or stimulus ( this observation is made more quantitative below ) . the gene - independent behavior immediately suggests that the rate parameters in the two - state picture are not determined by the details of molecular regulation of an individual promoter ( such as the binding and unbinding kinetics of a specific transcription factor ) or the topology of the individual gene network ( e.g. the presence or absence of feedback ) . instead , gene on / off switching is dominated by a process that acts in a similar manner on different genes , possibly exerting its influence at a genome - wide level ( see discussion below ) . thus , all genes expressed at a given level exhibit a similar transcriptional time series . note that this similarity in time - series characteristics does not necessarily mean that the actual activity of different genes is coordinated in time , i.e. that genes turn on and off in unison . it is interesting to note , however , that multiple copies of the same gene ( present when the bacterial chromosome replicates ) exhibit a positive , non - zero covariance ( figure s10 ) , suggesting that their temporal activity may indeed be correlated . as we discuss below , the observed universality in transcription burstiness readily explains previous observations made at the protein level ( and similar findings in yeast ) . we next used the experimental data of b(n ) and (n ) to ask what property of the transcriptional time series is modulated as gene expression level is varied . when comparing the experimental plots in figures 3a and 3b to the theoretical ones in figures 1d and 1e , we note that the observed mrna statistics is consistent with the assumption that expression level is changed by varying the rate at which the gene switches back to the off state ( off - rate koff ) , or in other words the duration of transcription bursts . specifically , note that b(n ) starts with a poisson - like behavior ( b ~ 1 ) and then increases as a sub - linear function of n. the observation can be made quantitative by fitting the experimental data for b(n ) and (n ) to the analytical expressions for the two - state model , under the scenario of varying koff ( see online methods ) . as seen in figure 3a , a good fit is obtained ( r = 0.81 ) . for comparison , trying to fit the observed data with the two alternative scenarios , modulating the gene on - rate kon or the transcription rate ktx , yielded inferior fits ( r = 7.9 10 and 0.58 , respectively ) . moreover , the scenario of varying koff yields a fit superior to the alternatives when compared on a promoter - by - promoter basis ( figures s11 and s12 ) . as a control , trying to fit a simulated collection of promoters with randomly selected kinetic parameters using the koff - modulation description also yielded a poor fit ( r = 0.085 , figure s13 ) . as an additional test for the validity of our parameter estimation , we performed detailed stochastic simulations of mrna kinetics and verified that the theoretical and experimental copy - number histograms are in agreement , beyond the mere values of n and ( figure s14 ) . the theoretical fit allows us to make the observation of gene - independence more quantitative : when comparing the data from individual promoters to the universal fit , we find that 6 of 7 data sets exhibit a correlation coefficient above 0.85 between data and theory ( figure s15 ) . the average deviation of a single - promoter data from the universal fit is ~33% ( figure s15 ) . fitting the experimental data to the scenario of koff - modulation allowed us to estimate the values of the three kinetic parameters governing mrna production : kon ( the rate of switching to the on state , which determines the frequency of bursts ) and ktx ( the rate of producing mrna while the gene is on)both of which are approximately constant for different genes and expression levels and koff ( the rate of switching back to the off state , which determines the duration of bursts ) which changes over more than 3 orders of magnitude when expression level is varied ( figure 3c ) . we note that , of these three parameters , the only one which has been estimated in the past is ktx , which corresponds to the maximal transcription initiation rate possible ( when a gene is constantly on ) . the value obtained from our single - cell measurements ( ktx = 0.23 0.11 s ) is in good agreement with values from the literature . we also note that kon and ktx exhibit a dependence on the bacterial growth rate ( figure s12 ) . the examination of mrna number statistics , though strongly indicating that koff alone is varied to control expression level , is limited by the fact that the process of transcription was not directly observed . to overcome this limitation and gain further support for the observation of koff modulation , we quantified the kinetics of mrna production from one promoter , plac / ara , in individual living cells . we used the ms2-gfp system , previously used to demonstrate transcriptional bursting in e. coli . briefly , cells were grown under the microscope in the presence of different levels of the inducers , iptg and arabinose . mrna production was followed in individual cells by measuring the intensity of fluorescent foci created when ms2-gfp binds to its rna recognition sequence . as expected , mrna kinetics was found to consist of periods of activity , where a random number of transcripts are produced , separated by periods of inactivity . measuring the mean durations of off and on periods , as well as the amount of mrna produced within each on period , allowed us in turn to estimate kon , koff , and ktx at a given gene activity level . as seen in figure 3d , the behavior of these kinetic parameters is consistent with the observations above : changing the level of mrna n is achieved by varying koff , while kon and ktx are kept approximately constant . we have thus seen that the discrete time - series of gene activity exhibits universal properties ; that is , the same kinetic parameters are common to different genes and environmental conditions . it is then natural to ask : can the specific choice of kinetic parameters optimize some function of the living cell and therefore be subject to evolutionary selection ? to address this question , we followed the approach of and considered the way gene activity is used by the cell to represent information about its environment . for example , the activity of the lactose promoter can be thought of as telling the cell how much lactose is present in its environment . we quantified the efficiency of information representation by the cell using shannon s mutual information , i(p , c ) , a function that measures how much information is transmitted to the output ( protein level , p ) about changes in the input stimulus , c ( for example , sugar concentration ) . in calculating i(p , c ) , we used the experimentally measured dose - response of the promoter , i.e. mean mrna number n as a function of stimulus c. the downstream production of protein was stochastically modeled using known parameters ( see supplementary note ) . importantly , a calculation using three different promoters studied in this work ( plac , pmarii , and pbiobfcd ) yielded almost identical results ( figure s16 and s17 ) . the mutual information i depends critically on the way the variance of mrna copy numbers , , changes with the mean n ( the statistics of the protein species follows the same scaling relations , up to a calculable factor , see supplementary note ) . to examine how the mutual information varies as a function of time - series parameters , we wrote in the phenomenological form /n = 1 + n/ ( such that the deviation of the burstiness b from the poisson case goes like the mean n to the power ) . by varying the parameters and , this functional form allowed us to approximate the behavior exhibited by the transcriptional time - series under the different modulation schemes ( see figure 1d above ) and under a broad range of kinetic parameters ; specifically , this form captures the (n ) behavior seen in our experiments ( figure 4a ) . we next calculated the mutual information ( maximized over possible inputs , see online methods ) as a function of the parameters ( , ) ( figure 4b ) , thus exploring the efficiency of information representation over the space of possible time - series characteristics . we found that the parameters describing the actual transcriptional time - series ( = 3.5 3.2 , = 0.64 0.06 ) are close to optimal they lie on a ridge in the map of i(, ) ( figure 4b ) . when plotting a histogram of i values obtained from a broad range of kinetic parameters ( figure 4c ) , one sees that the maximal mutual information of the actual time series ( i~2.5 bits , or discrimination of > 5 input levels ) , is significantly higher than the mean performance obtained by randomly choosing the time - series parameters ( ~0.68 bits ) . in other words , the specific parameters of the transcriptional time series , which are observed in experiment , are superior to most other possible parameter sets , in the sense of allowing the cell to best represent information about its environment through the discrete activity of its genes . we quantified the copy - number statistics of endogenous mrna using single - molecule fluorescence in situ hybridization ( smfish ) , following the method of , which we adapted for counting mrna in e. coli at single - transcript resolution ( see online methods ) . briefly , a set of ~5070 fluorescently labeled oligonucleotide probes , each 20 bases in length , was designed against the transcript - of - interest . probes were hybridized to fixed cells and imaged using epifluorescence microscopy . to estimate the number of mrna molecules from the gene - of - interest in a given cell , the total intensity of fluorescent foci in the cell was measured , yielding an estimate of the number of bound probes , in turn indicating the number of target mrna molecules . this approach follows the one previously used in live - cell studies of mrna kinetics using the ms2 system . figure 2 demonstrates the dynamic range and accuracy of measuring mrna copy - numbers using smfish , for the case of the plac promoter . mrna levels covering ~3 orders of magnitude ( ~0.160 molecules / cell ) could be measured . the smfish - based estimation of mrna numbers was in excellent agreement with measurements using quantitative pcr ( qpcr ) as well as with data from the literature . a similar comparison made in four other promoters yielded good agreement between smfish and other assays ( figures s2s5 ) . the smfish - based measurements allowed us to obtain the copy - number statistics of mrna transcripts from a gene - of - interest under a given growth condition . the mrna histograms were well described by a negative binomial distribution ( figure 2c ) , consistent with the prediction of the two - state model . in particular , the smfish data allowed us to accurately measure both the mean ( n ) and variance ( ) of mrna copy - number , and therefore calculate the burstiness parameter b = /n characterizing the transcriptional time - series . we used smfish to quantify mrna statistics from 20 promoters : plac , pgaletkm , pmarii , rrnbp1 , pbiobfcd , bacteriophage promoter pr , and 13 variants of the bacteriophage promoter prm ( see tables s1s3 ) . in cases where promoter activity is regulated by growth conditions ( e.g. the presence of a specific sugar or amino acid ) , a range of growth conditions was used so that the full range of mrna levels could be achieved ( see supplementary note ) . this ensemble of promoters allowed us to scan a range of expression levels ( ~0.0160 mrna / cell ) , different molecular mechanisms of transcription regulation ( activation , repression and combinations thereof ) and topologies of gene networks controlling gene activity ( such as the presence or absence of feedback ) ; see table s3 . all of these factors can conceivably affect the observed fluctuations in gene activity . in total , > 150 independent experiments were made , each one yielding the distribution of mrna copy - number from a given gene at a given stimulus level . to characterize the transcriptional time - series in the complete data set , we plot ( figures 3a and b ) the burstiness b and the noise from each experiment , as a function of the mean expression level n at that condition . the expression levels were corrected for the differences in gene copy number ( see figures s6 and s7 and supplementary note ) and mrna lifetime ( see table s4 , figures s8 and s9 and supplementary note ) , so that the characteristics of mrna production from a single - copy promoter can be examined . we first note that the cell - to - cell variability in mrna numbers is dominated by the inherent fluctuations of the two - state process ( intrinsic noise ) rather than by cell - to - cell difference in parameter values ( extrinsic noise ) . this is suggested by the following observations : ( i ) the noise decreases monotonically with n ( figure 3b ) , which is the typical behavior of intrinsic , but not of extrinsic , noise . ( ii ) in the limit of low n , 2n1 ( figure 3a ) , as expected for the intrinsic noise of a poisson process . that transcription is poissonian at very low expression level has been shown previously . ( iii ) in the limit of high n , decreases sharply rather than approach a plateau ( figure 3b ) . the observed dominance of intrinsic noise in mrna number fluctuations is consistent with previous observations , that extrinsic noise is an important factor at the level of the protein species , but not mrna . the most striking feature in figures 3a and 3b is that b and exhibit gene - independent behavior ; that is , the values from different genes and growth conditions show a clear trend , with a dependence on the expression level n alone . thus , the properties of the time - series seem to depend primarily on the mean mrna level , not on the specific gene or stimulus ( this observation is made more quantitative below ) . the gene - independent behavior immediately suggests that the rate parameters in the two - state picture are not determined by the details of molecular regulation of an individual promoter ( such as the binding and unbinding kinetics of a specific transcription factor ) or the topology of the individual gene network ( e.g. the presence or absence of feedback ) . instead , gene on / off switching is dominated by a process that acts in a similar manner on different genes , possibly exerting its influence at a genome - wide level ( see discussion below ) . thus , all genes expressed at a given level exhibit a similar transcriptional time series . note that this similarity in time - series characteristics does not necessarily mean that the actual activity of different genes is coordinated in time , i.e. that genes turn on and off in unison . it is interesting to note , however , that multiple copies of the same gene ( present when the bacterial chromosome replicates ) exhibit a positive , non - zero covariance ( figure s10 ) , suggesting that their temporal activity may indeed be correlated . as we discuss below , the observed universality in transcription burstiness readily explains previous observations made at the protein level ( and similar findings in yeast ) . we next used the experimental data of b(n ) and (n ) to ask what property of the transcriptional time series is modulated as gene expression level is varied . when comparing the experimental plots in figures 3a and 3b to the theoretical ones in figures 1d and 1e , we note that the observed mrna statistics is consistent with the assumption that expression level is changed by varying the rate at which the gene switches back to the off state ( off - rate koff ) , or in other words the duration of transcription bursts . specifically , note that b(n ) starts with a poisson - like behavior ( b ~ 1 ) and then increases as a sub - linear function of n. the observation can be made quantitative by fitting the experimental data for b(n ) and (n ) to the analytical expressions for the two - state model , under the scenario of varying koff ( see online methods ) . as seen in figure 3a , a good fit is obtained ( r = 0.81 ) . for comparison , trying to fit the observed data with the two alternative scenarios , modulating the gene on - rate kon or the transcription rate ktx , yielded inferior fits ( r = 7.9 10 and 0.58 , respectively ) . moreover , the scenario of varying koff yields a fit superior to the alternatives when compared on a promoter - by - promoter basis ( figures s11 and s12 ) . as a control , trying to fit a simulated collection of promoters with randomly selected kinetic parameters using the koff - modulation description also yielded a poor fit ( r = 0.085 , figure s13 ) . as an additional test for the validity of our parameter estimation , we performed detailed stochastic simulations of mrna kinetics and verified that the theoretical and experimental copy - number histograms are in agreement , beyond the mere values of n and ( figure s14 ) . the theoretical fit allows us to make the observation of gene - independence more quantitative : when comparing the data from individual promoters to the universal fit , we find that 6 of 7 data sets exhibit a correlation coefficient above 0.85 between data and theory ( figure s15 ) . the average deviation of a single - promoter data from the universal fit is ~33% ( figure s15 ) . fitting the experimental data to the scenario of koff - modulation allowed us to estimate the values of the three kinetic parameters governing mrna production : kon ( the rate of switching to the on state , which determines the frequency of bursts ) and ktx ( the rate of producing mrna while the gene is on)both of which are approximately constant for different genes and expression levels and koff ( the rate of switching back to the off state , which determines the duration of bursts ) which changes over more than 3 orders of magnitude when expression level is varied ( figure 3c ) . we note that , of these three parameters , the only one which has been estimated in the past is ktx , which corresponds to the maximal transcription initiation rate possible ( when a gene is constantly on ) . the value obtained from our single - cell measurements ( ktx = 0.23 0.11 s ) is in good agreement with values from the literature . we also note that kon and ktx exhibit a dependence on the bacterial growth rate ( figure s12 ) . the examination of mrna number statistics , though strongly indicating that koff alone is varied to control expression level , is limited by the fact that the process of transcription was not directly observed . to overcome this limitation and gain further support for the observation of koff modulation , we quantified the kinetics of mrna production from one promoter , plac / ara , in individual living cells . we used the ms2-gfp system , previously used to demonstrate transcriptional bursting in e. coli . briefly , cells were grown under the microscope in the presence of different levels of the inducers , iptg and arabinose . mrna production was followed in individual cells by measuring the intensity of fluorescent foci created when ms2-gfp binds to its rna recognition sequence . as expected , mrna kinetics was found to consist of periods of activity , where a random number of transcripts are produced , separated by periods of inactivity . measuring the mean durations of off and on periods , as well as the amount of mrna produced within each on period , allowed us in turn to estimate kon , koff , and ktx at a given gene activity level . as seen in figure 3d , the behavior of these kinetic parameters is consistent with the observations above : changing the level of mrna n is achieved by varying koff , while kon and ktx are kept approximately constant . we have thus seen that the discrete time - series of gene activity exhibits universal properties ; that is , the same kinetic parameters are common to different genes and environmental conditions . it is then natural to ask : can the specific choice of kinetic parameters optimize some function of the living cell and therefore be subject to evolutionary selection ? to address this question , we followed the approach of and considered the way gene activity is used by the cell to represent information about its environment . for example , the activity of the lactose promoter can be thought of as telling the cell how much lactose is present in its environment . we quantified the efficiency of information representation by the cell using shannon s mutual information , i(p , c ) , a function that measures how much information is transmitted to the output ( protein level , p ) about changes in the input stimulus , c ( for example , sugar concentration ) . in calculating i(p , c ) , we used the experimentally measured dose - response of the promoter , i.e. mean mrna number n as a function of stimulus c. the downstream production of protein was stochastically modeled using known parameters ( see supplementary note ) . importantly , a calculation using three different promoters studied in this work ( plac , pmarii , and pbiobfcd ) yielded almost identical results ( figure s16 and s17 ) . the mutual information i depends critically on the way the variance of mrna copy numbers , , changes with the mean n ( the statistics of the protein species follows the same scaling relations , up to a calculable factor , see supplementary note ) . to examine how the mutual information varies as a function of time - series parameters , we wrote in the phenomenological form /n = 1 + n/ ( such that the deviation of the burstiness b from the poisson case goes like the mean n to the power ) . by varying the parameters and , this functional form allowed us to approximate the behavior exhibited by the transcriptional time - series under the different modulation schemes ( see figure 1d above ) and under a broad range of kinetic parameters ; specifically , this form captures the (n ) behavior seen in our experiments ( figure 4a ) . we next calculated the mutual information ( maximized over possible inputs , see online methods ) as a function of the parameters ( , ) ( figure 4b ) , thus exploring the efficiency of information representation over the space of possible time - series characteristics . we found that the parameters describing the actual transcriptional time - series ( = 3.5 3.2 , = 0.64 0.06 ) are close to optimal they lie on a ridge in the map of i(, ) ( figure 4b ) . when plotting a histogram of i values obtained from a broad range of kinetic parameters ( figure 4c ) , one sees that the maximal mutual information of the actual time series ( i~2.5 bits , or discrimination of > 5 input levels ) , is significantly higher than the mean performance obtained by randomly choosing the time - series parameters ( ~0.68 bits ) . in other words , the specific parameters of the transcriptional time series , which are observed in experiment , are superior to most other possible parameter sets , in the sense of allowing the cell to best represent information about its environment through the discrete activity of its genes . multiple studies in recent years have demonstrated that gene activity is often bursty rather than poissonian , and can be described via a two - state model for mrna production . in this work we have extended and generalized these observations by describing how the transcriptional time - series in e. coli is modulated when gene expression level is varied . we found that promoter activity tends to be non - bursty at low expression levels ( at or below n ~ 1 molecules / cell ) ; the degree of burstiness , as characterized by the fano factor b = /n , then rises in a sub - linear manner with increasing gene activity . this behavior is consistent with varying of the gene off - rate as the means to change the expression level , while maintaining the gene on - rate and transcription rate constant . in other words , the duration of the transcription bursts is the main feature that changes as expression level is varied . importantly , this behavior is not gene- or input - specific ( although it can also be observed when examining a single gene , see figure s11 ) ; rather , it was observed in the complete ensemble of promoters and stimuli examined . we note that a more complex scenario , where multiple kinetic parameters are simultaneously varied , i s a lso consistent with the observed smfish data ( see supplementary note and figure s18 ) . however , such a scenario does not need to be invoked in order to explain the experimental data . the multi - parameter modulation scenario also appears inconsistent with the live - cell data ( figure 3d above ) . a number of past studies have characterized the noise level of multiple genes , using a library of fluorescent protein fusions . a study in yeast found that the squared coefficient of variation displayed a genome - wide trend of power - law dependence on mean expression level ( a similar trend was recently observed when examining different mutants of a single yeast promoter ) . moreover , by modeling the underlying kinetics , the authors in concluded that protein fluctuations were likely dominated by the mrna species , as assumed in our work . a recent genome - wide study in e. coli found that the fano factor increased monotonically with mean protein level . this observation is most easily explained by our findings of a gene - independent behavior of the transcriptional burst size ( figure 3a above ) . the authors in also performed measurement of mrna levels in single cells , which they analyzed using the assumption of poissonian kinetics . the measured values of mrna numbers per cell , as well as the range of expression levels , were significantly smaller than those observed in our study . in addition , the authors found no correlation between mrna and protein numbers from a given gene in individual cells . it is possible that the use of a single fluorescent probe per gene limited the accuracy of their measurement ( see e.g. figure s22 in ) and thus did not allow a quantitative characterization of cell - to - cell variability in mrna numbers . from an evolutionary point of view , we note that the expression level of a gene has been shown to be a phenotype subject to selection . more recently , a number of studies have suggested that , beyond the mean expression level , the degree of population heterogeneity ( noise ) in gene expression may also be subject to selection . here we estimated the mutual information between external stimulus and the transcriptional time - series and showed that the specific modulation scheme chosen by the cell is efficient in the sense of reliably representing , through the transcriptional time - series , the environment in which the cell resides . in quantifying this efficiency , we demonstrated how the properties of the transcriptional time - series itself , beyond merely the mean expression level , emerge as a meaningful phenotype subject to selection . we note that this new observation also extends and generalizes previous works , in which the burstiness of gene expression was suggested to affect the cellular phenotype . two important limitations of our work need to be mentioned : first , when describing gene activity we centered on the mrna species only , while leaving out the downstream production of proteins . in doing so we implicitly assumed that protein kinetics is enslaved by mrna kinetics to a sufficient degree such that the discrete , stochastic time - series of mrna production governs cell - to - cell heterogeneity . this assumption is supported by the observation that protein - number statistics closely reflect the properties of mrna statistics , as found here . second , by mainly using in situ hybridization to count mrna , we were able to obtain snapshots of cell populations but were naturally unable to follow the time - course of gene activity in individual cells ( with the exception of a single promoter ) . correlations in the gene - activity trajectories of individual cells have been shown to contain important information about the underlying gene regulatory network . extending the use of the ms2-based system to multiple promoters should allow the characterization of such temporal effects in the future . at this stage , there is no mechanistic , molecular - level understanding of what gives rise to the bursty behavior of gene activity in bacteria ; specifically , what the physiological nature of the gene on and off states is , and therefore also how the rates of switching between states can be varied in the individual cell or over the time course of evolution . the most common theoretical model used to explain two - state gene activity in bacteria involves the binding and unbinding of transcription factors at the promoter . however , our finding here , that the properties of the transcriptional time - series are gene - independent ( rather than gene - specific ) , suggest that the observed two - state kinetics involves gene - nonspecific features such as dna topology , rna polymerase dynamics or regulation by broad - target dna - binding proteins . interestingly , these types of mechanisms are reminiscent of those suggested to underlie non - poissonian transcription kinetics in eukaryotes , where burstiness is broadly ascribed to chromatin modifications . future studies will have to reveal whether the fact that transcription burstiness appears in both kingdoms reflects a similarity in underlying mechanisms , or instead results form the selection of an advantageous phenotype in different systems .

gene activity is described by the time - series of discrete , stochastic mrna production events . this transcriptional time - series exhibits intermittent , bursty behavior . one consequence of this temporal intricacy is that gene expression can be tuned by varying different features of the time - series . what schemes for varying the transcriptional time - series are observed in the cell ? are the observed properties of these time - series optimized for cellular function ? to address these questions , we characterize mrna copy - number statistics at single - molecule resolution from multiple escherichia coli promoters . we find that the degree of burstiness depends only on the gene expression level , while being independent of the details of gene regulation . the observed behavior is explained by the underlying variation in the duration of bursting events . using information theory , we find that the properties of the transcriptional time series allow the cell to efficiently map the extracellular concentration of inducer molecules to intracellular levels of mrna and proteins .

RESULTS Quantifying mRNA statistics at single-molecule resolution Burstiness exhibits universal behavior across different genes and conditions Expression level is varied by modulating the gene off rate The transcriptional time-series optimizes information representation by the cell DISCUSSION Supplementary Material

we quantified the copy - number statistics of endogenous mrna using single - molecule fluorescence in situ hybridization ( smfish ) , following the method of , which we adapted for counting mrna in e. coli at single - transcript resolution ( see online methods ) . to estimate the number of mrna molecules from the gene - of - interest in a given cell , the total intensity of fluorescent foci in the cell was measured , yielding an estimate of the number of bound probes , in turn indicating the number of target mrna molecules . figure 2 demonstrates the dynamic range and accuracy of measuring mrna copy - numbers using smfish , for the case of the plac promoter . the smfish - based measurements allowed us to obtain the copy - number statistics of mrna transcripts from a gene - of - interest under a given growth condition . in particular , the smfish data allowed us to accurately measure both the mean ( n ) and variance ( ) of mrna copy - number , and therefore calculate the burstiness parameter b = /n characterizing the transcriptional time - series . all of these factors can conceivably affect the observed fluctuations in gene activity . in total , > 150 independent experiments were made , each one yielding the distribution of mrna copy - number from a given gene at a given stimulus level . to characterize the transcriptional time - series in the complete data set , we plot ( figures 3a and b ) the burstiness b and the noise from each experiment , as a function of the mean expression level n at that condition . the expression levels were corrected for the differences in gene copy number ( see figures s6 and s7 and supplementary note ) and mrna lifetime ( see table s4 , figures s8 and s9 and supplementary note ) , so that the characteristics of mrna production from a single - copy promoter can be examined . we first note that the cell - to - cell variability in mrna numbers is dominated by the inherent fluctuations of the two - state process ( intrinsic noise ) rather than by cell - to - cell difference in parameter values ( extrinsic noise ) . the most striking feature in figures 3a and 3b is that b and exhibit gene - independent behavior ; that is , the values from different genes and growth conditions show a clear trend , with a dependence on the expression level n alone . thus , the properties of the time - series seem to depend primarily on the mean mrna level , not on the specific gene or stimulus ( this observation is made more quantitative below ) . the gene - independent behavior immediately suggests that the rate parameters in the two - state picture are not determined by the details of molecular regulation of an individual promoter ( such as the binding and unbinding kinetics of a specific transcription factor ) or the topology of the individual gene network ( e.g. note that this similarity in time - series characteristics does not necessarily mean that the actual activity of different genes is coordinated in time , i.e. we next used the experimental data of b(n ) and (n ) to ask what property of the transcriptional time series is modulated as gene expression level is varied . when comparing the experimental plots in figures 3a and 3b to the theoretical ones in figures 1d and 1e , we note that the observed mrna statistics is consistent with the assumption that expression level is changed by varying the rate at which the gene switches back to the off state ( off - rate koff ) , or in other words the duration of transcription bursts . as an additional test for the validity of our parameter estimation , we performed detailed stochastic simulations of mrna kinetics and verified that the theoretical and experimental copy - number histograms are in agreement , beyond the mere values of n and ( figure s14 ) . the theoretical fit allows us to make the observation of gene - independence more quantitative : when comparing the data from individual promoters to the universal fit , we find that 6 of 7 data sets exhibit a correlation coefficient above 0.85 between data and theory ( figure s15 ) . fitting the experimental data to the scenario of koff - modulation allowed us to estimate the values of the three kinetic parameters governing mrna production : kon ( the rate of switching to the on state , which determines the frequency of bursts ) and ktx ( the rate of producing mrna while the gene is on)both of which are approximately constant for different genes and expression levels and koff ( the rate of switching back to the off state , which determines the duration of bursts ) which changes over more than 3 orders of magnitude when expression level is varied ( figure 3c ) . the examination of mrna number statistics , though strongly indicating that koff alone is varied to control expression level , is limited by the fact that the process of transcription was not directly observed . to overcome this limitation and gain further support for the observation of koff modulation , we quantified the kinetics of mrna production from one promoter , plac / ara , in individual living cells . briefly , cells were grown under the microscope in the presence of different levels of the inducers , iptg and arabinose . as seen in figure 3d , the behavior of these kinetic parameters is consistent with the observations above : changing the level of mrna n is achieved by varying koff , while kon and ktx are kept approximately constant . we have thus seen that the discrete time - series of gene activity exhibits universal properties ; that is , the same kinetic parameters are common to different genes and environmental conditions . to address this question , we followed the approach of and considered the way gene activity is used by the cell to represent information about its environment . for example , the activity of the lactose promoter can be thought of as telling the cell how much lactose is present in its environment . we quantified the efficiency of information representation by the cell using shannon s mutual information , i(p , c ) , a function that measures how much information is transmitted to the output ( protein level , p ) about changes in the input stimulus , c ( for example , sugar concentration ) . the mutual information i depends critically on the way the variance of mrna copy numbers , , changes with the mean n ( the statistics of the protein species follows the same scaling relations , up to a calculable factor , see supplementary note ) . to examine how the mutual information varies as a function of time - series parameters , we wrote in the phenomenological form /n = 1 + n/ ( such that the deviation of the burstiness b from the poisson case goes like the mean n to the power ) . by varying the parameters and , this functional form allowed us to approximate the behavior exhibited by the transcriptional time - series under the different modulation schemes ( see figure 1d above ) and under a broad range of kinetic parameters ; specifically , this form captures the (n ) behavior seen in our experiments ( figure 4a ) . we next calculated the mutual information ( maximized over possible inputs , see online methods ) as a function of the parameters ( , ) ( figure 4b ) , thus exploring the efficiency of information representation over the space of possible time - series characteristics . we found that the parameters describing the actual transcriptional time - series ( = 3.5 3.2 , = 0.64 0.06 ) are close to optimal they lie on a ridge in the map of i(, ) ( figure 4b ) . when plotting a histogram of i values obtained from a broad range of kinetic parameters ( figure 4c ) , one sees that the maximal mutual information of the actual time series ( i~2.5 bits , or discrimination of > 5 input levels ) , is significantly higher than the mean performance obtained by randomly choosing the time - series parameters ( ~0.68 bits ) . in other words , the specific parameters of the transcriptional time series , which are observed in experiment , are superior to most other possible parameter sets , in the sense of allowing the cell to best represent information about its environment through the discrete activity of its genes . we quantified the copy - number statistics of endogenous mrna using single - molecule fluorescence in situ hybridization ( smfish ) , following the method of , which we adapted for counting mrna in e. coli at single - transcript resolution ( see online methods ) . to estimate the number of mrna molecules from the gene - of - interest in a given cell , the total intensity of fluorescent foci in the cell was measured , yielding an estimate of the number of bound probes , in turn indicating the number of target mrna molecules . figure 2 demonstrates the dynamic range and accuracy of measuring mrna copy - numbers using smfish , for the case of the plac promoter . the smfish - based measurements allowed us to obtain the copy - number statistics of mrna transcripts from a gene - of - interest under a given growth condition . in particular , the smfish data allowed us to accurately measure both the mean ( n ) and variance ( ) of mrna copy - number , and therefore calculate the burstiness parameter b = /n characterizing the transcriptional time - series . all of these factors can conceivably affect the observed fluctuations in gene activity . in total , > 150 independent experiments were made , each one yielding the distribution of mrna copy - number from a given gene at a given stimulus level . to characterize the transcriptional time - series in the complete data set , we plot ( figures 3a and b ) the burstiness b and the noise from each experiment , as a function of the mean expression level n at that condition . the expression levels were corrected for the differences in gene copy number ( see figures s6 and s7 and supplementary note ) and mrna lifetime ( see table s4 , figures s8 and s9 and supplementary note ) , so that the characteristics of mrna production from a single - copy promoter can be examined . we first note that the cell - to - cell variability in mrna numbers is dominated by the inherent fluctuations of the two - state process ( intrinsic noise ) rather than by cell - to - cell difference in parameter values ( extrinsic noise ) . the most striking feature in figures 3a and 3b is that b and exhibit gene - independent behavior ; that is , the values from different genes and growth conditions show a clear trend , with a dependence on the expression level n alone . thus , the properties of the time - series seem to depend primarily on the mean mrna level , not on the specific gene or stimulus ( this observation is made more quantitative below ) . the gene - independent behavior immediately suggests that the rate parameters in the two - state picture are not determined by the details of molecular regulation of an individual promoter ( such as the binding and unbinding kinetics of a specific transcription factor ) or the topology of the individual gene network ( e.g. note that this similarity in time - series characteristics does not necessarily mean that the actual activity of different genes is coordinated in time , i.e. we next used the experimental data of b(n ) and (n ) to ask what property of the transcriptional time series is modulated as gene expression level is varied . when comparing the experimental plots in figures 3a and 3b to the theoretical ones in figures 1d and 1e , we note that the observed mrna statistics is consistent with the assumption that expression level is changed by varying the rate at which the gene switches back to the off state ( off - rate koff ) , or in other words the duration of transcription bursts . as an additional test for the validity of our parameter estimation , we performed detailed stochastic simulations of mrna kinetics and verified that the theoretical and experimental copy - number histograms are in agreement , beyond the mere values of n and ( figure s14 ) . the theoretical fit allows us to make the observation of gene - independence more quantitative : when comparing the data from individual promoters to the universal fit , we find that 6 of 7 data sets exhibit a correlation coefficient above 0.85 between data and theory ( figure s15 ) . fitting the experimental data to the scenario of koff - modulation allowed us to estimate the values of the three kinetic parameters governing mrna production : kon ( the rate of switching to the on state , which determines the frequency of bursts ) and ktx ( the rate of producing mrna while the gene is on)both of which are approximately constant for different genes and expression levels and koff ( the rate of switching back to the off state , which determines the duration of bursts ) which changes over more than 3 orders of magnitude when expression level is varied ( figure 3c ) . the examination of mrna number statistics , though strongly indicating that koff alone is varied to control expression level , is limited by the fact that the process of transcription was not directly observed . to overcome this limitation and gain further support for the observation of koff modulation , we quantified the kinetics of mrna production from one promoter , plac / ara , in individual living cells . briefly , cells were grown under the microscope in the presence of different levels of the inducers , iptg and arabinose . as seen in figure 3d , the behavior of these kinetic parameters is consistent with the observations above : changing the level of mrna n is achieved by varying koff , while kon and ktx are kept approximately constant . we have thus seen that the discrete time - series of gene activity exhibits universal properties ; that is , the same kinetic parameters are common to different genes and environmental conditions . to address this question , we followed the approach of and considered the way gene activity is used by the cell to represent information about its environment . for example , the activity of the lactose promoter can be thought of as telling the cell how much lactose is present in its environment . we quantified the efficiency of information representation by the cell using shannon s mutual information , i(p , c ) , a function that measures how much information is transmitted to the output ( protein level , p ) about changes in the input stimulus , c ( for example , sugar concentration ) . the mutual information i depends critically on the way the variance of mrna copy numbers , , changes with the mean n ( the statistics of the protein species follows the same scaling relations , up to a calculable factor , see supplementary note ) . to examine how the mutual information varies as a function of time - series parameters , we wrote in the phenomenological form /n = 1 + n/ ( such that the deviation of the burstiness b from the poisson case goes like the mean n to the power ) . by varying the parameters and , this functional form allowed us to approximate the behavior exhibited by the transcriptional time - series under the different modulation schemes ( see figure 1d above ) and under a broad range of kinetic parameters ; specifically , this form captures the (n ) behavior seen in our experiments ( figure 4a ) . we next calculated the mutual information ( maximized over possible inputs , see online methods ) as a function of the parameters ( , ) ( figure 4b ) , thus exploring the efficiency of information representation over the space of possible time - series characteristics . we found that the parameters describing the actual transcriptional time - series ( = 3.5 3.2 , = 0.64 0.06 ) are close to optimal they lie on a ridge in the map of i(, ) ( figure 4b ) . when plotting a histogram of i values obtained from a broad range of kinetic parameters ( figure 4c ) , one sees that the maximal mutual information of the actual time series ( i~2.5 bits , or discrimination of > 5 input levels ) , is significantly higher than the mean performance obtained by randomly choosing the time - series parameters ( ~0.68 bits ) . in other words , the specific parameters of the transcriptional time series , which are observed in experiment , are superior to most other possible parameter sets , in the sense of allowing the cell to best represent information about its environment through the discrete activity of its genes . multiple studies in recent years have demonstrated that gene activity is often bursty rather than poissonian , and can be described via a two - state model for mrna production . in this work we have extended and generalized these observations by describing how the transcriptional time - series in e. coli is modulated when gene expression level is varied . we found that promoter activity tends to be non - bursty at low expression levels ( at or below n ~ 1 molecules / cell ) ; the degree of burstiness , as characterized by the fano factor b = /n , then rises in a sub - linear manner with increasing gene activity . this behavior is consistent with varying of the gene off - rate as the means to change the expression level , while maintaining the gene on - rate and transcription rate constant . in other words , the duration of the transcription bursts is the main feature that changes as expression level is varied . importantly , this behavior is not gene- or input - specific ( although it can also be observed when examining a single gene , see figure s11 ) ; rather , it was observed in the complete ensemble of promoters and stimuli examined . from an evolutionary point of view , we note that the expression level of a gene has been shown to be a phenotype subject to selection . more recently , a number of studies have suggested that , beyond the mean expression level , the degree of population heterogeneity ( noise ) in gene expression may also be subject to selection . here we estimated the mutual information between external stimulus and the transcriptional time - series and showed that the specific modulation scheme chosen by the cell is efficient in the sense of reliably representing , through the transcriptional time - series , the environment in which the cell resides . in quantifying this efficiency , we demonstrated how the properties of the transcriptional time - series itself , beyond merely the mean expression level , emerge as a meaningful phenotype subject to selection . two important limitations of our work need to be mentioned : first , when describing gene activity we centered on the mrna species only , while leaving out the downstream production of proteins . in doing so we implicitly assumed that protein kinetics is enslaved by mrna kinetics to a sufficient degree such that the discrete , stochastic time - series of mrna production governs cell - to - cell heterogeneity . this assumption is supported by the observation that protein - number statistics closely reflect the properties of mrna statistics , as found here . second , by mainly using in situ hybridization to count mrna , we were able to obtain snapshots of cell populations but were naturally unable to follow the time - course of gene activity in individual cells ( with the exception of a single promoter ) . correlations in the gene - activity trajectories of individual cells have been shown to contain important information about the underlying gene regulatory network . extending the use of the ms2-based system to multiple promoters should allow the characterization of such temporal effects in the future . at this stage , there is no mechanistic , molecular - level understanding of what gives rise to the bursty behavior of gene activity in bacteria ; specifically , what the physiological nature of the gene on and off states is , and therefore also how the rates of switching between states can be varied in the individual cell or over the time course of evolution . however , our finding here , that the properties of the transcriptional time - series are gene - independent ( rather than gene - specific ) , suggest that the observed two - state kinetics involves gene - nonspecific features such as dna topology , rna polymerase dynamics or regulation by broad - target dna - binding proteins .

alzheimer s disease ( ad ) is the most common type of dementia , affecting over 25 million people worldwide.1 at present , there are no treatments that can stop or reverse the progression of ad , and current medications such as cholinesterase inhibitors ( cheis ) are only considered to be symptomatic therapy . several meta - analyses have consistently documented modest effects of cheis on cognitive function.2,3 however , these meta - analyses have mainly assessed placebo - controlled randomized trials that demonstrated relatively short - term effects on cognitive function , neuropsychiatric symptoms , and global clinical assessments . because ethical constraints prohibit long - term placebo - controlled studies of cheis in ad , placebo - controlled trials of these drugs have generally not run for longer than 1 year . on the other hand , ad is a slowly progressive disease and patients can be expected to survive for an average of 810 years at the time of diagnosis,4 which means that long - term efficacy is clinically important . based on our literature search , only two relatively long - term studies ( 2 years ) have been conducted to assess the efficacy of cheis ( donepezil and galantamine ) in ad,5,6 which means there is little evidence regarding the long - term efficacy of these drugs . due to this lack of robust evidence , the american geriatric society has stated that the risk and benefits of long - term chei therapy are not well established.7 long - term open - label observational studies performed in the routine clinical setting can provide supplementary information to that demonstrated by placebo - controlled trials , particularly with regard to long - term efficacy . because open - label observational studies lack a placebo arm , detailed understanding of the natural disease trajectory in patients with ad is important for analysis of the data . several authors have proposed mathematical models for describing cognitive decline in untreated patients with ad.810 all these models suggest that there is a significant quadratic correlation between baseline cognitive function and the annual rate of cognitive decline in untreated ad cohorts.810 using such models , several studies have been conducted to assess the long - term efficacy of cheis for ad in comparison with the simulated untreated trajectory of cognitive decline and to establish models for prediction of expected changes in chei - treated patients.11,12 however , these studies involved prediction of group mean values rather than individual patient outcomes . therefore , the long - term benefits of chei treatment for individual ad patients have not been clarified . because of the modest effect of cheis on cognitive function in ad and considerable variability of the disease trajectory , it is difficult to judge whether chei therapy is beneficial on a patient - by - patient basis . to do so would require a reliable individual patient - based predictive model for cognitive outcomes in untreated ad . such a model could support clinical decisions , such as switching to another chei because the current medication was predicted to lack efficacy . we conducted a 72-week post - marketing surveillance study in the routine clinical setting to evaluate the efficacy and safety of galantamine therapy for ad based on the mini mental state examination ( mmse ) and the clinical global impression improvement scale ( cgi - i).13 this study was designed to investigate the following points : the long - term effect of galantamine on cognitive function ( mmse ) and on the clinical state ( cgi - i ) in patients with mild - to - moderate ad during a 72-week treatment period.the effect of galantamine on cognitive function in comparison with the untreated disease trajectory predicted by mendiondo s mathematical model9 at both the group and individual patient level.the safety and adverse effects of galantamine therapy in the routine clinical setting . the long - term effect of galantamine on cognitive function ( mmse ) and on the clinical state ( cgi - i ) in patients with mild - to - moderate ad during a 72-week treatment period . the effect of galantamine on cognitive function in comparison with the untreated disease trajectory predicted by mendiondo s mathematical model9 at both the group and individual patient level . the safety and adverse effects of galantamine therapy in the routine clinical setting . this was a 72-week multicenter observational open - label study of galantamine therapy for ad . patients meeting the enrollment criteria were men or women with mild - to - moderate ad who had recently commenced treatment with galantamine . the diagnosis of ad was done based on each physician s clinical judgment and severity of ad was defined based on functional assessment staging of alzheimer s disease and mmse . physicians were advised that all treatments and dose adjustments should be based on approved regimens , and management decisions were made at the attending physician s discretion according to routine practice . in general , for at least 4 weeks before being increased to 12 mg b.i.d . with dosage adjustment being based on the physician s assessment of the clinical response and tolerability . the study drug was not supplied to the patients and they received the medication as part of their usual care . the protocol of this study , including the ethical aspects , was assessed by an internal review board of janssen pharmaceutical ( named : research concept review board ) , and was approved by the pharmaceuticals and medical devices agency ( pmda ) . conducting this post - marketing surveillance was a part of mandatory actions determined and reviewed by pmda . the study was conducted in accordance with the japanese regulation ( ministry of health , labour and welfare ministerial ordinance no 171 ) of good post - marketing study practice . each site ( hospitals or clinic ) follows its own regulations or standards for obtaining of the irb approval , including taking either written or oral informed consent for participants at each site . a similar procedure is generally followed in japan for this type of naturalistic registry study . cognitive function was assessed by using the mmse14 and the global severity and/or change of the clinical condition was assessed by the cgi - i scale . the mmse and cgi - i scores were determined at baseline and after 4 , 12 , 24 , 36 , 48 , 60 , and 72 weeks of treatment with galantamine . adverse events ( aes ) and data on patients who discontinued treatment were also recorded during the study period . an electronic data capture system was used , allowing the majority of the data to be transcribed from source documents into the electronic case report forms by the attending physicians and transmitted securely to the sponsor . the natural course of cognitive decline without treatment was simulated by calculating changes of the mmse score using a previously reported model.9 the original model and 95% confidence limits reported by mendiondo et al was : annualchangeofthemmsescore=1/(ax2+bx+c),a=0.003340.00048,b=0.07300.0136,c=0.60130.0884x = baselinemmsescore because of the significant impact of age in this model , it was weighted according to the duration of cognitive decline for two age clusters as follows.9 weight<72yearsold=5.902/(5.90 + 8.23)=0.8351weight72yearsold=8.232/(5.90 + 8.23)=1.1649 the confidence interval ( ci ) of the model was adjusted by bonferroni s correction because of the significantly low probability that all of these three coefficients would take 95% confidence limits . < 72yearsold : annualchangeofthemmsescore=1/(ax2+bx+c),a72yearsold=0.003341.16490.00016,b72yearsold=0.007301.16490.0045333,c72yearsold=0.60131.16490.0294667 estimated 95% ci for 6 months and 1 year determined by these equations with different baseline mmse values are shown in table 1 . based on this table , the number of patients was determined who had mmse scores higher than the predicted upper limit of the ci ( better than untreated ) , within the ci range , or below the predicted lower limit of the ci ( worse than untreated ) after 1 year and after 1.5 years of galantamine treatment . the discontinuation rate was estimated by the kaplan meier method , and comparison of the discontinuation rate between patients on monotherapy versus those on combined therapy was performed with the log - rank test . comparison between simulated and actual changes of mmse scores was performed with a generalized linear mixed model ( glmm ) . the actual baseline mmse scores were used to calculate the predicted scores for each patient . to manage missing values , analysis was based on the observed cases ( oc ) at each scheduled visit and on data obtained by the last observation estimated forward ( loef ) method , which replaces missing values based on observed previous value and mendiondo s model . interactions of time with the type of data ( predicted untreated outcome , actual oc , or actual loef ) were modeled on glmm . when significant differences were detected , post - hoc comparisons were performed with the tukey kramer test . statistical analyses were conducted using r statistical software , version 3.1.0 ( foundation for statistical computing , vienna , austria ) and sas software , version 9.3 ( sas institute inc . , glmm was performed with the glimmix procedure of sas and other analyses were performed using r. this was a 72-week multicenter observational open - label study of galantamine therapy for ad . patients meeting the enrollment criteria were men or women with mild - to - moderate ad who had recently commenced treatment with galantamine . the diagnosis of ad was done based on each physician s clinical judgment and severity of ad was defined based on functional assessment staging of alzheimer s disease and mmse . physicians were advised that all treatments and dose adjustments should be based on approved regimens , and management decisions were made at the attending physician s discretion according to routine practice . in general , for at least 4 weeks before being increased to 12 mg b.i.d . with dosage adjustment being based on the physician s assessment of the clinical response and tolerability . the study drug was not supplied to the patients and they received the medication as part of their usual care . the protocol of this study , including the ethical aspects , was assessed by an internal review board of janssen pharmaceutical ( named : research concept review board ) , and was approved by the pharmaceuticals and medical devices agency ( pmda ) . conducting this post - marketing surveillance was a part of mandatory actions determined and reviewed by pmda . the study was conducted in accordance with the japanese regulation ( ministry of health , labour and welfare ministerial ordinance no 171 ) of good post - marketing study practice . each site ( hospitals or clinic ) follows its own regulations or standards for obtaining of the irb approval , including taking either written or oral informed consent for participants at each site . a similar procedure is generally followed in japan for this type of naturalistic registry study . cognitive function was assessed by using the mmse14 and the global severity and/or change of the clinical condition was assessed by the cgi - i scale . the mmse and cgi - i scores were determined at baseline and after 4 , 12 , 24 , 36 , 48 , 60 , and 72 weeks of treatment with galantamine . adverse events ( aes ) and data on patients who discontinued treatment were also recorded during the study period . an electronic data capture system was used , allowing the majority of the data to be transcribed from source documents into the electronic case report forms by the attending physicians and transmitted securely to the sponsor . the natural course of cognitive decline without treatment was simulated by calculating changes of the mmse score using a previously reported model.9 the original model and 95% confidence limits reported by mendiondo et al was : annualchangeofthemmsescore=1/(ax2+bx+c),a=0.003340.00048,b=0.07300.0136,c=0.60130.0884x = baselinemmsescore because of the significant impact of age in this model , it was weighted according to the duration of cognitive decline for two age clusters as follows.9 weight<72yearsold=5.902/(5.90 + 8.23)=0.8351weight72yearsold=8.232/(5.90 + 8.23)=1.1649 the confidence interval ( ci ) of the model was adjusted by bonferroni s correction because of the significantly low probability that all of these three coefficients would take 95% confidence limits . < 72yearsold : annualchangeofthemmsescore=1/(ax2+bx+c),a<72yearsold=0.003340.83510.00016,b<72yearsold=0.007300.83510.0045333,c<72yearsold=0.60130.83510.0294667 < 72yearsold : annualchangeofthemmsescore=1/(ax2+bx+c),a72yearsold=0.003341.16490.00016,b72yearsold=0.007301.16490.0045333,c72yearsold=0.60131.16490.0294667 estimated 95% ci for 6 months and 1 year determined by these equations with different baseline mmse values are shown in table 1 . based on this table , the number of patients was determined who had mmse scores higher than the predicted upper limit of the ci ( better than untreated ) , within the ci range , or below the predicted lower limit of the ci ( worse than untreated ) after 1 year and after 1.5 years of galantamine treatment . the discontinuation rate was estimated by the kaplan meier method , and comparison of the discontinuation rate between patients on monotherapy versus those on combined therapy was performed with the log - rank test . comparison between simulated and actual changes of mmse scores was performed with a generalized linear mixed model ( glmm ) . the actual baseline mmse scores were used to calculate the predicted scores for each patient . to manage missing values , analysis was based on the observed cases ( oc ) at each scheduled visit and on data obtained by the last observation estimated forward ( loef ) method , which replaces missing values based on observed previous value and mendiondo s model . interactions of time with the type of data ( predicted untreated outcome , actual oc , or actual loef ) were modeled on glmm . when significant differences were detected , post - hoc comparisons were performed with the tukey kramer test . statistical analyses were conducted using r statistical software , version 3.1.0 ( foundation for statistical computing , vienna , austria ) and sas software , version 9.3 ( sas institute inc . , glmm was performed with the glimmix procedure of sas and other analyses were performed using r. a total of 661 patients were enrolled in this study , of whom 642 were evaluable for safety and 554 for efficacy ( figure 1 ) . seventy subjects were removed from the efficacy dataset because of lack of sufficient baseline information . the baseline demographic and clinical characteristics are summarized in table 2 . in the safety analysis set , the mean standard deviation ( sd ) age was 79.107.20 years , the mean disease duration was 0.73 years , and 4.98% of the patients were hospitalized at study entry . among the subjects , 22.90% were using other anti - dementia drugs before study entry . according to the functional assessment of staging scale , most patients had mild ( 39.10% ) or moderate ( 47.66% ) ad at study entry , while the mean mmse score was 18.955.04 . the mean daily dose of galantamine was 15.014.76 mg and the mean duration of drug exposure was 343.44202.30 days . sixty - five patients ( 10.12% ) received memantine combined with galantamine ( memantine users ) . the discontinuation rate was ~20% at 12 weeks , 40% at 48 weeks , and 46.73% after 72 weeks ( figure 2 ) . patients discontinued the study because of safety problems ( 31.08% ) , transfer to another hospital ( 24.32% ) , loss of contact ( 19.93% ) , personal choice ( 11.15% ) , and other reasons ( 13.51% ) . post - hoc comparison between memantine users and non - users demonstrated a significantly lower discontinuation rate among memantine users than non - users ( log - rank test , p=0.0051 ; figure 2 ) . the mean change from baseline to 72 weeks was 0.71 according to oc analysis , 1.21 according to loef analysis , and 3.35 for the predicted outcome without treatment . glmm revealed a significant interaction between these three cohorts and time ( f(2,6427 ) = 14.63 , p<0.001 ) . a post - hoc tukey kramer test identified significant differences between the oc and predicted outcomes and between the loef and predicted outcomes at 36 weeks ( oc vs predicted : t ( 3,403 ) = 4.37 , p=0.003 ; loef vs predicted : t ( 3,403 ) = 3.96 , p=0.016 ) , at 48 weeks ( oc vs predicted : t ( 3,403 ) = 4.49 , p=0.0018 ; loef vs predicted : t ( 3,403 ) = 3.59 , p=0.0599 ) , at 60 weeks ( oc vs predicted : t ( 3,403 ) = 5.53 , p<0.001 ; loef vs predicted : t ( 3,403 ) = 4.16 , p=0.0074 ) , and at 72 weeks ( oc vs predicted : t ( 3,403 ) = 5.45 , p<0.0001 ; loef vs predicted : t ( 3,403 ) = 4.54 , p=0.0014 ) . table 3 displays the patients whose mmse scores were above , within , or below the predicted ci . after 1 year , oc analysis showed that 75.65% of patients had an mmse score significantly above the predicted value without treatment , as did 71.43% of patients at 1.5 years . according to loef analysis , 57.79% of patients had an mmse score significantly above the predicted value after 1 year and 55.75% at 1.5 years . the time course of the observation period in cgi - i is shown in figure 4 . at 1.5 years , 2.24% of patients were rated as very much improved , 10.45% were much improved , 33.21% were no change , 14.93% were minimally worse , 5.22% were much worse , and 1.12% were very much worse . the most frequently reported aes were nausea ( 5.30% ) , decreased appetite ( 3.43% ) , vomiting ( 2.49% ) , insomnia ( 1.40% ) , agitation ( 1.09% ) , dizziness ( 1.09% ) , and headache ( 1.09% ) . frequently reported saes occurring in at least two patients and the number of deaths are also listed in table 4 . twelve deaths ( 1.87% ) were reported during the study , with the causes being bronchial pneumonia ( n=1 , 0.16% ) , acute bronchitis ( n=1 , 0.16% ) , stomach cancer ( n=1 , 0.16% ) , aspiration pneumonitis ( n=1 , 0.16% ) , myocardial infarction / pneumonia ( n=1 , 0.16% ) , complete heart block ( n=1 , 0.16% ) , renal insufficiency ( n=1 , 0.16% ) , sepsis ( n=1 , 0.16% ) , drowning ( n=1 , 0.16% ) , and unknown ( n=3 , 0.47% ) . a total of 661 patients were enrolled in this study , of whom 642 were evaluable for safety and 554 for efficacy ( figure 1 ) . seventy subjects were removed from the efficacy dataset because of lack of sufficient baseline information . the baseline demographic and clinical characteristics are summarized in table 2 . in the safety analysis set , the mean standard deviation ( sd ) age was 79.107.20 years , the mean disease duration was 0.73 years , and 4.98% of the patients were hospitalized at study entry . among the subjects , 22.90% were using other anti - dementia drugs before study entry . according to the functional assessment of staging scale , most patients had mild ( 39.10% ) or moderate ( 47.66% ) ad at study entry , while the mean mmse score was 18.955.04 . the mean daily dose of galantamine was 15.014.76 mg and the mean duration of drug exposure was 343.44202.30 days . sixty - five patients ( 10.12% ) received memantine combined with galantamine ( memantine users ) . the discontinuation rate was ~20% at 12 weeks , 40% at 48 weeks , and 46.73% after 72 weeks ( figure 2 ) . patients discontinued the study because of safety problems ( 31.08% ) , transfer to another hospital ( 24.32% ) , loss of contact ( 19.93% ) , personal choice ( 11.15% ) , and other reasons ( 13.51% ) . post - hoc comparison between memantine users and non - users demonstrated a significantly lower discontinuation rate among memantine users than non - users ( log - rank test , p=0.0051 ; figure 2 ) . the mean change from baseline to 72 weeks was 0.71 according to oc analysis , 1.21 according to loef analysis , and 3.35 for the predicted outcome without treatment . glmm revealed a significant interaction between these three cohorts and time ( f(2,6427 ) = 14.63 , p<0.001 ) . a post - hoc tukey kramer test identified significant differences between the oc and predicted outcomes and between the loef and predicted outcomes at 36 weeks ( oc vs predicted : t ( 3,403 ) = 4.37 , p=0.003 ; loef vs predicted : t ( 3,403 ) = 3.96 , p=0.016 ) , at 48 weeks ( oc vs predicted : t ( 3,403 ) = 4.49 , p=0.0018 ; loef vs predicted : t ( 3,403 ) = 3.59 , p=0.0599 ) , at 60 weeks ( oc vs predicted : t ( 3,403 ) = 5.53 , p<0.001 ; loef vs predicted : t ( 3,403 ) = 4.16 , p=0.0074 ) , and at 72 weeks ( oc vs predicted : t ( 3,403 ) = 5.45 , p<0.0001 ; loef vs predicted : t ( 3,403 ) = 4.54 , p=0.0014 ) . table 3 displays the patients whose mmse scores were above , within , or below the predicted ci . after 1 year , oc analysis showed that 75.65% of patients had an mmse score significantly above the predicted value without treatment , as did 71.43% of patients at 1.5 years . according to loef analysis , 57.79% of patients had an mmse score significantly above the predicted value after 1 year and 55.75% at 1.5 years . the time course of the observation period in cgi - i is shown in figure 4 . at 1.5 years , 2.24% of patients were rated as very much improved , 10.45% were much improved , 33.21% were no change , 14.93% were minimally worse , 5.22% were much worse , and 1.12% were very much worse . the most frequently reported aes were nausea ( 5.30% ) , decreased appetite ( 3.43% ) , vomiting ( 2.49% ) , insomnia ( 1.40% ) , agitation ( 1.09% ) , dizziness ( 1.09% ) , and headache ( 1.09% ) . frequently reported saes occurring in at least two patients and the number of deaths are also listed in table 4 . twelve deaths ( 1.87% ) were reported during the study , with the causes being bronchial pneumonia ( n=1 , 0.16% ) , acute bronchitis ( n=1 , 0.16% ) , stomach cancer ( n=1 , 0.16% ) , aspiration pneumonitis ( n=1 , 0.16% ) , myocardial infarction / pneumonia ( n=1 , 0.16% ) , complete heart block ( n=1 , 0.16% ) , renal insufficiency ( n=1 , 0.16% ) , sepsis ( n=1 , 0.16% ) , drowning ( n=1 , 0.16% ) , and unknown ( n=3 , 0.47% ) . the present study was designed to assess the long - term efficacy of galantamine in patients with mild - to - moderate ad in comparison with the natural disease trajectory predicted using a mathematical model , as well as investigating the safety and tolerability of galantamine therapy for ad in the real - world clinical setting . we found that the mean mmse score improved over 24 weeks and was stable up to 1 year . furthermore , there was a long - term beneficial effect on cognitive function measured by the mmse compared with the predicted outcome without treatment . when efficacy was assessed on a patient - by - patient basis , about 71% of the patients showed significantly better cognitive function at 72 weeks compared with the predicted outcome . in agreement with the changes of the mmse score , assessment of the cgi - i score also showed that galantamine treatment was effective for maintaining the clinical state ( better or unchanged ) in almost 80% of the patients at 72 weeks . taken together , these findings suggest that galantamine therapy could be beneficial for 70%80% of ad patients . since about 10% of the patients received memantine in addition to galantamine , we also analyzed the data after excluding memantine users , but we found that the changes of the mmse score were essentially the same ( data not shown ) . on the other hand , the indication for memantine therapy is moderate - to - severe ad in japan and the mean baseline mmse score of memantine users was significantly lower than that of non - users ( mean sd : 16.795.41 for memantine users vs 19.134.97 for memantine non - users , t-(427 ) = 2.43 , p=0.02 ) . the lower dropout rate of memantine users in the present study is inconsistent with the results of a previous study that suggested baseline disease severity and age were associated with dropout from ad trials.15 however , we have no adequate explanations for such a discrepancy at present . safety evaluation revealed that all of the reported aes were known events , such as gastrointestinal symptoms . accordingly , both efficacy and safety data supported the validity of long - term galantamine therapy for mild - to - moderate ad . this study not only demonstrated a long - term beneficial effect of galantamine therapy for ad at the group level , but also revealed that galantamine maintained cognitive function of individual patients in the routine clinical setting . published consensus recommendations and clinical practice guidelines advocate an individualized approach to ad , but there is a need for more evidence that long - term therapy is worthwhile.16,17 as shown in table 1 , our present findings could be useful for assisting physicians and the relatives of patients when deciding treatment options . a previous study established a patient - level ad model and simulation method by applying beta regression to the alzheimer s disease assessment scale cognitive subscale ( adas - cog).18 while this beta regression model employing adas - cog could be suitable for clinical trials of disease - modifying drugs , it is complicated and adas - cog is not usually employed in the routine clinical setting . on the other hand , simulating changes of the mmse score by using mendiondo s model is simple and can be easily applied to individual patients in routine practice . similar to the present results , two previous placebo - controlled studies have demonstrated long - term beneficial effects of chei therapy.6,19 on the other hand , the alzheimer s disease neuroimaging initiative ( adni ) and the australian imaging , biomarkers and lifestyle study obtained opposing results , with greater cognitive decline amongst ad patients taking cheis compared with those not using cheis.20 unlike the aforementioned two randomized controlled trials ( rcts ) showing a favorable effect of cheis , the adni study was not a randomized trial , so treatment selection bias should be taken into consideration when interpreting its results ( ie , physicians might have selectively prescribed cheis for patients with more progressive ad ) . as mentioned above , the present study demonstrated similar efficacy to that found in a previous 2-year placebo - controlled rct of galantamine,6 but there were some discrepancies between our results and previous findings . although similar patient factors were considered to be associated with cognitive decline ( baseline mmse and age ) , the previous study demonstrated a more rapid decrease of mmse scores in patients receiving galantamine treatment compared with our results . it is possible that survival bias contributed to the favorable effect of galantamine in the present study . observational studies have an inherent risk of survival bias due to dropout and high dropout rates are a problem in all long - term studies of ad . in this study , the completion rate was actually higher than in the previous study ; however , it should be taken into consideration as different criteria for the termination : previous study has been terminated due to a data safety monitoring board - recommended early study termination . according to wallin et al , the reported 1-year completion rate ranges widely from 26% to 89%.12 the completion rate was 53% at 1.5 years in the present study , which is average for this type of study . when changes of the mmse score were assessed by oc analysis , the mmse score was evaluated at 72 weeks in only 38.2% of the patients ; so , there is a potential risk of survival bias . since the last observation carried forward method seems to be inappropriate for analysis of mmse decline in patients with a progressive disease like ad , we predicted mmse decline based on mendiondo s model and applied the results to dropouts as a supplementary analysis . the results of our group level loef analysis were essentially the same as those of oc analysis . on the other hand , there were an increased number of patients within the disease trajectory due to the relatively high discontinuation rate . however , > 50% of patients still showed significantly better cognitive function compared with the predicted outcome . while we can not completely exclude survival bias , we believe that our data indicate a long - term beneficial effect of galantamine treatment on cognitive function . indeed , a previous long - term randomized head - to - head comparison of galantamine with donepezil and an observational study with a relatively high completion rate demonstrated similar mmse changes to those in our study , with stabilization of mmse scores after 1 year of treatment.12,21 another important difference between the present findings and those of the previous 2-year study of galantamine therapy6 is that the decline of mmse scores predicted by mendiondo s model was more rapid than that observed in the placebo group of the previous study . because the factors associated with mendiondo s model ( age and baseline mmse score ) were similar in both studies , this discrepancy should need to be explained . one possible explanation is the placebo effect in patients allocated to the placebo arm in the previous study . since mendiondo investigated the natural history of ad in an observational study without pharmacological intervention for dementia , the placebo effect on mmse changes should have been negligible in their data . on the other hand , mmse changes of participants in the previous placebo - controlled rct would probably be modulated by the placebo effect and this might have contributed to the discrepancy with our present findings . another possibility is that mendiondo s mathematical model overestimates cognitive decline in untreated patients due to general advances in medicine since the 1980s , when the data set for the mathematical model was acquired . for example , an association between vascular risk factors , such as hypertension , diabetes , and cardiovascular disease , and the pathogenesis and/or progression of ad received attention only since around 2000.22 in elderly people , vascular risk factors are currently managed better than in the 1980s . this could affect the disease trajectory of ad and lead to overestimation of cognitive decline in untreated patients due to changes in the healthcare environment . indeed , a previous pooled analysis of studies on donepezil conducted between 1990 and 1999 revealed that patients allocated to placebo therapy showed slower cognitive decline in more recent trials compared with older trials , despite having more comorbidities.23 furthermore , our previous network meta - analysis of chei therapy revealed an increasing placebo effect on cognitive function over time across various drugs of this class.24 these results suggest that there may have been possible overestimation of the speed of cognitive decline based on mendiondo s model in the present study . however , other studies conducted in the 2000s have revealed similar or more rapid decline of mmse scores than the present data.5,25 for example , a study of the natural history of ad over 3 years reported by holmes and lovestone demonstrated annual decline of the mmse score by 3.4 points ( sd 3.5 : median = 3.0 ) in patients with mild - to - moderate disease.25 this reported rate of decline is larger than the decline simulated by mendiondo s model in this study , but holmes and lovestone investigated a relatively small sample.25 further research is needed to establish a contemporary disease trajectory model of ad for evaluating the efficacy of chei therapy and better management of ad patients first , the diagnosis of ad was done based on physician s clinical judgment rather than the strict diagnostic criteria . one would be concerned about the diagnostic accuracy of this study ; however , the purpose of the study is to show effectiveness of galantamine in the real world . in this context , diagnosis based on physician s clinical judgment seems to be suitable for the purpose of this study . second , several important patient characteristics associated with ad progression and risk were not investigated , including apolipoprotein e ( apoe ) alleles and education level . while the influence of the 4 allele on the risk of ad and its age of onset is generally consistent in most studies , there have been widely varying reports with regard to whether different apoe alleles influence the rate of cognitive decline following the onset of dementia.26 mendiondo s model does not require information on apoe alleles and we do not consider the lack of allele data to be a critical flaw . a lower education level is also considered to be a risk factor for developing ad and rapid progression.9,27 however , wattmo et al reported an opposite effect of education level on the long - term outcome of ad , with a high education level being a risk factor for faster cognitive decline.28 thus , the relationship between education and disease progression remains to be clarified . furthermore , the influence of education level on mendiondo s model is smaller than that of age , so lack of education level data in this study should not be a critical flaw . this 1.5-year observational study revealed long - term efficacy of galantamine for maintaining cognitive function in ad patients based on changes of the mmse score , and also showed that galantamine therapy was generally safe and well tolerated . importantly , individual analysis demonstrated that galantamine was beneficial for > 70% of ad patients .

backgroundlong - term maintenance of cognitive function is an important goal of treatment for alzheimer s disease ( ad ) , but evidence about the long - term efficacy of cholinesterase inhibitors is sparse . to evaluate the long - term efficacy and safety of galantamine for ad in routine clinical practice , we conducted a 72-week post - marketing surveillance study . the effect of galantamine on cognitive function was estimated in comparison with a simulated disease trajectory.patients and methodspatients with mild - to - moderate ad received flexible dosing of galantamine ( 1624 mg / day ) during this study . cognitive function was assessed by the mini mental state examination ( mmse ) and the clinical status was determined by the clinical global impression - improvement ( cgi - i ) . changes of the mmse score without treatment were estimated in each patient using mendiondo s model . generalized linear mixed model analysis was performed to compare the simulated mmse scores with the actual scores.resultsof the 661 patients who were enrolled , 642 were evaluable for safety and 554 were assessed for efficacy . the discontinuation rate was 46.73% . cognitive decline indicated by the mean change of actual mmse scores was significantly smaller than the simulated decline . individual analysis demonstrated that > 70% of patients had better actual mmse scores than their simulated scores . significant improvement of cgi - i was also observed during the observation period . adverse events occurred in 28.5% of patients and were serious in 8.41% . the reported events generally corresponded with the safety profile of galantamine in previous studies.conclusionthese findings support the long - term efficacy of galantamine for maintaining cognitive function and the clinical state in ad patients . treatment with galantamine was generally safe . importantly , this study revealed that galantamine improved cognitive function above the predicted level in > 70% of the patients .

Introduction Patients and methods Study design Outcome assessment Models of the natural cognitive decline Statistical analysis Results Baseline demographic and clinical characteristics Discontinuation rate Long-term effect of galantamine on cognitive function Safety Discussion Conclusion

alzheimer s disease ( ad ) is the most common type of dementia , affecting over 25 million people worldwide.1 at present , there are no treatments that can stop or reverse the progression of ad , and current medications such as cholinesterase inhibitors ( cheis ) are only considered to be symptomatic therapy . because ethical constraints prohibit long - term placebo - controlled studies of cheis in ad , placebo - controlled trials of these drugs have generally not run for longer than 1 year . based on our literature search , only two relatively long - term studies ( 2 years ) have been conducted to assess the efficacy of cheis ( donepezil and galantamine ) in ad,5,6 which means there is little evidence regarding the long - term efficacy of these drugs . due to this lack of robust evidence , the american geriatric society has stated that the risk and benefits of long - term chei therapy are not well established.7 long - term open - label observational studies performed in the routine clinical setting can provide supplementary information to that demonstrated by placebo - controlled trials , particularly with regard to long - term efficacy . several authors have proposed mathematical models for describing cognitive decline in untreated patients with ad.810 all these models suggest that there is a significant quadratic correlation between baseline cognitive function and the annual rate of cognitive decline in untreated ad cohorts.810 using such models , several studies have been conducted to assess the long - term efficacy of cheis for ad in comparison with the simulated untreated trajectory of cognitive decline and to establish models for prediction of expected changes in chei - treated patients.11,12 however , these studies involved prediction of group mean values rather than individual patient outcomes . therefore , the long - term benefits of chei treatment for individual ad patients have not been clarified . because of the modest effect of cheis on cognitive function in ad and considerable variability of the disease trajectory , it is difficult to judge whether chei therapy is beneficial on a patient - by - patient basis . we conducted a 72-week post - marketing surveillance study in the routine clinical setting to evaluate the efficacy and safety of galantamine therapy for ad based on the mini mental state examination ( mmse ) and the clinical global impression improvement scale ( cgi - i).13 this study was designed to investigate the following points : the long - term effect of galantamine on cognitive function ( mmse ) and on the clinical state ( cgi - i ) in patients with mild - to - moderate ad during a 72-week treatment period.the effect of galantamine on cognitive function in comparison with the untreated disease trajectory predicted by mendiondo s mathematical model9 at both the group and individual patient level.the safety and adverse effects of galantamine therapy in the routine clinical setting . the long - term effect of galantamine on cognitive function ( mmse ) and on the clinical state ( cgi - i ) in patients with mild - to - moderate ad during a 72-week treatment period . the effect of galantamine on cognitive function in comparison with the untreated disease trajectory predicted by mendiondo s mathematical model9 at both the group and individual patient level . the safety and adverse effects of galantamine therapy in the routine clinical setting . this was a 72-week multicenter observational open - label study of galantamine therapy for ad . patients meeting the enrollment criteria were men or women with mild - to - moderate ad who had recently commenced treatment with galantamine . the protocol of this study , including the ethical aspects , was assessed by an internal review board of janssen pharmaceutical ( named : research concept review board ) , and was approved by the pharmaceuticals and medical devices agency ( pmda ) . cognitive function was assessed by using the mmse14 and the global severity and/or change of the clinical condition was assessed by the cgi - i scale . the mmse and cgi - i scores were determined at baseline and after 4 , 12 , 24 , 36 , 48 , 60 , and 72 weeks of treatment with galantamine . adverse events ( aes ) and data on patients who discontinued treatment were also recorded during the study period . the natural course of cognitive decline without treatment was simulated by calculating changes of the mmse score using a previously reported model.9 the original model and 95% confidence limits reported by mendiondo et al was : annualchangeofthemmsescore=1/(ax2+bx+c),a=0.003340.00048,b=0.07300.0136,c=0.60130.0884x = baselinemmsescore because of the significant impact of age in this model , it was weighted according to the duration of cognitive decline for two age clusters as follows.9 weight<72yearsold=5.902/(5.90 + 8.23)=0.8351weight72yearsold=8.232/(5.90 + 8.23)=1.1649 the confidence interval ( ci ) of the model was adjusted by bonferroni s correction because of the significantly low probability that all of these three coefficients would take 95% confidence limits . based on this table , the number of patients was determined who had mmse scores higher than the predicted upper limit of the ci ( better than untreated ) , within the ci range , or below the predicted lower limit of the ci ( worse than untreated ) after 1 year and after 1.5 years of galantamine treatment . the discontinuation rate was estimated by the kaplan meier method , and comparison of the discontinuation rate between patients on monotherapy versus those on combined therapy was performed with the log - rank test . comparison between simulated and actual changes of mmse scores was performed with a generalized linear mixed model ( glmm ) . the actual baseline mmse scores were used to calculate the predicted scores for each patient . to manage missing values , analysis was based on the observed cases ( oc ) at each scheduled visit and on data obtained by the last observation estimated forward ( loef ) method , which replaces missing values based on observed previous value and mendiondo s model . , glmm was performed with the glimmix procedure of sas and other analyses were performed using r. this was a 72-week multicenter observational open - label study of galantamine therapy for ad . patients meeting the enrollment criteria were men or women with mild - to - moderate ad who had recently commenced treatment with galantamine . the protocol of this study , including the ethical aspects , was assessed by an internal review board of janssen pharmaceutical ( named : research concept review board ) , and was approved by the pharmaceuticals and medical devices agency ( pmda ) . the study was conducted in accordance with the japanese regulation ( ministry of health , labour and welfare ministerial ordinance no 171 ) of good post - marketing study practice . cognitive function was assessed by using the mmse14 and the global severity and/or change of the clinical condition was assessed by the cgi - i scale . the mmse and cgi - i scores were determined at baseline and after 4 , 12 , 24 , 36 , 48 , 60 , and 72 weeks of treatment with galantamine . adverse events ( aes ) and data on patients who discontinued treatment were also recorded during the study period . the natural course of cognitive decline without treatment was simulated by calculating changes of the mmse score using a previously reported model.9 the original model and 95% confidence limits reported by mendiondo et al was : annualchangeofthemmsescore=1/(ax2+bx+c),a=0.003340.00048,b=0.07300.0136,c=0.60130.0884x = baselinemmsescore because of the significant impact of age in this model , it was weighted according to the duration of cognitive decline for two age clusters as follows.9 weight<72yearsold=5.902/(5.90 + 8.23)=0.8351weight72yearsold=8.232/(5.90 + 8.23)=1.1649 the confidence interval ( ci ) of the model was adjusted by bonferroni s correction because of the significantly low probability that all of these three coefficients would take 95% confidence limits . based on this table , the number of patients was determined who had mmse scores higher than the predicted upper limit of the ci ( better than untreated ) , within the ci range , or below the predicted lower limit of the ci ( worse than untreated ) after 1 year and after 1.5 years of galantamine treatment . the discontinuation rate was estimated by the kaplan meier method , and comparison of the discontinuation rate between patients on monotherapy versus those on combined therapy was performed with the log - rank test . comparison between simulated and actual changes of mmse scores was performed with a generalized linear mixed model ( glmm ) . the actual baseline mmse scores were used to calculate the predicted scores for each patient . to manage missing values , analysis was based on the observed cases ( oc ) at each scheduled visit and on data obtained by the last observation estimated forward ( loef ) method , which replaces missing values based on observed previous value and mendiondo s model . , glmm was performed with the glimmix procedure of sas and other analyses were performed using r. a total of 661 patients were enrolled in this study , of whom 642 were evaluable for safety and 554 for efficacy ( figure 1 ) . in the safety analysis set , the mean standard deviation ( sd ) age was 79.107.20 years , the mean disease duration was 0.73 years , and 4.98% of the patients were hospitalized at study entry . the mean daily dose of galantamine was 15.014.76 mg and the mean duration of drug exposure was 343.44202.30 days . the discontinuation rate was ~20% at 12 weeks , 40% at 48 weeks , and 46.73% after 72 weeks ( figure 2 ) . the mean change from baseline to 72 weeks was 0.71 according to oc analysis , 1.21 according to loef analysis , and 3.35 for the predicted outcome without treatment . table 3 displays the patients whose mmse scores were above , within , or below the predicted ci . after 1 year , oc analysis showed that 75.65% of patients had an mmse score significantly above the predicted value without treatment , as did 71.43% of patients at 1.5 years . according to loef analysis , 57.79% of patients had an mmse score significantly above the predicted value after 1 year and 55.75% at 1.5 years . the time course of the observation period in cgi - i is shown in figure 4 . a total of 661 patients were enrolled in this study , of whom 642 were evaluable for safety and 554 for efficacy ( figure 1 ) . in the safety analysis set , the mean standard deviation ( sd ) age was 79.107.20 years , the mean disease duration was 0.73 years , and 4.98% of the patients were hospitalized at study entry . according to the functional assessment of staging scale , most patients had mild ( 39.10% ) or moderate ( 47.66% ) ad at study entry , while the mean mmse score was 18.955.04 . the mean daily dose of galantamine was 15.014.76 mg and the mean duration of drug exposure was 343.44202.30 days . the discontinuation rate was ~20% at 12 weeks , 40% at 48 weeks , and 46.73% after 72 weeks ( figure 2 ) . the mean change from baseline to 72 weeks was 0.71 according to oc analysis , 1.21 according to loef analysis , and 3.35 for the predicted outcome without treatment . table 3 displays the patients whose mmse scores were above , within , or below the predicted ci . after 1 year , oc analysis showed that 75.65% of patients had an mmse score significantly above the predicted value without treatment , as did 71.43% of patients at 1.5 years . according to loef analysis , 57.79% of patients had an mmse score significantly above the predicted value after 1 year and 55.75% at 1.5 years . the time course of the observation period in cgi - i is shown in figure 4 . the present study was designed to assess the long - term efficacy of galantamine in patients with mild - to - moderate ad in comparison with the natural disease trajectory predicted using a mathematical model , as well as investigating the safety and tolerability of galantamine therapy for ad in the real - world clinical setting . furthermore , there was a long - term beneficial effect on cognitive function measured by the mmse compared with the predicted outcome without treatment . when efficacy was assessed on a patient - by - patient basis , about 71% of the patients showed significantly better cognitive function at 72 weeks compared with the predicted outcome . in agreement with the changes of the mmse score , assessment of the cgi - i score also showed that galantamine treatment was effective for maintaining the clinical state ( better or unchanged ) in almost 80% of the patients at 72 weeks . since about 10% of the patients received memantine in addition to galantamine , we also analyzed the data after excluding memantine users , but we found that the changes of the mmse score were essentially the same ( data not shown ) . on the other hand , the indication for memantine therapy is moderate - to - severe ad in japan and the mean baseline mmse score of memantine users was significantly lower than that of non - users ( mean sd : 16.795.41 for memantine users vs 19.134.97 for memantine non - users , t-(427 ) = 2.43 , p=0.02 ) . accordingly , both efficacy and safety data supported the validity of long - term galantamine therapy for mild - to - moderate ad . this study not only demonstrated a long - term beneficial effect of galantamine therapy for ad at the group level , but also revealed that galantamine maintained cognitive function of individual patients in the routine clinical setting . published consensus recommendations and clinical practice guidelines advocate an individualized approach to ad , but there is a need for more evidence that long - term therapy is worthwhile.16,17 as shown in table 1 , our present findings could be useful for assisting physicians and the relatives of patients when deciding treatment options . on the other hand , simulating changes of the mmse score by using mendiondo s model is simple and can be easily applied to individual patients in routine practice . similar to the present results , two previous placebo - controlled studies have demonstrated long - term beneficial effects of chei therapy.6,19 on the other hand , the alzheimer s disease neuroimaging initiative ( adni ) and the australian imaging , biomarkers and lifestyle study obtained opposing results , with greater cognitive decline amongst ad patients taking cheis compared with those not using cheis.20 unlike the aforementioned two randomized controlled trials ( rcts ) showing a favorable effect of cheis , the adni study was not a randomized trial , so treatment selection bias should be taken into consideration when interpreting its results ( ie , physicians might have selectively prescribed cheis for patients with more progressive ad ) . although similar patient factors were considered to be associated with cognitive decline ( baseline mmse and age ) , the previous study demonstrated a more rapid decrease of mmse scores in patients receiving galantamine treatment compared with our results . it is possible that survival bias contributed to the favorable effect of galantamine in the present study . when changes of the mmse score were assessed by oc analysis , the mmse score was evaluated at 72 weeks in only 38.2% of the patients ; so , there is a potential risk of survival bias . since the last observation carried forward method seems to be inappropriate for analysis of mmse decline in patients with a progressive disease like ad , we predicted mmse decline based on mendiondo s model and applied the results to dropouts as a supplementary analysis . however , > 50% of patients still showed significantly better cognitive function compared with the predicted outcome . while we can not completely exclude survival bias , we believe that our data indicate a long - term beneficial effect of galantamine treatment on cognitive function . indeed , a previous long - term randomized head - to - head comparison of galantamine with donepezil and an observational study with a relatively high completion rate demonstrated similar mmse changes to those in our study , with stabilization of mmse scores after 1 year of treatment.12,21 another important difference between the present findings and those of the previous 2-year study of galantamine therapy6 is that the decline of mmse scores predicted by mendiondo s model was more rapid than that observed in the placebo group of the previous study . because the factors associated with mendiondo s model ( age and baseline mmse score ) were similar in both studies , this discrepancy should need to be explained . indeed , a previous pooled analysis of studies on donepezil conducted between 1990 and 1999 revealed that patients allocated to placebo therapy showed slower cognitive decline in more recent trials compared with older trials , despite having more comorbidities.23 furthermore , our previous network meta - analysis of chei therapy revealed an increasing placebo effect on cognitive function over time across various drugs of this class.24 these results suggest that there may have been possible overestimation of the speed of cognitive decline based on mendiondo s model in the present study . however , other studies conducted in the 2000s have revealed similar or more rapid decline of mmse scores than the present data.5,25 for example , a study of the natural history of ad over 3 years reported by holmes and lovestone demonstrated annual decline of the mmse score by 3.4 points ( sd 3.5 : median = 3.0 ) in patients with mild - to - moderate disease.25 this reported rate of decline is larger than the decline simulated by mendiondo s model in this study , but holmes and lovestone investigated a relatively small sample.25 further research is needed to establish a contemporary disease trajectory model of ad for evaluating the efficacy of chei therapy and better management of ad patients first , the diagnosis of ad was done based on physician s clinical judgment rather than the strict diagnostic criteria . one would be concerned about the diagnostic accuracy of this study ; however , the purpose of the study is to show effectiveness of galantamine in the real world . while the influence of the 4 allele on the risk of ad and its age of onset is generally consistent in most studies , there have been widely varying reports with regard to whether different apoe alleles influence the rate of cognitive decline following the onset of dementia.26 mendiondo s model does not require information on apoe alleles and we do not consider the lack of allele data to be a critical flaw . a lower education level is also considered to be a risk factor for developing ad and rapid progression.9,27 however , wattmo et al reported an opposite effect of education level on the long - term outcome of ad , with a high education level being a risk factor for faster cognitive decline.28 thus , the relationship between education and disease progression remains to be clarified . furthermore , the influence of education level on mendiondo s model is smaller than that of age , so lack of education level data in this study should not be a critical flaw . this 1.5-year observational study revealed long - term efficacy of galantamine for maintaining cognitive function in ad patients based on changes of the mmse score , and also showed that galantamine therapy was generally safe and well tolerated . importantly , individual analysis demonstrated that galantamine was beneficial for > 70% of ad patients .

computed tomography ( ct ) has become a key medical examination technique for patient management due to its outstanding diagnostic capabilities . however , the use of x - ray radiation is resulting in the slightly enhanced effective doses even in single ct examination , ranging from 2.5 to dozen msv , in comparison to those from natural ionizing radiation sources ( world average value of 2.4 msv ) . the recent well statistically based epidemiological studies undoubtedly proved a positive association between the radiation dose from ct scans and leukaemia and other tumours in children [ 1 , 2 ] . therefore , the dose reduction in ct has become a top priority for all radiological practices . apart from a spectacular progress in the development of the new generation ct machines with reduced radiation exposure and improvement in the ct standardized protocols , which also substantially decreases the doses during these examinations , the use of the bismuth containing elastomeric shields for these purpose has been also recently strongly recommended . a review of published dosimetry investigations of bismuth shield techniques has been reported by kim et al . . as it is evident from that review , several studies demonstrated that bismuth shields can effectively reduce the radiation dose without degrading image quality and it is a valuable tool to reduce radiation risk in children . a bismuth shield primary function is to remove the lower energy photons contributing to the doses in the surface tissues . however , the use of the bismuth shields has been questioned because of the emission of the scattered additional photons from such shield , which may influence on the quality of the ct scans [ 7 , 8 ] . one of the important source of such scattered radiation are fluorescence photons coming from excitation of heavy metal atoms after absorption of the incoming x - ray radiation . the fluorescence radiation from the bismuth atoms can be reduced by an addition of the second metal additive with slightly lower atomic number , for example tungsten . however , tungsten after excitation emits fluorescence radiation with photon energies in the range of 60 kev , which may also influence both : on the quality of ct scans and surface tissue doses . therefore , sometimes a third metal components , particularly gadolinium is added to the elastomeric shields . the objective of our present study was to evaluate the optimal metal concentration of bi , w and gd in the rubber composites in order to achieve the highest dose reduction factor ( drf ) for such shields . the eight new types of elastomeric composites have been synthesized to evaluate the relation between heavy metal fraction and attenuation efficiency of the x - ray radiation by the shields . especially the influence of the gd concentration on shielding properties of the shields was investigated . for these purposes , eight different samples of elastomeric shields containing bi ( 1535 % ) , w ( 15 % ) , gd ( 024 % ) and sb ( 3 % ) have been prepared . all samples of the elastomeric composites have been prepared after vulcanisation of the natural rubber and heavy metal oxides of bi2o3 , wo3 , gd2o3 and sb2o3 . the samples were synthesized in institute of polymer and dye technology of lodz university of technology . samples were prepared in the form of 140 80 mm sheets and ca 1 mm thickness . the shielding properties evaluation procedure was based on the measurement of the photons intensity out coming from the shield in the different energy spectrum ranges . these photons were induced within the shield by heavy metal atoms ( bi , w , gd and sb ) excitation from the external co-57 source . the measurements were taken within the photon energy range of 20140 kev.table 1mass fractions of elastomeric composites , phrsamplenr ( g)zno ( phr)sulphur ( phr)mbt ( phr)stearic acid ( phr)bi ( bi2o3 ) ( phr)w ( wo3 ) ( phr)gd ( gd2o3 ) ( phr)sb ( sb2o3 ) ( phr)nru1005122gr1100512262.8 ( 70.0)gr2100512290.9 ( 101.3)39.0 ( 49.2)gr31005122117.1 ( 130.5)50.3 ( 63.4)27.5 ( 31.7)gr41005122132.8 ( 148.1)57.0 ( 71.9)31.2 ( 36.0)12.4 ( 14.8)nrbg08100512232.0 ( 35.67)32.0 ( 40.3)17.2 ( 19.8)6.6 ( 7.8)nrbg16100512239.1 ( 43.6)39.1 ( 49.3)41.8 ( 48.1)8.3 ( 9.9)nrbg24100512250.1 ( 55.8)50.0 ( 63.0)80.7 ( 93.0)10.6 ( 12.7 ) nr natural rubber , mbt 2-mercaptobenzothiazole , phr parts per hundred rubber mass fractions of elastomeric composites , phr nr natural rubber , mbt 2-mercaptobenzothiazole , phr parts per hundred rubber the radiation source used for experimental procedure was co-57 closed isotopic source ( polatom , poland ) , emitting two main groups of photons of energy 122.1 ( 85.6 % emission probability ) and 136.5 kev ( 10.7 % ) . radiation detection system used was a coaxial hpge detector ( canberra , gx3020 ) with thin beryllium window , housed in 10 cm thick lead shield lined inside with 1 mm of copper . the resolution of the detector was 0.9 kev for the 122.1 kev peak , and its relative efficiency was 30 % for the 1.33 mev peak . the data were recorded ( each spectrum over 1,800 s ) and processed using genie 2000 software from canberra . the recorded spectra were quantitatively analyzed within the following energy ranges 2035 , 3555 , 5570 , 7090 and 90140 kev . the energy ranges were chosen according to the occurrence of the main x - ray fluorescence photon emission ranges of sb , gd , w and bi respectively [ 12 , 13 ] , and co-57 emission for the range 90140 kev . relative intensity of the x - ray fluorescence emission photons was generally weak comparing with the main 122.1 and 136.5 kev photons of co-57 , so for the weak x - ray fluorescence signals it was necessary to perform correction for the background radiation . prior to the measurements of the protective shields , detection system and method were evaluated according to the procedure based on the measurements of the standardized lead plates described previously elsewhere . calculations were performed using xcom software available from the national institute of standards and technology ( nist ) website [ 14 , 15 ] . the composition data for the human tissue ( soft ) used ( h 10.20 % , c 14.30 % , n 3.40 % , o 70.80 % , na 0.20 % , p 0.30 % , s 0.30 % , cl 0.20 % , k 0.30 % ) to calculate the mass attenuation coefficient and dose reduction factor were also taken from the nist database and based on the data included in the icru 44 report . next to the measurements of the x - ray fluorescence radiation , x - ray attenuation properties of the investigated composites were measured according to the pn - en 61331 - 1:2003 standard by determination of their lead equivalents . the comparative kerma rate in air measurement method was applied using standardized lead foil as a reference material . as a x - ray source gulmay x - ray calibration system 300 kv was used . samples were measured for 4 x - ray photons energies , 45 , 57 , 79 and 104 kev of ~50 % relative width at 1.5 m distance from the x - ray source . air kerma rate was measured with ionization chamber ( open type , model m23361 , ptw freiburg ) connected to a reference electrometer unidos e ( t 10008 type , ptw freiburg ) . lead equivalents for the investigated composites were determined by comparison of attenuation factors obtained for the measured samples with reference curves generated using the standardized lead foil . the main aim of the presented studies was optimization of the heavy metal concentrations in the protective shields previously described by us . the main concept of the elastomeric shields for ct examination was utilization of the heavy metal additives with gradually decreasing atomic number , capable to attenuate efficiently x - ray fluorescence radiation generated within the shield itself . metal concentration data of the elastomeric shields are presented in table 1 ( by components used during synthesis ) and in table 2 ( by element , recalculation based on the data in table 1 ) . two sets of composites were used in experimental procedure . the gr series ( gr1gr4 ) , and nrbg series with constant bi , w and sb concentrations and gradually increasing fraction of gd additive . additionally , beside of the gr and nrbg composites , the raw sample of the pure rubber matrix were used ( nru ) for comparison.table 2elemental composition of the bi w sb composite shieldssample codethickness ( cm)thickness ( g / cm)density ( g / cm)mass fractionhcnosznsbgdwbinru0.0980.0970.9970.10930.81760.00150.00990.02520.0365gr10.0930.1331.4390.06680.49960.00090.04610.01540.02230.3488gr20.1050.01981.8840.04620.34520.00060.08330.01060.01540.14980.3488gr30.0980.2192.2480.03580.26800.00050.09490.00820.01200.08200.14980.3488gr40.0930.2182.3570.03160.23620.00040.10090.00730.01050.03250.08200.14970.3489nrbg080.1150.1911.6580.05630.42100.00080.07970.01300.01880.03070.08040.14970.1498nrbg160.1050.2031.9320.04610.34470.00060.09120.01060.01540.03170.16010.14980.1499nrbg240.0940.2202.3430.03590.26880.00050.10250.00830.01200.03180.24120.14930.1496 elemental composition of the bi sb composite shields generation of the x - ray fluorescence radiation within the shields used for ct examination can be described by the first order linear differential equation ( eq . 1 ) [ 9 , 12 , 13 , 18].1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{d}}r_{\text{f } } \ ; = \;\omega bc_{me}\left ( { \sum\limits_{i } { r_{\gamma i}^{{^{\text{o } } } } } e^ { { - \mu_{i } x } } \tau_{i } } \right){\text{d}}x - r_{\text{f } } \mu_{\text{f } } { \text{d}}x , $ $ \end{document}drf=bcmeirioe-ixidx - rffdx , where rf and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r_{\gamma i}^{{^{\text{o } } } } $ $ \end{document}rio represents the induced x - ray fluorescence radiation intensity and initial intensities of the excitation photons from the co-57 source at 122.1 and 136.5 kev , respectively ; i is the photoelectric absorption coefficient for a given photon energy and metal additive , in cm / g ; i and f are the mass attenuation coefficients for excitation photons and the secondary fluorescence radiation , respectively , in cm / g ; and b represent the k fluorescence yield and branching ratio for the transition of a specific x - ray emission photon energy ; cme is the concentration of the metal additive in the bulk material ; and dx represents the shield surface density increment in g / cm . solving eq . 1 leads to a well known two exponential formula ( eq . 2 ) , where in case of co-57 source , two components should be taken into consideration ( due to emission of two groups of photons : 122.1 and 136.5 kev).2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r_{\text{f } } = \ , \frac{{r_{\gamma 1}^{o } \tau_{1 } \omega bc_{\text{me } } } } { { \mu_{\text{f } } - \mu_{1 } } } \left ( { e^ { { - \mu_{1 } x } } - e^ { { - \mu_{\text{f } } x } } } \right ) + \frac{{r_{\gamma 2}^{o } \tau_{2 } \omega bc_{\text{me } } } } { { \mu_{\text{f } } - \mu_{2 } } } \left ( { e^ { { - \mu_{2 } x } } - e^ { { - \mu_{\text{f } } x } } } \right ) $ $ \end{document}rf = r1o1bcmef-1e-1x - e-fx+r2o2bcmef-2e-2x - e-fx equation 2 with good approximation describes behavior of the x - ray fluorescence radiation induced within the shield , that is its intensity versus thickness of the shield . for a given experimental conditions ( constant geometry , photon flux , detector efficiency ) and for particular shield ( constant composition and exactly defined 1 , 2 and f ) one can observe a saturation curve of emission intensity versus thickness or curve with maximum which position is dependent on the 1 , 2 and f coefficients . figure 1a d present x - ray fluorescence emission intensities of the investigated shields within the specific photon emission energy ranges for sb ( 2035 kev ) , gd ( 3550 kev ) , w ( 5570 kev ) and bi ( 7090 kev ) versus mass thickness of the particular composite . the measured photon emission intensities include scattered radiation and are background corrected in each particular energy range . although background correction , external shield around the measurement system and application of beam collimator it was not possible to avoid completely the weak signals in each energy region originating probably from the external shield and collimator materials . what can be clearly seen , behavior of the two series of investigated composites is significantly different . in all measured energy ranges x - ray fluorescence radiation emission is much stronger for samples containing higher fraction of bi additive ( fig . it is expected as the bi additive is the main component of the gr shields and is responsible for generation of the x - ray fluorescence radiation within the shield . effect of the addition of lower z elements ( w , gd and sb ) is clearly visible in gr series samples , where intensity of the x - ray fluorescence radiation gradually decreases when w , w + gd and w + gd + sb additives are incorporated into the elastomeric matrix ( fig . moreover , the shifts of the emission maxima towards lower thickness can be observed when moving to composite with higher heavy metal fraction , from gr1 ( bi only ) to gr4 ( bi + w + gd + sb ) . one can expect , that increasing bi concentration would result in higher attenuation effect within the shield , but from the other hand it can be also expected , that shields with higher heavy metal content ( especially bi ) will generate x - ray fluorescence radiation in higher extent , what is observed as higher emission intensity within the 7090 kev region . this is clearly visible when we take into account dependence of the x - ray fluorescence radiation emission intensity versus shield thickness for the second series of the investigated composites ( nrbg series ) . nrbg samples consist of decreased concentration of bi fraction ( xbi = 0.15 ) as compared with the gr samples ( xbi = 0.35 ) , whereas w and sb fraction were constant , xw = 0.08 and xsb = 0.03 respectively . the only one variable was gd fraction ( xgd = 0.08 , 0.16 and 0.24 for nrbg08 , nrbg16 and nrbg24 respectively ) . the effect of decreasing bi concentration is visible as an effective reduction of x - ray fluorescence radiation intensity in all analyzed energy regions ( fig . 1a d ) . increasing concentration of gd additive from xgd = 0.080.24 results in further slight reduction of bi emission intensities within the 7090 kev energy range ( fig . generally , it can be explained , that lower emission intensities within the energy ranges of 2035 and 3555 kev is the result of lower fraction of scattered x - ray fluorescence radiation of bi and w origin.fig . 1relative x - ray fluorescence radiation intensities induced in elastomeric composites in a specific energy ranges for : a sb emission ( 2035 kev ) , b gd emission ( 3555 kev ) , c w emission ( 5570 kev ) and d bi emission ( 7090 kev ) relative x - ray fluorescence radiation intensities induced in elastomeric composites in a specific energy ranges for : a sb emission ( 2035 kev ) , b gd emission ( 3555 kev ) , c w emission ( 5570 kev ) and d bi emission ( 7090 kev ) the final evaluation of the investigated shields was based on the calculation of the dose reduction factor ( drf ) , defined as the ratio of dose deposited in the examined tissue with and without application of the protective shield ( eq . 3).3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{drf } } = \frac{d}{{d_{\text{o } } } } = \frac{{\sum\limits_{i } { r_{i } \mu_{\text{ts } } e_{i } } } } { { \sum\limits_{i } { r_{i}^{\text{o } } \mu_{\text{ts } } e_{i } } } } , $ $ \end{document}drf = ddo=iritseiiriotsei , where ri denotes radiation flux ( in 1/cms ) , ts the mass attenuation coefficient for the tissue being irradiated ( in cm / g ) and ei the energy of the absorbed photons ( in kev ) . drf can be a number from the range of 01 , and the lower drf value corresponds to more efficient protective effect of the shield . for calculation of drf simplified model was applied , in which dose delivered to the tissue by photons from the specific energy range was calculated assuming average photon energy ei and average tissue mass attenuation coefficient ts . the most efficient protective effect was achieved for composites with decreased concentration of bi additive from xbi = 0.350.15 , with only slight influence of the gd concentration . the lowest drf value ( 0.47 ) , with 53 % dose reduction capability was observed for nrbg24 composite ( thickness of the sample ca . 2 mm and 0.44 g / cm ) . from the practical reasons , 2 mm shield thickness is still acceptable.table 3dose reduction factors ( drf ) for examined compositescompositeadditive(s ) d ( g / cm ) ( 1 layer)drf drf d ( g / cm ) ( 2 layers)drf drf nru0.0970.920.920.1940.910.91gr1bi ( 0.35)0.1330.910.910.2660.790.79gr2bi + w ( 0.35 + 0.15)0.1980.760.760.3960.580.58gr3bi + w + gd ( 0.35 + 0.15 + 0.08)0.2190.710.710.4380.490.50gr4bi + w + gd + sb ( 0.35 + 0.15 + 0.08 + 0.03)0.2180.730.730.4360.510.51nrbg08bi + w + gd + sb ( 0.15 + 0.15 + 0.08 + 0.03)0.1910.760.760.3820.610.61nrbg16bi + w + gd + sb ( 0.15 + 0.15 + 0.16 + 0.03)0.2030.720.720.4060.540.54nrbg24bi + w + gd + sb ( 0.15 + 0.15 + 0.24 + 0.03)0.2200.650.650.4400.470.46 x - ray fluorescence radiation of bi , w , gd and sb involved only for 122.1 and 136.5 kev co-57 photons dose reduction factors ( drf ) for examined composites x - ray fluorescence radiation of bi , w , gd and sb involved only for 122.1 and 136.5 kev co-57 photons taking into account that total intensity of x - ray photons escaping from the shield is the sum of the excited fluorescence radiation ( rf ) and primary photons not absorbed in the shield ( ra ) one can write that ri = ra + rfi . the primary radiation flux , ra , passing the shield of thickness x can be described by the well known exponential dependence , ra = rexp(x ) . from the described previously eq . 2 and assuming for further simplification that only first part of that equation is important , one can write that intensity of the generated x - ray fluorescence radiation , within the whole energy range , can be roughly expressed by the eq . 4.4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r_{{{\text{f}}i } } = \frac{{r^{\text{o } } \tau \omega bc_{\text{me } } } } { { \mu_{{{\text{f}}i } } - \mu } } \left ( { e^ { - \mu x } - e^ { { - \mu_{{{\text{f}}i } } x } } } \right ) . then we can consider drf coefficient also as shielding properties parameter in terms of both primary , penetrating radiation and that generated x - ray fluorescence radiation . according to eq . 3 and introduced above simplifications , after recalculation we can write , that drf is a function presented by eq . 5.5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{drf } } = e^ { - \mu x } + \mathop \sum \limits_{i } \frac{{\tau_{i } \omega_{i } b_{i } c_{{{\text{me}}i } } } } { { \mu_{{{\text{f}}i } } - \mu } } \left ( { e^ { - \mu x } - e^ { { - \mu_{fi } x } } } \right)\frac{{e_{i } } } { { e^{\text{o } } } } . validity of such approach for drf calculation has been checked for comparison of the proper lead equivalent thicknesses for investigated shields . calculations confirmed that the highest contribution to the dose absorbed is due to the primary radiation with only slight contribution of x - ray fluorescence generated within the shield ( below 10 % of the total photon flux escaping the shield ) . the results of measured and calculated lead equivalents values are presented in table 4 . in most cases experimentally determined lead equivalents well correspond to values obtained by calculations with higher deviations observed for gr2 sample and 79 kev photons . measured lead equivalents thicknesses are in good correlation with calculated drf values , where the same tendency is observed in protective properties sequence of the investigated elastomers , from nrbg24 to nru ( compare tables 3 , 4 ; fig . 2).table 4measured and calculated lead equivalents for investigated elastomeric shieldscompositelead equivalents ( in mmpb ) for photons energy ( kev)45 57 79 104 45 57 79 104 nru0.00320.00410.00420.00360.00290.00400.00710.0030gr10.04200.04200.03700.03800.04500.04600.04900.0450gr20.06600.07200.07600.06900.08500.08700.15000.0870gr30.10000.12000.12000.10000.10000.13000.21000.1000gr40.12000.13000.14000.11000.11000.14000.21000.1100nrbg080.06200.07800.08400.06300.06200.08800.15000.0590nrbg160.08400.12000.12000.08300.07200.13000.19000.0700nrbg240.12000.17000.18000.12000.08500.17000.25000.0840 experimental values values calculatedfig . 2dependence of the measured lead equivalents ( in mmpb ) versus x - ray photons energy for investigated elastomeric shields ; relative error of each measurement 2 % measured and calculated lead equivalents for investigated elastomeric shields dependence of the measured lead equivalents ( in mmpb ) versus x - ray photons energy for investigated elastomeric shields ; relative error of each measurement 2 % another very interesting observation was made when comparing drf values with the ratio of the total x - ray fluorescence radiation to total intensity . 3 . two completely distinct behaviors of the gr and nrbg series are observed . in case of gr series one can observe opposite effect of rf / ro and drf : decreasing drf ( desired effect ) is assisted by simultaneous increasing rf / ro ratio ( higher fraction of x - ray fluorescence radiation , undesired effect ) , whereas in case of nrbg series composites ( nrbg08 , nrbg16 and nrbg24 ) decreasing drf is assisted by simultaneous decreasing of rf / ro ratio resulting in the lower fraction of induced x - ray fluorescence radiation . the same effect as for nrbg series samples is observed also for the raw nru sample.fig . 3correlation between drf and relative total x - ray fluorescence intensity for high bi fraction samples ( gr series , x bi = 0.35 ) and low bi content ( nrbg series , x bi = 0.15 ) ; nru ( reference ) sample without heavy metal additives for comparison correlation between drf and relative total x - ray fluorescence intensity for high bi fraction samples ( gr series , x bi = 0.35 ) and low bi content ( nrbg series , x bi = 0.15 ) ; nru ( reference ) sample without heavy metal additives for comparison the dependence of photon emission intensity within the 90140 kev energy range vs. shield thickness is in good agreement with the exponential absorption law ( data not shown ) and the measured average mass attenuation coefficients are close to these calculated using xcom software and based on the elemental composition of the investigated samples . the relative error of the measured and calculated attenuation coefficients did not exceed 20 % . eight new composites ( seven containing heavy metal additives and reference elastomer matrix sample ) for ct examination procedures were investigated towards their usability for shielding purposes . the best shielding properties were achieved for elastomers containing lower concentration of bi ( xbi = 0.15 vs. 0.35 ) and variable fraction of gd additive ( xgd = 0.080.24 ) . shielding performance of the examined elastomeric composites were determined for both characteristic x - ray fluorescence emission lines and scattered radiation . such approach lead to a more reliable results in comparison with traditional method when only energetically narrow beam of photons is considered for a total dose delivered to the tissue . the most important fraction of the induced x - ray fluorescence photons was that originating from bi and w additive . x - ray fluorescence radiation induced in gd and sb additives is of lower importance , as it constitutes of only small fraction of the total x - ray fluorescence radiation generated within the shield . due to this fact , decreasing of bi concentration ( from xbi = 0.350.15 ) in the shields results in significant lowering of the total x - ray fluorescence radiation intensity both in the 5570 ( w ) and 7090 kev ( bi ) energy regions as well as in the lower energy regions of gd and sb excitations . calculations of the drf values led us to conclusion that the best shielding properties exhibit nrbg24 composite . next to these observations , both methods of verification of shielding properties of the investigated composites indicate for the analogical order of the protective shields performance .

eight elastomeric composites ( nru , gr1gr4 , nrbg08nrbg24 ) containing mixtures of different proportions of heavy metal additives ( bi , w , gd and sb ) have been synthesized and examined as protective shields . the nru sample was a pure rubber matrix and served as a reference sample for heavy metal modified composites . experimental procedure used for evaluation of the composite shields and their attenuation properties was based on the utilization of hpge spectrometry and analysis of x - ray fluorescence radiation intensity of the heavy metal additives in the following energy ranges for : sb ( 2035 kev ) , gd ( 3555 kev ) , w ( 5570 kev ) and bi ( 7090 kev ) . the main contributor to the induced x - ray fluorescence radiation within the shield is bi additive and the intensity of the x - ray radiation generated within the energy range of 7090 kev strongly depends on its concentration . it was found that decreasing concentration of the bi fraction from 0.35 ( gr samples ) to 0.15 ( nrbg samples ) results in significant lowering bi x - ray fluorescence radiation within the 7090 kev energy range . secondary effect of decreasing bi concentration was efficient diminishing excitation processes for lower z heavy metal additives ( w , gd and sb , gr vs. nrbg samples ) . as the final quality parameter of the shielding properties for the examined elastomers , dose reduction factor ( drf ) coefficients were calculated for each shield . it was found , that the best shielding properties are observed for composites with lower bi concentration ( 0.15 vs. 0.35 bi mass fraction ) with only slight further improvement of their parameters ( drf ) with increasing of gd concentration ( gd mass fraction 0.08 , 0.16 and 0.24 ) . the most efficient dose reduction composite was found to be nrbg24 elastomer with drf value 0.47 ( 53 % dose reduction ) for ca . 2 mm and 0.44 g / cm2 layer thickness .

Introduction Materials and methods Results and discussion Conclusions

the objective of our present study was to evaluate the optimal metal concentration of bi , w and gd in the rubber composites in order to achieve the highest dose reduction factor ( drf ) for such shields . the eight new types of elastomeric composites have been synthesized to evaluate the relation between heavy metal fraction and attenuation efficiency of the x - ray radiation by the shields . for these purposes , eight different samples of elastomeric shields containing bi ( 1535 % ) , w ( 15 % ) , gd ( 024 % ) and sb ( 3 % ) have been prepared . the shielding properties evaluation procedure was based on the measurement of the photons intensity out coming from the shield in the different energy spectrum ranges . these photons were induced within the shield by heavy metal atoms ( bi , w , gd and sb ) excitation from the external co-57 source . the measurements were taken within the photon energy range of 20140 kev.table 1mass fractions of elastomeric composites , phrsamplenr ( g)zno ( phr)sulphur ( phr)mbt ( phr)stearic acid ( phr)bi ( bi2o3 ) ( phr)w ( wo3 ) ( phr)gd ( gd2o3 ) ( phr)sb ( sb2o3 ) ( phr)nru1005122gr1100512262.8 ( 70.0)gr2100512290.9 ( 101.3)39.0 ( 49.2)gr31005122117.1 ( 130.5)50.3 ( 63.4)27.5 ( 31.7)gr41005122132.8 ( 148.1)57.0 ( 71.9)31.2 ( 36.0)12.4 ( 14.8)nrbg08100512232.0 ( 35.67)32.0 ( 40.3)17.2 ( 19.8)6.6 ( 7.8)nrbg16100512239.1 ( 43.6)39.1 ( 49.3)41.8 ( 48.1)8.3 ( 9.9)nrbg24100512250.1 ( 55.8)50.0 ( 63.0)80.7 ( 93.0)10.6 ( 12.7 ) nr natural rubber , mbt 2-mercaptobenzothiazole , phr parts per hundred rubber mass fractions of elastomeric composites , phr nr natural rubber , mbt 2-mercaptobenzothiazole , phr parts per hundred rubber the radiation source used for experimental procedure was co-57 closed isotopic source ( polatom , poland ) , emitting two main groups of photons of energy 122.1 ( 85.6 % emission probability ) and 136.5 kev ( 10.7 % ) . the energy ranges were chosen according to the occurrence of the main x - ray fluorescence photon emission ranges of sb , gd , w and bi respectively [ 12 , 13 ] , and co-57 emission for the range 90140 kev . relative intensity of the x - ray fluorescence emission photons was generally weak comparing with the main 122.1 and 136.5 kev photons of co-57 , so for the weak x - ray fluorescence signals it was necessary to perform correction for the background radiation . next to the measurements of the x - ray fluorescence radiation , x - ray attenuation properties of the investigated composites were measured according to the pn - en 61331 - 1:2003 standard by determination of their lead equivalents . the main concept of the elastomeric shields for ct examination was utilization of the heavy metal additives with gradually decreasing atomic number , capable to attenuate efficiently x - ray fluorescence radiation generated within the shield itself . additionally , beside of the gr and nrbg composites , the raw sample of the pure rubber matrix were used ( nru ) for comparison.table 2elemental composition of the bi w sb composite shieldssample codethickness ( cm)thickness ( g / cm)density ( g / cm)mass fractionhcnosznsbgdwbinru0.0980.0970.9970.10930.81760.00150.00990.02520.0365gr10.0930.1331.4390.06680.49960.00090.04610.01540.02230.3488gr20.1050.01981.8840.04620.34520.00060.08330.01060.01540.14980.3488gr30.0980.2192.2480.03580.26800.00050.09490.00820.01200.08200.14980.3488gr40.0930.2182.3570.03160.23620.00040.10090.00730.01050.03250.08200.14970.3489nrbg080.1150.1911.6580.05630.42100.00080.07970.01300.01880.03070.08040.14970.1498nrbg160.1050.2031.9320.04610.34470.00060.09120.01060.01540.03170.16010.14980.1499nrbg240.0940.2202.3430.03590.26880.00050.10250.00830.01200.03180.24120.14930.1496 elemental composition of the bi sb composite shields generation of the x - ray fluorescence radiation within the shields used for ct examination can be described by the first order linear differential equation ( eq . 1 ) [ 9 , 12 , 13 , 18].1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{d}}r_{\text{f } } \ ; = \;\omega bc_{me}\left ( { \sum\limits_{i } { r_{\gamma i}^{{^{\text{o } } } } } e^ { { - \mu_{i } x } } \tau_{i } } \right){\text{d}}x - r_{\text{f } } \mu_{\text{f } } { \text{d}}x , $ $ \end{document}drf=bcmeirioe-ixidx - rffdx , where rf and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r_{\gamma i}^{{^{\text{o } } } } $ $ \end{document}rio represents the induced x - ray fluorescence radiation intensity and initial intensities of the excitation photons from the co-57 source at 122.1 and 136.5 kev , respectively ; i is the photoelectric absorption coefficient for a given photon energy and metal additive , in cm / g ; i and f are the mass attenuation coefficients for excitation photons and the secondary fluorescence radiation , respectively , in cm / g ; and b represent the k fluorescence yield and branching ratio for the transition of a specific x - ray emission photon energy ; cme is the concentration of the metal additive in the bulk material ; and dx represents the shield surface density increment in g / cm . 2 ) , where in case of co-57 source , two components should be taken into consideration ( due to emission of two groups of photons : 122.1 and 136.5 kev).2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r_{\text{f } } = \ , \frac{{r_{\gamma 1}^{o } \tau_{1 } \omega bc_{\text{me } } } } { { \mu_{\text{f } } - \mu_{1 } } } \left ( { e^ { { - \mu_{1 } x } } - e^ { { - \mu_{\text{f } } x } } } \right ) + \frac{{r_{\gamma 2}^{o } \tau_{2 } \omega bc_{\text{me } } } } { { \mu_{\text{f } } - \mu_{2 } } } \left ( { e^ { { - \mu_{2 } x } } - e^ { { - \mu_{\text{f } } x } } } \right ) $ $ \end{document}rf = r1o1bcmef-1e-1x - e-fx+r2o2bcmef-2e-2x - e-fx equation 2 with good approximation describes behavior of the x - ray fluorescence radiation induced within the shield , that is its intensity versus thickness of the shield . figure 1a d present x - ray fluorescence emission intensities of the investigated shields within the specific photon emission energy ranges for sb ( 2035 kev ) , gd ( 3550 kev ) , w ( 5570 kev ) and bi ( 7090 kev ) versus mass thickness of the particular composite . it is expected as the bi additive is the main component of the gr shields and is responsible for generation of the x - ray fluorescence radiation within the shield . effect of the addition of lower z elements ( w , gd and sb ) is clearly visible in gr series samples , where intensity of the x - ray fluorescence radiation gradually decreases when w , w + gd and w + gd + sb additives are incorporated into the elastomeric matrix ( fig . one can expect , that increasing bi concentration would result in higher attenuation effect within the shield , but from the other hand it can be also expected , that shields with higher heavy metal content ( especially bi ) will generate x - ray fluorescence radiation in higher extent , what is observed as higher emission intensity within the 7090 kev region . this is clearly visible when we take into account dependence of the x - ray fluorescence radiation emission intensity versus shield thickness for the second series of the investigated composites ( nrbg series ) . nrbg samples consist of decreased concentration of bi fraction ( xbi = 0.15 ) as compared with the gr samples ( xbi = 0.35 ) , whereas w and sb fraction were constant , xw = 0.08 and xsb = 0.03 respectively . the effect of decreasing bi concentration is visible as an effective reduction of x - ray fluorescence radiation intensity in all analyzed energy regions ( fig . increasing concentration of gd additive from xgd = 0.080.24 results in further slight reduction of bi emission intensities within the 7090 kev energy range ( fig . generally , it can be explained , that lower emission intensities within the energy ranges of 2035 and 3555 kev is the result of lower fraction of scattered x - ray fluorescence radiation of bi and w origin.fig . 1relative x - ray fluorescence radiation intensities induced in elastomeric composites in a specific energy ranges for : a sb emission ( 2035 kev ) , b gd emission ( 3555 kev ) , c w emission ( 5570 kev ) and d bi emission ( 7090 kev ) relative x - ray fluorescence radiation intensities induced in elastomeric composites in a specific energy ranges for : a sb emission ( 2035 kev ) , b gd emission ( 3555 kev ) , c w emission ( 5570 kev ) and d bi emission ( 7090 kev ) the final evaluation of the investigated shields was based on the calculation of the dose reduction factor ( drf ) , defined as the ratio of dose deposited in the examined tissue with and without application of the protective shield ( eq . the most efficient protective effect was achieved for composites with decreased concentration of bi additive from xbi = 0.350.15 , with only slight influence of the gd concentration . the lowest drf value ( 0.47 ) , with 53 % dose reduction capability was observed for nrbg24 composite ( thickness of the sample ca . from the practical reasons , 2 mm shield thickness is still acceptable.table 3dose reduction factors ( drf ) for examined compositescompositeadditive(s ) d ( g / cm ) ( 1 layer)drf drf d ( g / cm ) ( 2 layers)drf drf nru0.0970.920.920.1940.910.91gr1bi ( 0.35)0.1330.910.910.2660.790.79gr2bi + w ( 0.35 + 0.15)0.1980.760.760.3960.580.58gr3bi + w + gd ( 0.35 + 0.15 + 0.08)0.2190.710.710.4380.490.50gr4bi + w + gd + sb ( 0.35 + 0.15 + 0.08 + 0.03)0.2180.730.730.4360.510.51nrbg08bi + w + gd + sb ( 0.15 + 0.15 + 0.08 + 0.03)0.1910.760.760.3820.610.61nrbg16bi + w + gd + sb ( 0.15 + 0.15 + 0.16 + 0.03)0.2030.720.720.4060.540.54nrbg24bi + w + gd + sb ( 0.15 + 0.15 + 0.24 + 0.03)0.2200.650.650.4400.470.46 x - ray fluorescence radiation of bi , w , gd and sb involved only for 122.1 and 136.5 kev co-57 photons dose reduction factors ( drf ) for examined composites x - ray fluorescence radiation of bi , w , gd and sb involved only for 122.1 and 136.5 kev co-57 photons taking into account that total intensity of x - ray photons escaping from the shield is the sum of the excited fluorescence radiation ( rf ) and primary photons not absorbed in the shield ( ra ) one can write that ri = ra + rfi . 2 and assuming for further simplification that only first part of that equation is important , one can write that intensity of the generated x - ray fluorescence radiation , within the whole energy range , can be roughly expressed by the eq . calculations confirmed that the highest contribution to the dose absorbed is due to the primary radiation with only slight contribution of x - ray fluorescence generated within the shield ( below 10 % of the total photon flux escaping the shield ) . in case of gr series one can observe opposite effect of rf / ro and drf : decreasing drf ( desired effect ) is assisted by simultaneous increasing rf / ro ratio ( higher fraction of x - ray fluorescence radiation , undesired effect ) , whereas in case of nrbg series composites ( nrbg08 , nrbg16 and nrbg24 ) decreasing drf is assisted by simultaneous decreasing of rf / ro ratio resulting in the lower fraction of induced x - ray fluorescence radiation . 3correlation between drf and relative total x - ray fluorescence intensity for high bi fraction samples ( gr series , x bi = 0.35 ) and low bi content ( nrbg series , x bi = 0.15 ) ; nru ( reference ) sample without heavy metal additives for comparison correlation between drf and relative total x - ray fluorescence intensity for high bi fraction samples ( gr series , x bi = 0.35 ) and low bi content ( nrbg series , x bi = 0.15 ) ; nru ( reference ) sample without heavy metal additives for comparison the dependence of photon emission intensity within the 90140 kev energy range vs. shield thickness is in good agreement with the exponential absorption law ( data not shown ) and the measured average mass attenuation coefficients are close to these calculated using xcom software and based on the elemental composition of the investigated samples . the best shielding properties were achieved for elastomers containing lower concentration of bi ( xbi = 0.15 vs. 0.35 ) and variable fraction of gd additive ( xgd = 0.080.24 ) . x - ray fluorescence radiation induced in gd and sb additives is of lower importance , as it constitutes of only small fraction of the total x - ray fluorescence radiation generated within the shield . due to this fact , decreasing of bi concentration ( from xbi = 0.350.15 ) in the shields results in significant lowering of the total x - ray fluorescence radiation intensity both in the 5570 ( w ) and 7090 kev ( bi ) energy regions as well as in the lower energy regions of gd and sb excitations .

covalently cross - linked hydrogels are applied as cell culture templates , absorbent materials , nonfouling coatings , contact lenses , and drug delivery vehicles . owing to high water content , reasonable transport of small molecules , and robust mechanical properties , covalently cross - linked hydrogels are particularly attractive materials for a broad array of biological and cellular applications . these reticulated polymer networks are formed by chemical cross - linking of hydrophilic macromolecules , such as synthetically derived poly(ethylene glycol ) ( peg ) or poly(vinyl alcohol ) and naturally derived hyaluronic acid , gelatin , or alginate , often mildly and in the presence of cells . despite the prevalence of hydrogels in the biomedical sciences , the manner by which the cross - linking mechanism and resultant microarchitecture of the hydrogel influences the macroscopic properties ( e.g. , strength , toughness , and degradation ) is still not fully elucidated . a better understanding of the structure function relationship in hydrogel performance would enable improved rational design of materials for a range of targeted applications . cross - linked , synthetic hydrogels have been formed traditionally through a free - radical initiated chain polymerization of telechelic monomers ( e.g. , diacrylated peg or 2-hydroxy ethyl methacrylate copolymerized with diethylene glycol dimethacrylate ) . in this manner , hydrogels have been fabricated rapidly with tunable material properties and have been functionalized with adhesion peptides and degradation sites . however , radical initiated chain polymerizations are limited in that they are inhibited by oxygen , proceed with complex kinetics , can be damaging to nucleic acids and proteins , and inherently introduce inhomogeneities into the network structure . these inhomogeneities compromise the material properties as stress is focused on weak portions of the network , reducing the macroscopic integrity of the hydrogel . recent work has focused on the formation of cross - linked hydrogels with more ideal and homogeneous microstructures to improve network cooperativity and increase hydrogel mechanical integrity . this has been achieved through the step polymerization of complementary , end - terminated comonomers . originally , hubbell and co - workers demonstrated the formation of step - polymerized hydrogels by cross - linking thiol and electron - poor , vinyl functionalized peg - based molecules for drug delivery and cell encapsulation . this paradigm has been extended to fabricate gels utilizing several different step growth reactions and associated functional groups , including the copper - catalyzed , huisgen azide alkyne coupling of functionalized peg - based comonomers , the coupling of propylamine terminated peg with succinimidyl glutarate terminated peg , and the photoinitiated thiol ene coupling of norbornene functionalized peg with dithiol peptides . uniquely , deforest et al . demonstrated the formation of step - polymerized hydrogels through the copper - free , strain promoted azide alkyne cycloaddition ( spaac ) , forming hydrogels in a bio - orthogonal and cytocompatible manner . seminal mechanical analyses of step - polymerized gels have found that these networks possess increased tensile extension as compared to chain - polymerized analogues , while sans data have demonstrated that these networks , although still not perfectly ideal , possess fewer heterogeneities in the network microstructure . while differences between chain and step polymerization mechanisms and resultant hydrogels are clear , there is little literature on the direct comparison of mechanics and degradation between chain - polymerized and step - polymerized hydrogels . function relationship of hydrogels through direct comparisons between chain- and step - polymerized hydrogels with similar chemical structures but profoundly different network connectivities , which will enable the rational design and application of unique hydrogel - based materials . furthermore , there is a growing interest in controlling the material properties of both step- and chain - polymerized hydrogels dynamically and in a user - defined fashion using cleavable chemistries whose degradation can be triggered exogenously . toward this end , recent work has presented a class of photodegradable hydrogels whose physical and chemical properties can be modified by light postfabrication with full spatial and temporal control . photodegradable hydrogels are appropriate for a myriad of applications in the biomedical and materials sciences . within the tissue engineering field there is a particular interest in designing cytocompatible , photodegradable hydrogels that allow the experimenter to control the extracellular microenvironment in the presence of cells in 3d and in real time . meanwhile , the drug delivery community is exploiting photodegradable hydrogels to release factors at specific locations and at precise times . for photodegradable hydrogels to be utilized most effectively in the broad range of applications , a precise and predictable understanding of how irradiation and network structure influence degradation - induced changes in material properties is required . in addition , photodegradation suggests unique opportunities to perform experiments that might provide a better understanding as to how network structure influences material properties during temporally regulated changes to the hydrogel structure . this work presents the synthesis and characterization of hydrogel networks that are formed by both chain and step polymerizations of a single photodegradable peg - based macromolecular precursor as model systems to understand differences in both mechanical properties and degradation between the resultant network structures . the formation and associated material properties of the hydrogels are investigated and compared . furthermore , the photolabile linker in the hydrogel is employed to compare and contrast the photodegradation - induced changes in the two gels . a previously developed statistical - kinetic model of photodegradation is adapted and expanded to describe the degradation of step growth networks . this model accurately describes degradation differences between hydrogels formed by chain and step growth mechanisms , elucidating aspects of the structure function relationship in hydrogel photodegradation . in all , the material chemistry enables a more robust understanding of how network connectivity and gel architecture influence properties and degradation , and this fundamental understanding should translate into an improved design of hydrogel cells carriers and drug delivery vehicles for biomedical applications . all reagents were purchased from sigma - aldrich and used as received except as otherwise noted . a photolabile , acrylate functionalized monomer , poly(ethylene glycol ) diphotodegradable acrylate ( pegdipda ) , was synthesized according to previous published protocols . briefly , an acrylated , o - nitrobenzyl ether was synthesized and coupled to poly(ethylene glycol ) bisamine ( mn 3400 da ; laysan bio inc . ) to generate a photoresponsive monomer that is capable of forming both chain- and step - polymerized networks . four - arm poly(ethylene glycol ) macromolecules ( mn 10 kda and mn 5 kda ; jenkem technology usa ) functionalized with thiol end groups ( peg4sh ) were synthesized according to a previously published protocol . chain - polymerized hydrogels were fabricated by copolymerizing pegdipda with monoacrylated poly(ethylene glycol ) ( mn 400 da , pega ; monomer - polymer dajac laboratories ) via redox - initiated , free - radical chain polymerization . stock solutions of the gel - forming precursors were prepared : 49 mm pegdipda in pbs , 1 m pega in pbs , 2 m ammonium persulfate ( aps ) in pbs , and 2 m tetramethylethylenediamine ( temed ) in pbs . three chain - polymerized hydrogels were fabricated for this work by varying the ratio of pegdipda to pega at a constant total polymer wt % of 15 wt % . pegdipda and pega were combined in pbs at final solution concentrations of 26.5 mm and 105 mm , respectively to form gel a. pegdipda and pega were combined in pbs at final solution concentrations of 17.2 mm and 200 mm , respectively to form gel b. pegdipda and pega were combined in pbs at final solution concentrations of 12.3 mm and 250 mm , respectively to form gel c. to initiate polymerization , aps and then temed were added to each solution while vortexing at final solution concentrations of 0.2 and 0.1 m , respectively . the solutions were reacted for 7 min to achieve complete polymerization , upon which the gels were swelled in pbs . gels were formed in situ on a parallel - plate shear rheometer ( 50 m thick ; ta instruments ares 4400 ) or between glass slides separated by 0.5 1.5 mm thick silicon rubber gaskets . step - polymerized hydrogels were fabricated by copolymerizing pegdipda with thiol - functionalized , four - arm poly(ethylene glycol ) ( peg4sh ; mn 5k or 10k ) via base - catalyzed , michael - addition . stock solutions of the gel - forming precursors were prepared : 49 mm pegdipda in pbs ph 8.0 , 20 mm peg4sh 10k in pbs ph 8.0 , 40 mm peg4sh 5k in pbs ph 8.0 , and 1 m triethanolamine ( teoha ) in pbs three step - polymerized hydrogels were fabricated for this work by varying the molecular weight of the peg4sh ( 5k or 10k ) and altering the ratio of acrylates to thiols at a constant total polymer wt % of 10 wt % . pegdipda and peg4sh 10k were combined in pbs ph 8.0 at final solution concentrations of 11 mm and 5.5 mm ( r = 1 ) , respectively to form gel d. pegdipda and peg4sh 10k were combined in pbs ph 8.0 at final solution concentrations of 9.8 mm and 6.0 mm ( r = 0.83 ) , respectively to form gel e. pegdipda and peg4sh 5k were combined in pbs ph 8.0 at final solution concentrations of 15.2 mm and 7.6 mm ( r = 1 ) , respectively to form gel f. to accelerate polymerization , teoha was added to each solution while vortexing at a final solution concentration of 0.3 m. the solution were reacted for 25 min to achieve complete polymerization , upon which the gels were swelled in pbs . gels were formed in situ on a parallel - plate shear rheometer ( 50 m thick ; ta instruments ares 4400 ) or between glass slides separated by 0.51.5 mm thick silicon rubber gaskets . in situ polymerization was quantified with time sweep tests on gelling solutions in a parallel - plate shear rheometer ( ta instruments ares 4400 ; 8.0 mm diameter and 0.05 mm height ) . time sweep tests were conducted at 10 rad / s with 10% strain , which was determined to be in the linear viscoelastic regime for both chain- and step - polymerized hydrogels . polymerization was followed until the shear storage modulus ( g ) reached a plateau ( n = 3 for each gel type ) . young s modulus was reported as three times the shear storage modulus based on the poisson ratio for peg - based hydrogels . for each gel type , gel samples ( n = 6 ) were swollen and weighed in the equilibrium swollen state . the gels were subsequently lyophilized to remove the water weight from the samples and the dry weight was measured . the ratio of the equilibrium swollen weight to the dry weight was used to calculate q , the mass swelling ratio . the volumetric swelling ratio , q , was then calculated from the mass swelling ratio . tensile testing of chain- and step - polymerized hydrogels ( n = 3 for each gel type ) was performed in uniaxial extension with a materials tester ( mts synergie 100 ) with a 10 n load head . swollen hydrogels were cut into 5 mm 25 mm rectangles , and the width , length , and thickness of each sample was measured with digital calipers prior to analysis . each sample was fixed on the materials tester by compression clamps at the top and bottom of the sample ( 5 mm from each end of the gel ) , and the local environment was kept humidified during the analyses . the initial separation distance was measured with digital calipers , and a constant strain rate of 0.15 mm / mm / min was applied to the sample to failure . the load , stress , strain , and elongation values recorded were used to calculate the stress and strain from the measured dimensions of each sample . the percent strain at failure was calculated as the final extension divided by the initial separation distance multiplied by 100 , and the toughness was calculated by numerically integrating for the area under the stress strain curve . to determine the shear strain to yield for each of the hydrogels , hydrogel samples ( n = 3 for each gel type ) once the gels reached complete polymerization , a strain sweep was performed from 1% to 1000% strain at a constant frequency of 2.5 rad / s while monitoring the storage modulus ( g ) and the loss modulus ( g ) values . shear strain to yield was characterized as the strain at which the value of the storage modulus fell below the value of the loss modulus . the kinetics of the photodegradation reaction in both chain- and step - polymerized hydrogels was quantified by irradiating ( = 365 nm ; i0 = 20 mw / cm ) in situ polymerized gels on a parallel - plate shear rheometer ( ta instruments ares 4400 ) and following the modulus evolution as a function of irradiation time . the normalized modulus g/ g0 is proportional to the normalized number density of elastically active network strands /0 , where is the number density of elastically active network strands , for each gel system . as irradiation cleaves bonds within the nbe moiety in the pegdipda molecule , elastically active network strands are broken and based on polymer physics and photoreaction kinetics : wherehere , is the quantum yield of the nbe moiety ; is the molar absorptivity of the nbe moiety at the wavelength of irradiation ( = 4300 l mol cm for = 365 nm ) ; i0 is the incident irradiation intensity ( w cm ) ; na is avogadro s number ; h is the planck constant ; is the frequency at the wavelength of irradiation ; keff is the effective rate constant by gathering all variables except for i0 . a linear fit of ln(g/g0 ) , as measured by the rheometry experiments , as a function of irradiation time was employed to calculate k and , thus , keff for both chain- and step - polymerized hydrogels . even though these gels are optically thick ( a > 0.1 ) , similar methods to use rheometry to quantify photodegradation kinetics in other optically thick gels have been verified by nmr on optically thin samples , demonstrating that this is a viable technique for assessing degradation kinetics . collimated light was delivered from an omnicure s1000 with an internal 365 nm filter through a liquid filled light guide and collimating lens . irradiation intensities for all degradation experiments were measured with a calibrated radiometer ( model il1400a , international light , inc . , newburyport , ma ) , and attenuation of light by the rheometer or photomasks was accounted for by increasing the incident light intensity so that the transmitted light was at the desired intensity . photopatterns ( 400 m wide black lines spaced by 400 m ) were originally drawn in adobe illustrator and printed on mylar ( advance reproductions , north andover , ma ) . swollen chain- and step - polymerized gels ( 10 mm 10 mm 1 mm ) were aligned under the channel patterns and surrounded by pbs to maintain hydration and facilitate dissolution of degraded products during patterning . the gels were then exposed to collimated 365 nm light at 10 mw / cm for up to 30 min ( omnicure s1000 with 365 nm filter , liquid filled light guide , and collimating lens , exfo ) . depths of the patterned channels were verified with a profilometer ( stylus profiler , dektak 6 m ) . a statistical - kinetic model of photodegradation in chain - polymerized networks was applied to model the erosion depth as a function of time for the chain - polymerized hydrogels in this work . this model was extended to describe photodegradation in step growth networks by altering the statistical assumptions of network connectivity to account for the differences in network structure . furthermore , as the time scale of erosion is much faster for step - polymerized hydrogels than chain - polymerized an additional dissolution assumption was included . briefly , this states that eroded products at the surface of the gel do not instantly diffuse out of the light path , but diffuse through the pbs solution in the light path to a solution sink at the original surface of the gel . by including this simple assumption , the statistical - kinetic model was able to describe the erosion depth as a function of irradiation time in both chain- and step - polymerized hydrogels . chain - polymerized ( cp ) hydrogels were formed by reacting the tetrafunctional pegdipda with a difunctional comonomer , pega , under redox - initiated free - radical chain polymerization . step - polymerized ( sp ) hydrogels were formed by reacting the difunctional pegdipda with a tetrafunctional comonomer , peg4sh , through a base - catalyzed michael addition . in each case , the network formation occurred through the chemical bonding of the acrylate - functionalized pegdipda . in the chain polymerization each acrylate is difunctional allowing the pegdipda to serve as a tetrafunctional cross - linker , whereas in the step polymerization each acrylate is monofunctional extending the elastically active chains between the tetrafunctional peg4sh cross - linkers . previous studies have shown that the network microstructure of peg gels formed by chain polymerization is comprised of dense polyacrylate kinetic chains connected by peg cross - links . these heterogeneities exist on the length scale of the peg cross - linker , while further heterogeneities form as radical initiation stochastically leads to regions of increased cross - linking density on the micrometer scale . in contrast , peg hydrogels formed by step polymerizations have been shown to possess fewer heterogeneities on all length scales . these heterogeneities are limited generally to cyclization and dangling ends . in this manner , the chain polymerization ( cp ) of pegdipda formed a heterogeneous network structure , while the step polymerization ( sp ) formed a more ideal network structure ( figure 1 ) . chain - polymerized and step - polymerized hydrogels were formed with the same photolabile monomer , pegdipda . chain - polymerized hydrogels ( cp gels ) were fabricated through the copolymerization of pegdipda with pega via free - radical polymerization , resulting in a heterogeneous network structure . step - polymerized hydrogels ( sp gels ) were fabricated through the copolymerization of pegdidpa with peg4sh via michael - addition polymerization . in this study , three chain - polymerized ( a c ) and three step - polymerized ( d f ) hydrogels were fabricated from an array of macromolecular solutions ( table 1 ) . it is difficult to generate chain- and step - polymerized hydrogels with directly comparable properties as the formation mechanisms lead to differences in the length of the network chains , the degree of cyclization , and swelling behavior . therefore , a range of materials was tested to study the effect of formation mechanism ( chain or step polymerization ) on mechanical properties , such as tensile strain to failure , tensile toughness , and shear strain to yield . the formulations for chain - polymerized ( a c ) and step - polymerized ( d f ) hydrogels are detailed in the materials and methods section . peg4sh is presented as concentration ( mm ) and molecular weight ( mn in daltons ) . hydrogels were formed for each gel system in situ on a parallel plate rheometer to quantify the young s modulus ( e ) ( table 1 ) and time to complete polymerization . all hydrogels formed in less than 25 min with a young s modulus ranging from 10 to 20 kpa . in all cases , the total polymerization time can be tuned by altering the initial macromer concentration and the initiator concentrations ( ammonium persulfate / temed for the chain polymerization and triethanolamine / ph for the step polymerization ) . ( data not shown ) the volumetric swelling ratio ( q ) for the hydrogels ranged from 12 to 20 . it has been suggested that the increased homogeneity and network cooperativity of sp hydrogels results in an increase in mechanical integrity , specifically tensile strain to break , as compared to cp hydrogels . here , network cooperativity is used to describe the ability of multiple network chains within a gel to distribute mechanical stress cooperatively over the network chains . to compare the tensile properties of the chain- and step - polymerized peg hydrogels studied in this work , the percent strains to failure for cp gels were 33 4% , 33 5% , and 20 3% for a , b , and c , respectively . whereas , the percent strains to failure for sp gels were 129 11% , 87 15% , and 112 6% for d , e , and f , respectively ( table 1 ; figure 2a , b ) . these data indicated that , in all cases , the sp gels were more ductile than the cp gels . further analysis of the tensile testing data revealed that sp gels possessed increased tensile toughness compared to cp gels ( table 1 ; figure 2a , c ) . specifically , the tensile toughness of the sp gels were 4.1 0.2 , 6.0 1.4 , and 14.5 2.0 kpa for d , e , and f , respectively , while the tensile toughness for the cp gels were 2.2 0.2 , 1.3 0.3 , and 0.5 0.2 kpa for a , b , and c , respectively . in addition to the tensile testing , strain sweeps on in situ polymerized hydrogels were conducted to investigate the shear strain to yield for each of the samples . the sp gels exhibited increased shear strain to yield in all cases as compared to the cp gels ( table 1 ; figure 2d ) , 420 40 , 500 70 , and 290 70% for sp gels d , e , and f , respectively , and 89 6 , 130 1 , and 93 4% for cp gels , a , b , and c , respectively . mechanical analysis of chain- and step - polymerized hydrogels . ( a ) uniaxial extension of cp and sp gels was conducted to measure the percent strain to failure and modulus of toughness from the stress strain curves . ( b ) the average percent strain to failure was increased for all sp gels ( dashed bars , d f ) as compared to cp gels ( solid bars , a c ) . f ) also possessed increased tensile toughnesses as compared to cp gels ( solid bars , a c ) . ( d ) the yield behavior of the hydrogels was analyzed on a parallel plate rheometer to determine the shear strain to yield for each sample . as with the tensile analyses , the sp gels ( dashed bars , d f ) demonstrated increased shear strains to yield as compared to cp gels ( solid bars , a , it was observed that mechanical integrity was improved for hydrogels formed by step polymerization as compared to chain polymerization . these differences in material properties were conferred by the network structure , specifically the increased network cooperativity and decreased heterogeneity in the sp hydrogel , and suggest that applications that require more ductile or tough materials should employ sp hydrogels . in addition to mechanical integrity , network connectivity directly relates to the diffusion of macromolecules through the hydrogel network and ideal gels should facilitate more uniform diffusion as compared to heterogeneous gels . finally , these data suggest that mechanical stresses were translated anisotropically in heterogeneous , cp gels , which may be important for mechanical stimulation or differentiation of mammalian cells . the cp and sp gels were formed from the same photolabile monomer , pegdipda , rendering them photodegradable . the degradation is facilitated by the o - nitrobenzyl ether ( nbe ) moieties that reside within the pegdipda monomer ( figure 1 ) and undergo an irreversible cleavage in the presence of light ( one - photon , = 320436 nm ; two - photon , = 740 nm ) . on account of this property , light was able to cleave bonds within the materials , resulting in the breakage of elastically active network strands and , ultimately , erosion of the gel with light exposure ( figure 3a , b ) . for the analysis of photodegradation in chain- and step - polymerized hydrogels , a representative cp gel ( formulation b ) and a representative sp gel ( formulation d ) were analyzed and compared . prior to erosion , photodegradation led to an exponential decrease in the shear storage modulus ( figure 3c ) , which was governed by the inherent rate of photocleavage of the nbe moiety , keff . as both of the gels contained the same nbe moiety in the network backbone , it was predicted that the initial cleavage rate of elastically active strands , measured as a decrease in shear storage modulus , would be the same for both the cp and sp gels . the cleavage rate , keff , for the cp gel was 0.0140 0.0012 s and the cleavage rate for the sp gel was 0.0142 0.0012 s. these effective cleavage rates were not statistically different and were in agreement with previously reported cleavage rates for similar nbe moieties . ( a and b ) the o - nitrobenzyl ether moieties ( orange ring structures ) in pegdipda undergo an irreversible cleavage in response to irradiation ( one - photon , 320436 nm ; two - photon , 740 nm ) , breaking elastically active network strands in the hydrogel backbone . in this manner , light can be employed to degrade and , ultimately , erode the cp and sp hydrogels . ( c ) owing to the inclusion of the same photolabile monomer into the network backbone , the initial effective cleavage kinetic constant , defined as the negative slope of ln(g / g0 ) as a function of irradiation time divided by the incident irradiation intensity , was similar for the cp ( black , formulation b ) and sp ( gray , formulation d ) gels . to investigate how network structure influences mass loss and erosion rates of the cp and sp gels , physical channels were eroded into the surfaces of both gels . while rheometry results indicated that the inherent rate of photodegradation is independent of network structure , the erosion rates for the representative cp and sp gels diverged even at short time scales ( figure 4a ) . statistical - kinetic models of photodegradation and erosion in chain - polymerized and step - polymerized hydrogels were applied to describe the depth of channel formation as a function of time to elucidate how network connectivity leads to dramatic differences in pattern formation rate . in both cases , the simple statistical - kinetic model captured the observed erosion behavior ( figure 4a ) , which indicates that the statistical - kinetic model includes the relevant physics of erosion in cp and sp photodegradable gels . these results demonstrate that the lower network connectivity observed in sp gels leads to an increased rate of erosion . for these experiments , the assumption of dissolution of erosion byproducts was accounted for in the rapidly degrading step - polymerized gels ( see materials and methods ) . ( a ) the erosion depth of photopatterned channels as a function of irradiation time was plotted for the cp ( black , formulation b ) and sp ( gray , formulation d ) photodegradable hydrogels . a statistical - kinetic photodegradation model ( solid and dashed lines for cp and sp , respectively ) based on the photocleavage reaction and network connectivity agreed well with experimental data over the 30 min exposure time . ( b ) critical extent of nbe moieties that need to be cleaved to reach reverse gelation governs the rate at which features can be patterned into photodegradable gels . prg is a function of network connectivity in both cp and sp gels . here , prg(n ) is plotted for cp gels ( solid black line ) and prg(fa ) is plotted for sp gels with fb = 2 ( gray circles ) , fb = 3 ( gray squares ) , and fb = 4 ( gray triangles ) . in both of these models , the critical parameter that dictates the erosion rate is the critical fraction of cleaved nbe species , prg , which governs reverse gelation . here , reverse gelation refers to the critical extent of bonds cleaved that causes the insoluble gel to erode completely into soluble polymer chains ( figure 3a , b ) . the network structure of the representative sp gel ( formulation b ) resulted in a prg = 0.42 while the representative cp gel ( formulation d ) resulted in a prg = 0.77 . a critical time scale , tc , was defined as the time to reach reverse gelation at the surface of a photodegradable hydrogel and is a function of prg:1where , keff is the effective kinetic constant of cleavage of the nbe moiety ; i0 is the intensity of the incident irradiation . since the cleavage reaction followed first - order kinetics with the same effective kinetic constant in both gels and each was exposed to the same incident irradiation , the difference in prg alone determined the difference in erosion time constants , tc = 490 s for the cp gel and tc = 180 s for sp gel . the critical erosion time scale , tc , governed not only the time to erode the surface of the gel , but also the rate at which erosion progresses through the depth of the gel . a critical length scale , zc , was defined from the beer - lamber law:2here i is the molar absorptivity of the nbe moiety ; ci is the concentration of the nbe moiety . a rate for which the erosion progressed through the gel was calculated as the critical length scale of photodegradation divided by the critical time scale of photodegradation:3 owing to the differences in the prg and the concentration of nbe moieties in the cp and sp gels , the rate of erosion was significantly faster for the sp gel as compared to the cp gel . the simple scaling analysis predicted an erosion rate of 3.6 and 18.4 m / min compared to experimental values of 4.4 0.1 and 18.6 2.0 m / min for the cp and sp gels , respectively . the above analysis of the relationship between erosion rate and prg holds for the specific chain - polymerized and step - polymerized hydrogels in this manuscript as well as for gels formed with the same network connectivity , i.e. , the same prg . however , more broadly , the equations hold for the general class of photodegradable hydrogels for which the network structure and physical parameters are known . specifically , step - polymerized gels have been formed from peg monomers with varying functionality leading to different network connectivity . for instance , the cross - linking of an octafunctional , thiol - terminated peg with a tetrafunctional , vinyl sulfone - terminated peg would form a network with different connectivity than a tetrafunctional , thiol - terminated peg and a trifunctional , vinyl differences in network connectivity are directly related to prg and , ultimately , the rate of erosion . the reverse gelation point for step - polymerized hydrogels , formed from two complementary monomers , has been adapted from classical derivations by flory and rehner that describe network formation in step growth polymerizations:4where , fa is the functionality of the a - terminated monomer ; fb is the functionality of the b - terminated monomer ; and r is the stoichiometric ratio of a to b. this derivation based on the flory rehner theory assumes complete reaction of all functional end groups in the polymer network without loops , dangling ends , or entanglements . therefore , real systems , such as the sp gels in this work , will have an effective prg lower than the ideal calculation as loops , dangling ends , and entanglements form during polymerization . the reverse gelation point for chain - polymerized hydrogels has been adapted from classical derivations of macosko and miller:5where , n is the number of cross - linking molecules per polyacrylate kinetic chain , which is determined by the polymerization conditions and monomer formulation . equations 4 and 5 indicate how network connectivity relates to prg , which can be related to the rate of erosion in photodegradable hydrogels ( eq 3 ) . figure 4b illustrates how prg is related to the monomers or polymerization conditions for both chain- and step - polymerized hydrogels ( r was assumed to be unity for all step polymerization conditions ; figure 4b ) . for a multifunctional monomer reacting with a difunctional monomer through step polymerization ( figure 4b , gray circles ) , however , chain polymerizations typically result in an n of 10100 , while it is difficult to synthesize multifunctional monomers beyond a functionality of 8 for step polymerizations ( fa 8) . therefore , to achieve reverse gelation points that are similar to common chain - polymerized formulations , one can copolymerize multifunctional monomers ( fa = 38 ) with trifunctional or tetrafunctional complementary monomers ( figure 4b ; gray squares and triangles , respectively ) . this analysis demonstrates how network structure relates to the rates of erosion or feature generation in photodegradable hydrogels . by exploiting the rapid erosion of step - polymerized hydrogels formed by the copolymerization of complementary tetrafunctional and difunctional monomers , photodegrading hydrogels were designed for the controlled release entrapped factors and cells , as well as geometric patterning of cell culture microwells . further , the increased prg for cp gels is advantageous to generate materials with broad anisotropic elasticities in the x y or z - dimensions as the gel remains intact at a lower cross - linking density than the sp gels . photodegradable hydrogels were fabricated by both chain and step polymerization from the same photolabile monomer , pegdipda . compared to chain - polymerized gels , step - polymerized hydrogels possessed increased mechanical integrity , as quantified by ductility , tensile toughness , and shear strain to yield . increases in mechanical integrity were attributed to increased homogeneity and network cooperativity possessed in step - polymerized hydrogels as compared to the relatively heterogeneous chain - polymerized gels . light - induced degradation and erosion was demonstrated in both the chain - polymerized and step - polymerized gels . the inherent kinetic constant of photodegradation was the same in the two systems as both gels possess the same o - nitrobenzyl ether moiety in their backbones , while the rate of erosion was much faster in step - polymerized hydrogels on account of the relatively lower network connectivity . taken together , these studies illustrate the utility of photodegradable hydrogels polymerized by either chain or step growth polymerization and provide quantitative tools for designing unique photodegradable gels and predicting their degradation and erosion , critical parameters for regulating cell fate , tissue regeneration , and drug release among many other biomedical applications .

the relationship between polymeric hydrogel microstructure and macroscopic properties is of specific interest to the materials science and polymer science communities for the rational design of materials for targeted applications . specifically , research has focused on elucidating the role of network formation and connectivity on mechanical integrity and degradation behavior . here , we compared the mechanical properties of chain- and step - polymerized , photodegradable hydrogels . increased ductility , tensile toughness , and shear strain to yield were observed in step - polymerized hydrogels , as compared to the chain - polymerized gels , indicating that increased homogeneity and network cooperativity in the gel backbone improves mechanical integrity . furthermore , the ability to degrade the hydrogels in a controlled fashion with light was exploited to explore how hydrogel microstructure influences photodegradation and erosion . here , the decreased network connectivity at the junction points in the step - polymerized gels resulted in more rapid erosion . finally , a relationship between the reverse gelation threshold and erosion rate was developed for the general class of photodegradable hydrogels . in all , these studies further elucidate the relationship between hydrogel formation and microarchitecture with macroscale behavior to facilitate the future design of polymer networks and degradable hydrogels , as well as photoresponsive materials such as cell culture templates , drug delivery vehicles , responsive coatings , and anisotropic materials .

Introduction Materials and Methods Results and Discussion Conclusion

covalently cross - linked hydrogels are applied as cell culture templates , absorbent materials , nonfouling coatings , contact lenses , and drug delivery vehicles . despite the prevalence of hydrogels in the biomedical sciences , the manner by which the cross - linking mechanism and resultant microarchitecture of the hydrogel influences the macroscopic properties ( e.g. a better understanding of the structure function relationship in hydrogel performance would enable improved rational design of materials for a range of targeted applications . recent work has focused on the formation of cross - linked hydrogels with more ideal and homogeneous microstructures to improve network cooperativity and increase hydrogel mechanical integrity . originally , hubbell and co - workers demonstrated the formation of step - polymerized hydrogels by cross - linking thiol and electron - poor , vinyl functionalized peg - based molecules for drug delivery and cell encapsulation . demonstrated the formation of step - polymerized hydrogels through the copper - free , strain promoted azide alkyne cycloaddition ( spaac ) , forming hydrogels in a bio - orthogonal and cytocompatible manner . seminal mechanical analyses of step - polymerized gels have found that these networks possess increased tensile extension as compared to chain - polymerized analogues , while sans data have demonstrated that these networks , although still not perfectly ideal , possess fewer heterogeneities in the network microstructure . while differences between chain and step polymerization mechanisms and resultant hydrogels are clear , there is little literature on the direct comparison of mechanics and degradation between chain - polymerized and step - polymerized hydrogels . function relationship of hydrogels through direct comparisons between chain- and step - polymerized hydrogels with similar chemical structures but profoundly different network connectivities , which will enable the rational design and application of unique hydrogel - based materials . furthermore , there is a growing interest in controlling the material properties of both step- and chain - polymerized hydrogels dynamically and in a user - defined fashion using cleavable chemistries whose degradation can be triggered exogenously . meanwhile , the drug delivery community is exploiting photodegradable hydrogels to release factors at specific locations and at precise times . for photodegradable hydrogels to be utilized most effectively in the broad range of applications , a precise and predictable understanding of how irradiation and network structure influence degradation - induced changes in material properties is required . this work presents the synthesis and characterization of hydrogel networks that are formed by both chain and step polymerizations of a single photodegradable peg - based macromolecular precursor as model systems to understand differences in both mechanical properties and degradation between the resultant network structures . furthermore , the photolabile linker in the hydrogel is employed to compare and contrast the photodegradation - induced changes in the two gels . in all , the material chemistry enables a more robust understanding of how network connectivity and gel architecture influence properties and degradation , and this fundamental understanding should translate into an improved design of hydrogel cells carriers and drug delivery vehicles for biomedical applications . to generate a photoresponsive monomer that is capable of forming both chain- and step - polymerized networks . chain - polymerized hydrogels were fabricated by copolymerizing pegdipda with monoacrylated poly(ethylene glycol ) ( mn 400 da , pega ; monomer - polymer dajac laboratories ) via redox - initiated , free - radical chain polymerization . three chain - polymerized hydrogels were fabricated for this work by varying the ratio of pegdipda to pega at a constant total polymer wt % of 15 wt % . stock solutions of the gel - forming precursors were prepared : 49 mm pegdipda in pbs ph 8.0 , 20 mm peg4sh 10k in pbs ph 8.0 , 40 mm peg4sh 5k in pbs ph 8.0 , and 1 m triethanolamine ( teoha ) in pbs three step - polymerized hydrogels were fabricated for this work by varying the molecular weight of the peg4sh ( 5k or 10k ) and altering the ratio of acrylates to thiols at a constant total polymer wt % of 10 wt % . time sweep tests were conducted at 10 rad / s with 10% strain , which was determined to be in the linear viscoelastic regime for both chain- and step - polymerized hydrogels . tensile testing of chain- and step - polymerized hydrogels ( n = 3 for each gel type ) was performed in uniaxial extension with a materials tester ( mts synergie 100 ) with a 10 n load head . each sample was fixed on the materials tester by compression clamps at the top and bottom of the sample ( 5 mm from each end of the gel ) , and the local environment was kept humidified during the analyses . to determine the shear strain to yield for each of the hydrogels , hydrogel samples ( n = 3 for each gel type ) once the gels reached complete polymerization , a strain sweep was performed from 1% to 1000% strain at a constant frequency of 2.5 rad / s while monitoring the storage modulus ( g ) and the loss modulus ( g ) values . the kinetics of the photodegradation reaction in both chain- and step - polymerized hydrogels was quantified by irradiating ( = 365 nm ; i0 = 20 mw / cm ) in situ polymerized gels on a parallel - plate shear rheometer ( ta instruments ares 4400 ) and following the modulus evolution as a function of irradiation time . a linear fit of ln(g/g0 ) , as measured by the rheometry experiments , as a function of irradiation time was employed to calculate k and , thus , keff for both chain- and step - polymerized hydrogels . , newburyport , ma ) , and attenuation of light by the rheometer or photomasks was accounted for by increasing the incident light intensity so that the transmitted light was at the desired intensity . swollen chain- and step - polymerized gels ( 10 mm 10 mm 1 mm ) were aligned under the channel patterns and surrounded by pbs to maintain hydration and facilitate dissolution of degraded products during patterning . a statistical - kinetic model of photodegradation in chain - polymerized networks was applied to model the erosion depth as a function of time for the chain - polymerized hydrogels in this work . this model was extended to describe photodegradation in step growth networks by altering the statistical assumptions of network connectivity to account for the differences in network structure . furthermore , as the time scale of erosion is much faster for step - polymerized hydrogels than chain - polymerized an additional dissolution assumption was included . by including this simple assumption , the statistical - kinetic model was able to describe the erosion depth as a function of irradiation time in both chain- and step - polymerized hydrogels . in the chain polymerization each acrylate is difunctional allowing the pegdipda to serve as a tetrafunctional cross - linker , whereas in the step polymerization each acrylate is monofunctional extending the elastically active chains between the tetrafunctional peg4sh cross - linkers . chain - polymerized and step - polymerized hydrogels were formed with the same photolabile monomer , pegdipda . chain - polymerized hydrogels ( cp gels ) were fabricated through the copolymerization of pegdipda with pega via free - radical polymerization , resulting in a heterogeneous network structure . in this study , three chain - polymerized ( a c ) and three step - polymerized ( d f ) hydrogels were fabricated from an array of macromolecular solutions ( table 1 ) . it is difficult to generate chain- and step - polymerized hydrogels with directly comparable properties as the formation mechanisms lead to differences in the length of the network chains , the degree of cyclization , and swelling behavior . therefore , a range of materials was tested to study the effect of formation mechanism ( chain or step polymerization ) on mechanical properties , such as tensile strain to failure , tensile toughness , and shear strain to yield . the formulations for chain - polymerized ( a c ) and step - polymerized ( d f ) hydrogels are detailed in the materials and methods section . in all cases , the total polymerization time can be tuned by altering the initial macromer concentration and the initiator concentrations ( ammonium persulfate / temed for the chain polymerization and triethanolamine / ph for the step polymerization ) . it has been suggested that the increased homogeneity and network cooperativity of sp hydrogels results in an increase in mechanical integrity , specifically tensile strain to break , as compared to cp hydrogels . to compare the tensile properties of the chain- and step - polymerized peg hydrogels studied in this work , the percent strains to failure for cp gels were 33 4% , 33 5% , and 20 3% for a , b , and c , respectively . specifically , the tensile toughness of the sp gels were 4.1 0.2 , 6.0 1.4 , and 14.5 2.0 kpa for d , e , and f , respectively , while the tensile toughness for the cp gels were 2.2 0.2 , 1.3 0.3 , and 0.5 0.2 kpa for a , b , and c , respectively . in addition to the tensile testing , strain sweeps on in situ polymerized hydrogels were conducted to investigate the shear strain to yield for each of the samples . the sp gels exhibited increased shear strain to yield in all cases as compared to the cp gels ( table 1 ; figure 2d ) , 420 40 , 500 70 , and 290 70% for sp gels d , e , and f , respectively , and 89 6 , 130 1 , and 93 4% for cp gels , a , b , and c , respectively . mechanical analysis of chain- and step - polymerized hydrogels . ( b ) the average percent strain to failure was increased for all sp gels ( dashed bars , d f ) as compared to cp gels ( solid bars , a c ) . ( d ) the yield behavior of the hydrogels was analyzed on a parallel plate rheometer to determine the shear strain to yield for each sample . as with the tensile analyses , the sp gels ( dashed bars , d f ) demonstrated increased shear strains to yield as compared to cp gels ( solid bars , a , it was observed that mechanical integrity was improved for hydrogels formed by step polymerization as compared to chain polymerization . in addition to mechanical integrity , network connectivity directly relates to the diffusion of macromolecules through the hydrogel network and ideal gels should facilitate more uniform diffusion as compared to heterogeneous gels . on account of this property , light was able to cleave bonds within the materials , resulting in the breakage of elastically active network strands and , ultimately , erosion of the gel with light exposure ( figure 3a , b ) . for the analysis of photodegradation in chain- and step - polymerized hydrogels , a representative cp gel ( formulation b ) and a representative sp gel ( formulation d ) were analyzed and compared . ( c ) owing to the inclusion of the same photolabile monomer into the network backbone , the initial effective cleavage kinetic constant , defined as the negative slope of ln(g / g0 ) as a function of irradiation time divided by the incident irradiation intensity , was similar for the cp ( black , formulation b ) and sp ( gray , formulation d ) gels . while rheometry results indicated that the inherent rate of photodegradation is independent of network structure , the erosion rates for the representative cp and sp gels diverged even at short time scales ( figure 4a ) . statistical - kinetic models of photodegradation and erosion in chain - polymerized and step - polymerized hydrogels were applied to describe the depth of channel formation as a function of time to elucidate how network connectivity leads to dramatic differences in pattern formation rate . for these experiments , the assumption of dissolution of erosion byproducts was accounted for in the rapidly degrading step - polymerized gels ( see materials and methods ) . in both of these models , the critical parameter that dictates the erosion rate is the critical fraction of cleaved nbe species , prg , which governs reverse gelation . here , reverse gelation refers to the critical extent of bonds cleaved that causes the insoluble gel to erode completely into soluble polymer chains ( figure 3a , b ) . since the cleavage reaction followed first - order kinetics with the same effective kinetic constant in both gels and each was exposed to the same incident irradiation , the difference in prg alone determined the difference in erosion time constants , tc = 490 s for the cp gel and tc = 180 s for sp gel . a rate for which the erosion progressed through the gel was calculated as the critical length scale of photodegradation divided by the critical time scale of photodegradation:3 owing to the differences in the prg and the concentration of nbe moieties in the cp and sp gels , the rate of erosion was significantly faster for the sp gel as compared to the cp gel . the simple scaling analysis predicted an erosion rate of 3.6 and 18.4 m / min compared to experimental values of 4.4 0.1 and 18.6 2.0 m / min for the cp and sp gels , respectively . the above analysis of the relationship between erosion rate and prg holds for the specific chain - polymerized and step - polymerized hydrogels in this manuscript as well as for gels formed with the same network connectivity , i.e. however , more broadly , the equations hold for the general class of photodegradable hydrogels for which the network structure and physical parameters are known . specifically , step - polymerized gels have been formed from peg monomers with varying functionality leading to different network connectivity . the reverse gelation point for step - polymerized hydrogels , formed from two complementary monomers , has been adapted from classical derivations by flory and rehner that describe network formation in step growth polymerizations:4where , fa is the functionality of the a - terminated monomer ; fb is the functionality of the b - terminated monomer ; and r is the stoichiometric ratio of a to b. this derivation based on the flory rehner theory assumes complete reaction of all functional end groups in the polymer network without loops , dangling ends , or entanglements . the reverse gelation point for chain - polymerized hydrogels has been adapted from classical derivations of macosko and miller:5where , n is the number of cross - linking molecules per polyacrylate kinetic chain , which is determined by the polymerization conditions and monomer formulation . figure 4b illustrates how prg is related to the monomers or polymerization conditions for both chain- and step - polymerized hydrogels ( r was assumed to be unity for all step polymerization conditions ; figure 4b ) . therefore , to achieve reverse gelation points that are similar to common chain - polymerized formulations , one can copolymerize multifunctional monomers ( fa = 38 ) with trifunctional or tetrafunctional complementary monomers ( figure 4b ; gray squares and triangles , respectively ) . by exploiting the rapid erosion of step - polymerized hydrogels formed by the copolymerization of complementary tetrafunctional and difunctional monomers , photodegrading hydrogels were designed for the controlled release entrapped factors and cells , as well as geometric patterning of cell culture microwells . further , the increased prg for cp gels is advantageous to generate materials with broad anisotropic elasticities in the x y or z - dimensions as the gel remains intact at a lower cross - linking density than the sp gels . compared to chain - polymerized gels , step - polymerized hydrogels possessed increased mechanical integrity , as quantified by ductility , tensile toughness , and shear strain to yield . increases in mechanical integrity were attributed to increased homogeneity and network cooperativity possessed in step - polymerized hydrogels as compared to the relatively heterogeneous chain - polymerized gels . light - induced degradation and erosion was demonstrated in both the chain - polymerized and step - polymerized gels . the inherent kinetic constant of photodegradation was the same in the two systems as both gels possess the same o - nitrobenzyl ether moiety in their backbones , while the rate of erosion was much faster in step - polymerized hydrogels on account of the relatively lower network connectivity . taken together , these studies illustrate the utility of photodegradable hydrogels polymerized by either chain or step growth polymerization and provide quantitative tools for designing unique photodegradable gels and predicting their degradation and erosion , critical parameters for regulating cell fate , tissue regeneration , and drug release among many other biomedical applications .

on - site wastewater treatment systems ( owts ) are widely utilized to treat and dispose of household wastewater in areas where centralised reticulated sewerage systems are unavailable . septic tanks , aerobic treatment units ( atus ) , and composting toilets with greywater treatment are among the more commonly used owts in australia and elsewhere . in the 1990s it was estimated that approximately 20% of australian households rely on septic systems for domestic wastewater management . by 2001 , it was estimated that more than 1 million owts were installed in australia , with the greatest distribution in new south wales ( 300,000 ) , followed by victoria ( 250,000 ) and queensland ( 250,000 ) . in western australia ( wa ) itself figure 1 represents the numbers of owts installations while the percentages on the figure indicate the proportion of unsewered properties in each state across australia . these figures are likely to increase along with australian population growth , the continuous development of rural and unsewered urban regions , and the acceptance that owts are a cost effective and long - term option for meeting public health and environmental health goals if properly installed , operated , and maintained . current average in australia is 2.6 people per household . assuming each person produces approximately 200 l of wastewater per day a total of approximately 31 tons of nitrogen , 8.4 tons of phosphorus , and 70 10 faecal coliform organisms will be discharged to the owts . nutrients and pathogens are released into the environment when owts fail and the public health and environmental consequences could be significant considering that owts failure in australia has been reported high , reaching up to 40% . undoubtedly , poorly performing or failing owts have economic impacts on the owners and pose environmental impacts due to nutrients that may disrupt ecosystem balance . furthermore , poorly performed or failed owts can contribute to eutrophication of sensitive water bodies and create algae blooms . failing owts are one of the main causes of streams , lakes , rivers , wetlands , and groundwater contamination due to the release of nutrients and pathogens into the environment . various studies worldwide and in australia have shown that poorly performing or failing owts have resulted in degradation of soil and water quality [ 3 , 6 , 7 ] . furthermore , effluent from underperformed or failed owts may pose a public health risk , particularly to susceptible population group such as children , the elderly , or people with compromised immune system . contamination of drinking water or recreational waters by failing owts may increase concentration of pathogens that may impact on human health . untreated effluent lying in gutters or pooling in public places provides an environment for the transmission of diseases , such as those listed below : gastroenteritis from bacteria such as salmonella or viruses such as rotavirus or protozoa ( namely giardia),bacterial dysentery , typhoid fever from salmonella , polio from enterovirus , infectious hepatitis ( hepatitis a ) causing liver disease , skin infections from viruses , bacteria , and fungi . gastroenteritis from bacteria such as salmonella or viruses such as rotavirus or protozoa ( namely giardia ) , typhoid fever from salmonella , polio from enterovirus , infectious hepatitis ( hepatitis a ) causing liver disease , skin infections from viruses , bacteria , and fungi . pooling off effluent creates a nuisance by emitting offensive odours thus affecting the amenity of an area . spray irrigation of untreated greywater or septic tank effluent can result in aerosol transmission of diseases causing organisms . irrigation of effluent or greywater water onto food crops can cause diseases such as gastroenteritis or serious life - threatening diseases . contact with lawn areas recently irrigated with untreated wastewater may also result in skin infections or gastroenteritis via direct or indirect ingestion of organisms . swimming in water contaminated with effluent may result in skin , ear , and eye infections or gastroenteritis . similarly , consuming shellfish from effluent contaminated water may result in diseases such as hepatitis a . however the main chemical hazard of poorly performing owts is nitrate . elevated nitrogen levels in drinking water supplies impacted by wastewater contamination may result in irregular haemoglobin levels or methemoglobinemia in children . poor performance or failures of owts are often associated with systems not situated and constructed correctly . in particular , areas with low permeability soils and/or high water tables present the greatest challenge in wa . failure of many onsite systems is generally not due to inherent flaws in system technology but rather due to inappropriate sitting and construction design or their operation and management . other contributor factors to poor performance or failure of owts are poor public awareness , poor local authority guidance , continuous wastewater management issues , poor maintenance procedures , and inadequate governance . the following causes have been identified as the major reasons for owts failure : lack of area on the properties for the treatment and disposal of wastewater , undersized or inadequate wastewater treatment units or absorption trenches , poor maintenance , such as failure to desludge septic tanks , a change in occupancy from occasional to permanent , inappropriate location of absorption trenches , that is , in areas that are poorly drained , subject to flooding or high seasonal water table , poor knowledge of operation and maintenance procedures by homeowners / occupiers . lack of area on the properties for the treatment and disposal of wastewater , undersized or inadequate wastewater treatment units or absorption trenches , poor maintenance , such as failure to desludge septic tanks , a change in occupancy from occasional to permanent , inappropriate location of absorption trenches , that is , in areas that are poorly drained , subject to flooding or high seasonal water table , poor knowledge of operation and maintenance procedures by homeowners / occupiers . there is an increasing concern with the cumulative environmental impacts and local public health risk of failing or inadequately designed owts in unsewered areas . therefore , at the planning stage , it is important to ensure that inappropriate development does not proceed with an expectation that reticulated sewerage will come at a later date to solve problems being incurred . in areas where reticulated sewerage is not available , on - site disposal of wastewater is required . an application to construct or install an apparatus for the treatment of sewage in accordance with the health ( treatment of sewage and disposal of effluent and liquid waste ) regulations 1974 is required to be lodged to the local government ( lg ) in the first instance . if it is intended that the proposed owts will handle less than 540 l / day of wastewater and the building to be serviced is a single dwelling , the lg will process the application . if the wastewater volume generated is greater than 540 l / day and the building being serviced is not a single dwelling , the lg will assess the application , prepare a local government report , and forward the application to the dohwa for approval . the dohwa is also responsible for the approval of apparatus for sewage treatment within the state and it provides technical information to assist lgs and the community . these legislations and guidelines for owts in unsewered areas in wa are listed and briefly described below . sewage in the act includes sewage from faecal waste , urine from toilets , and water from shower , bath , laundry , and kitchen . it necessitates the collection , treatment , and disposal of sewage and greywater to ensure public health safety . health ( treatment of sewage and disposal of effluent and liquid waste ) regulations 1974 . applications for the installation of owts are only approved in the cases where sewer connections are not available . this code of practice supports the long - term reuse of greywater in both sewered and unsewered areas in wa . dohwa assesses and approves applications when multiple dwellings or commercial premises intend to use greywater for in - house end - uses , premises to reuse greywater via spray irrigation , or premises to reuse greywater via subsoil irrigation that produce more than 5,000 l of greywater daily . code of practice for the design , manufacture , installation , and operation of aerobic treatment units ( atus ) . this code of practice is published under the provisions of the health act 1911 ( wa ) and to be used simultaneously with the health ( treatment of sewage and disposal of effluent and liquid waste ) regulations 1974 . it provides guidance on the design , construction , installation , and operation of atu to serve single dwellings . the code of practice specifies the conditions necessary for on - site wastewater systems ( ows ) manufacturers who wish to sell their systems to the public or to use the systems for treating and disposing sewage . applicants are required to demonstrate that the ows comply with relevant australian standards and other regulations through documentations submitted to the dohwa . the dohwa is currently adopting standards australia and standards new zealand ( as / nzs ) 1547:2000 on - site domestic wastewater management ( as / nzs 1547.2012 ) . this standard is performance based and it guides system design based on site and soil assessment , with the aim of achieving sustainable performance . the dohwa is currently preparing the code of practice for on - site wastewater management with endorsement of as / nzs 1547 . it is expected that this code of practice will supersede the current codes and regulations . the policies encourage all new developments and subdivisions in wa to connect to reticulated sewerage as the most efficient , socially equitable , and safe method of wastewater disposal . however , in situations where connection to reticulated sewerage is not possible or practicable , the policies set out the on - site wastewater disposal requirements that proposals for developments and subdivisions must meet , in order to obtain the dohwa approval [ 17 , 18 ] . sewage in the act includes sewage from faecal waste , urine from toilets , and water from shower , bath , laundry , and kitchen . it necessitates the collection , treatment , and disposal of sewage and greywater to ensure public health safety . health ( treatment of sewage and disposal of effluent and liquid waste ) regulations 1974 . applications for the installation of owts are only approved in the cases where sewer connections are not available . this code of practice supports the long - term reuse of greywater in both sewered and unsewered areas in wa . dohwa assesses and approves applications when multiple dwellings or commercial premises intend to use greywater for in - house end - uses , premises to reuse greywater via spray irrigation , or premises to reuse greywater via subsoil irrigation that produce more than 5,000 code of practice for the design , manufacture , installation , and operation of aerobic treatment units ( atus ) . this code of practice is published under the provisions of the health act 1911 ( wa ) and to be used simultaneously with the health ( treatment of sewage and disposal of effluent and liquid waste ) regulations 1974 . it provides guidance on the design , construction , installation , and operation of atu to serve single dwellings . the code of practice specifies the conditions necessary for on - site wastewater systems ( ows ) manufacturers who wish to sell their systems to the public or to use the systems for treating and disposing sewage . applicants are required to demonstrate that the ows comply with relevant australian standards and other regulations through documentations submitted to the dohwa . the dohwa is currently adopting standards australia and standards new zealand ( as / nzs ) 1547:2000 on - site domestic wastewater management ( as / nzs 1547.2012 ) . this standard is performance based and it guides system design based on site and soil assessment , with the aim of achieving sustainable performance . the dohwa is currently preparing the code of practice for on - site wastewater management with endorsement of as / nzs 1547 . it is expected that this code of practice will supersede the current codes and regulations . the policies encourage all new developments and subdivisions in wa to connect to reticulated sewerage as the most efficient , socially equitable , and safe method of wastewater disposal . however , in situations where connection to reticulated sewerage is not possible or practicable , the policies set out the on - site wastewater disposal requirements that proposals for developments and subdivisions must meet , in order to obtain the dohwa approval [ 17 , 18 ] . this paper aims to review the installations and failures of owts in wa from 1997 to 2011 . recommendations to the dohwa and local government in regard to the administration of owts are also presented . a literature review related to owts legislation , standards , and approval process was undertaken . following on from this , data on the numbers of owts installed in wa was collected from two sources : centralised dohwa data management system and data provided by lg from their own data systems . as explained above , the lg is responsible for the approval of owts producing less than 540 l / day of wastewater while nondomestic and commercial owts producing more than 540 l / day are referred to the dohwa for approval . inspections of installed owts are the responsibility of the lg environmental health officer ( eho ) . the eho is required to inspect the owts and ensure that installation complies with the approval and conditions issued either by lg or by dohwa . the dohwa database collected the following information : applicant 's details , date of application , dwelling address and purpose , type of apparatus installed , and date of inspection . the database was created in 1997 and in order to match the information with the dohwa database , data from 1997 onwards was requested to lg . data related to the numbers of owts approved by lg was obtained via emails that were distributed in august 2012 to 140 lgs across wa . there were 140 lgs across wa at the time the information was requested . attached with the email was a microsoft excel 2007 spreadsheet containing two tables that lg environmental health officer ( eho ) filled out in order to record the number of installations approved . tables 1 and 2 contain overall numbers of owts by year for periods 19972011 approved by lg and dohwa , respectively . all data was checked and analysed in microsoft excel 2007 . at the end of the analysed period in 2011 , the dohwa conducted a survey to gather data from various lgs in wa on the primary causes of owts failures . the survey in the form of microsoft excel 2007 spreadsheet was sent out to 140 lgs across wa . the information collected in the survey included the type of owts and the reasons for system failures . follow - up interviews via phone calls were conducted to correct any errors in the data . overall , 28 lgs ( out of 140 ) provided data on the numbers of owts installations ( table 3 ) . nine of the lgs were located within perth urban areas while 19 others were in the outer suburban areas . based on the data obtained , more than 12,500 applications were approved between the periods of 1997 to 2011 . the highest number of applications was recorded in 2008 with over 1,000 applications , while the lowest was observed in 1997 with less than 500 applications . based on the data collected from the dohwa database from 1997 to 2011 , the dohwa approved over 3,700 owts applications , with an average of 247 applications annually . the highest number of applications was noted in 1999 , with over 350 applications whereas the lowest was observed in 1997 , with 128 applications . the number of owts approved by the dohwa from january 1997 to december 2011 is shown in figure 2 . east pilbara and derby were the two regions with the highest numbers of owts approvals , with 304 and 293 approvals , respectively , whereas most of perth urban areas ( e.g. , 73 applications in stirling ) recorded relatively low number of installations ( figure 2 ) . this is expected because communities with higher population density in metropolitan and urban areas are more likely to be connected to reticulated sewerage . the proportions of owts installed in wa regional areas are larger than in urban areas . however , the risks of contamination due to failed systems are higher in urban / metro areas due to the increased density of owts . moreover , increasing population density increases the likelihood of release of contaminants such as nitrogen to the environment in owts failure . east pilbara , for example , had the highest number of owts approvals between the periods of 1997 and 2011 , with a total of 304 installations , whereas city of stirling has a total of 73 approvals . however , according to the australian bureau of statistics ( abs ) the population density of these two lgs is quite different . east pilbara with an area of over 379,000 km had a total population of 11,950 in 2011 , giving it a population density of 0.031 people per km . on the other hand , city of stirling had a total population of over 200,000 with a relatively smaller area of 105.2 km , giving it a population density of 1,958 people per km . data collected from 1997 to 2011 have revealed that conventional septic tanks account for 76.5% and 63.7% of all wastewater systems approved by lg and dohwa , respectively . due to its simplicity and affordable cost , this is despite the fact that atu is becoming more prevalent . as evident from figure 3 , atu has become more popular in wa over the last decade due to its ability to produce high quality effluent , as well as its capacity to contain and reuse treated effluent on lawns or garden beds via surface or subsurface irrigation . figures 4 and 5 indicate that lg and dohwa have approved more than 1,800 ( 15% ) and over 800 ( 23% ) of atu installations , respectively , during the study period of 1997 to 2011 . the proportions of owts types approved by lg and dohwa are shown in figures 4 and 5 . more than 80% of the other systems were simply described as other without any description or explanation , whereas the remaining 10% corresponds to 2% holding tanks , 4% greywater system , and 4% nutrient removal system . on the other hand , composting was the type described as other in the owts approved by dohwa during the study period of 1997 to 2011 . as mentioned in section 4 , the dohwa conducted a survey in 2011 to collect information from various lgs in wa on the major causes of owts failures . out of the 21 lgs that responded back to the survey , 6 were located within perth metro areas while the remaining 15 lgs were in the outer suburban areas . based on the data collected , there were a total of 53 owts failures , with shire of dundas and shire of derby / west kimberley reporting the highest number of failures at 11 and 6 owts failures , respectively . the most common reasons for owts failures in the survey include groundwater and surface water ingress , systems not installed properly , unsuitable soil type , and undersized systems . several lgs also mentioned other factors that contributed to owts failures , including increase in wastewater volume , root invasion , illegal installation , unauthorised tampering , cross connection to stormwater disposal , undersized systems , and unauthorised materials ( such as fats , oils , and yeasts ) . septic systems remain a preferred option for onsite wastewater management in unsewered urban and rural regions of wa . however , the use of other types of owts such as atu and composting toilets with greywater treatment has increased over the years . given that wa climate is characterised by mild winters and very hot dry summers , there is an increased tendency to improve wastewater quality to achieve fit for purpose nonpotable quality to alleviate the pressure on scarce water resources . as significant amount of effluent is generated , owts have to be installed and monitored properly in order to prevent biological and nutrient contaminants from entering surface water and groundwater . throughout wa , over 12,500 owts applications were approved by 28 lgs during the period of 1997 to 2011 , representing 20% of the total number of existing lgs . while 20% of lgs managed to provide statistics of their owts approvals , the remaining could not produce any information mainly due to the absence of and difficulties retrieving data . additionally , more than 3,700 applications were approved through the dohwa within the same time period , with east pilbara and derby being the two regions with the highest number of approvals . as expected , the number of applications was generally higher in regional areas compared to urban areas as most urban areas are connected to centralized sewerage systems . currently , national and state guidelines are available to ensure adequate construction , installation , and management of owts in order to protect public health . however , the survey conducted in 2011 indicated that a number of owts are either performing poorly or failing with about 48% of owts failures in wa , which is due to groundwater and surface water ingress . other reasons include incorrect system installation , unsuitable soil type , undersized system , increasing wastewater volume , hydrostatic pressure , high groundwater , root invasion , cross connection to stormwater disposal , and a small percentage of unauthorized tampering . although technology is improving over time and some owts are capable of producing better effluent quality , the problems identified in the dohwa survey are also reported elsewhere . as such , there is a need to assess site and soil conditions rather than relying solely on technology . failing or poorly performing systems often lead to various environmental and public health issues [ 1 , 4 , 5 ] . it is common that a slow passage of raw septic tank effluent through 0.5 to 0.6 m of soil results in a high degree of purification . therefore it is necessary to ensure that there is a minimum of 0.5 to 0.6 m of soil below the base of a trench before high permeable fissured rock , soil , or a groundwater table . in accordance with the as / nzs 1547:2000 , the 600 mm soil absorptive zone is required for filtering , isolating , and absorbing wastewater , microorganisms , nutrients , and partials . thus , to reduce the potential for owts failure due to ground and surface water ingress , implementation of 600 mm absorptive zone is recommended . population density and number of installed owts need to be addressed in more detail . identifying and comparing areas with high owts installations in wa in addition , it is essential to ensure that a fair and consistent approach to dealing with malfunctioning owts is taken by lgs under the guidance from the dohwa . inadequate management strategies and the lack of coordinate institutional arrangements in relation to owts have been previously reported in australia . for instance , in a survey of 48 septic tanks , it was found that 72% of dispersal fields were soggy and ( by inference ) ineffective , 67% required solids removal , 8% needed structural repairs , 6% had insufficient capacity , and 4% were incorrectly sited . also , a study of owts performance in south australia ( sa ) indicates that 22% of atus contains indicator bacteria at levels higher than the maximum acceptable levels specified in the sa 's regulatory guidelines . collecting data of owts installations was conducted by the dohwa via emails , relying on individual lg across wa to provide data on the number of owts installed during the study period . however , as previously mentioned , data regarding the number of approved owts applications by lg was only collected from 20% of the existing lgs in wa ( 28 out of 140 ) . while responses were not expected from many of the metropolitan lgs as they have infill sewerage systems it is acknowledged that response was not received from some lgs with owts . a follow - up on the lg that failed to respond to the email sent out by the dohwa staff enquiring about the numbers of owts approvals indicates inadequate number of staff to review the applications or lack of adequate records to respond to the survey . among those who responded , however , several ehos found it difficult to complete the task due to lack of simple and efficient methods to collate / retrieve the information . several reasons for the lack of available data from lgs include the following.records were not available , due to lack of general file , lg in remote locations , and lack of full time ehos.records were not easily retrieved as data stored in offsite storage.information on owts was not recorded in the early years.for some lgs approval , inspection , and ongoing management for owts were conducted in a reactive way rather than on a proactive manner.ehos do not have the time to collate information because owts are only one part of their several duties.systems described as records were not available , due to lack of general file , lg in remote locations , and lack of full time ehos . information on owts was not recorded in the early years . for some lgs approval , inspection , and ongoing management for owts ehos do not have the time to collate information because owts are only one part of their several duties . lack of appropriate administration system to record owts approvals and installations is a significant drawback in the management of owts in wa . without any feasible management strategies to assess and maintain the performance of owts in each region , the resulting environmental and public health risks associated with poor system performance would increase . hence , there is a need to provide a generic approach for a standardized , common system that will allow each lg to record the number of owts applications that have been approved and to store information related to the approvals in a clear and standard record system . furthermore , there is a need for a multigovernance approach that can address the importance of an effective and robust planning and development of sites . government agencies need to understand their roles and responsibilities in the management of owts when assessing applications for land - use or other developments to ensure that all potential effects arising from such developments are considered prior to approvals . it is important that the site assessment for on - site wastewater management be carried out in the early stage of planning phase . this site assessment procedure should be applied at the rezoning or subdivision stages of the planning process . this will help to develop a management regime for owts that can minimize health risks and environmental impacts and enhance owts 's long - term sustainability . based on the findings reported above , to improve water quality from owts , the dohwa should do the following.it should provide a standard management tool to all lgs for record of owts including gps location . database should be able to record information on installation , operation , maintenance , and any other inspection requirements particularly for atus or systems achieving a secondary treated quality effluent.it should implement a soil absorption zone path length of at least 600 mm to reduce the potential for owts failure due to ground and surface water ingress.it should provide ehos with on - site wastewater management training based on as 1547 and current legislations as part of their professional development and an important measure of quality assurance for the industry , government , and general public.it should produce educational materials to owners of owts to facilitate the understanding of how owts work , how to identify signs of systems failures , and why inadequate maintenance can increase the system failures and the risk to humans and the environment . it should provide a standard management tool to all lgs for record of owts including gps location . database should be able to record information on installation , operation , maintenance , and any other inspection requirements particularly for atus or systems achieving a secondary treated quality effluent . it should implement a soil absorption zone path length of at least 600 mm to reduce the potential for owts failure due to ground and surface water ingress . it should provide ehos with on - site wastewater management training based on as 1547 and current legislations as part of their professional development and an important measure of quality assurance for the industry , government , and general public . it should produce educational materials to owners of owts to facilitate the understanding of how owts work , how to identify signs of systems failures , and why inadequate maintenance can increase the system failures and the risk to humans and the environment . as recommendations , the lgs should do the following.they should implement a risk management program to minimise failures , to rapidly identify and to address failures when problems occur . it is considered that a tolerable rate of system failure is smaller than 5% annually.they should implement a planning strategy for owts in the early stage of any development to identify the resources that are required to ensure adequate owts performance.they should implement site inspection strategies that are predominantly risk based , taking account of sensitive receptors , and should be integrated with other regulatory inspections as appropriate.they should distribute educational materials produced by the dohwa or lgs to owners of owts and provide advices to applicants on owts - related matters when required.they should update the register of installed owts in each lg jurisdiction and implement an inspection regime to identify legacy sites that will require remediation work . inspection can initially be targeted at the highest risk areas or owts with previous history of failures and complaints.in the case of low - risk sites , a variety of nonroutine inspection strategies and in particular proxy ( e.g. , monitoring of water quality ) , third party , engagement , and incentive strategies may be implemented . these strategies can involve working closely with home owners or stakeholders to ensure that those who are responsible for owts understand how to comply with the regulations and are encouraged to do so.they should embark on a series of activities to raise awareness and compliance related to the operation and maintenance of owts . information campaigns may include the communication of international best practice to water services australia ( wsa ) and specific local guidance to nongovernment organizations ( ngos ) and homeowners.they should conduct follow - up site inspections of approved owts that focus specifically on operation and maintenance . priority should be given to known areas of risk or environmental sensitive areas such as owts close to rivers and lakes . inspections will vary in both type and frequency as the inspection program develops over time . they should implement a risk management program to minimise failures , to rapidly identify and to address failures when problems occur . it is considered that a tolerable rate of system failure is smaller than 5% annually . they should implement a planning strategy for owts in the early stage of any development to identify the resources that are required to ensure adequate owts performance . they should implement site inspection strategies that are predominantly risk based , taking account of sensitive receptors , and should be integrated with other regulatory inspections as appropriate . they should distribute educational materials produced by the dohwa or lgs to owners of owts and provide advices to applicants on owts - related matters when required . they should update the register of installed owts in each lg jurisdiction and implement an inspection regime to identify legacy sites that will require remediation work . inspection can initially be targeted at the highest risk areas or owts with previous history of failures and complaints . in the case of low - risk sites , a variety of nonroutine inspection strategies and in particular proxy ( e.g. , monitoring of water quality ) , third party , engagement , and incentive strategies may be implemented . these strategies can involve working closely with home owners or stakeholders to ensure that those who are responsible for owts understand how to comply with the regulations and are encouraged to do so . they should embark on a series of activities to raise awareness and compliance related to the operation and maintenance of owts . information campaigns may include the communication of international best practice to water services australia ( wsa ) and specific local guidance to nongovernment organizations ( ngos ) and homeowners . they should conduct follow - up site inspections of approved owts that focus specifically on operation and maintenance . priority should be given to known areas of risk or environmental sensitive areas such as owts close to rivers and lakes . inspections will vary in both type and frequency as the inspection program develops over time .

on - site wastewater treatment systems ( owts ) are widely used in western australia ( wa ) to treat and dispose of household wastewater in areas where centralized sewerage systems are unavailable . septic tanks , aerobic treatment units ( atus ) , and composting toilets with greywater systems are among the most well established and commonly used owts . however , there are concerns that some owts installed in wa are either performing below expected standards or failing . poorly performing owts are often attributed to inadequate installation , inadequate maintenance , poor public awareness , insufficient local authority resources , ongoing wastewater management issues , or inadequate adoption of standards , procedures , and guidelines . this paper is to review the installations and failures of owts in wa . recommendations to the department of health western australia ( dohwa ) and local government ( lg ) in regard to management strategies and institutional arrangements of owts are also highlighted .

1. Introduction 2. Impacts of Poorly Performing OWTS 3. Aim 4. Methods 5. Results 6. Discussion 7. Conclusions and Recommendations

on - site wastewater treatment systems ( owts ) are widely utilized to treat and dispose of household wastewater in areas where centralised reticulated sewerage systems are unavailable . septic tanks , aerobic treatment units ( atus ) , and composting toilets with greywater treatment are among the more commonly used owts in australia and elsewhere . in the 1990s it was estimated that approximately 20% of australian households rely on septic systems for domestic wastewater management . by 2001 , it was estimated that more than 1 million owts were installed in australia , with the greatest distribution in new south wales ( 300,000 ) , followed by victoria ( 250,000 ) and queensland ( 250,000 ) . in western australia ( wa ) itself figure 1 represents the numbers of owts installations while the percentages on the figure indicate the proportion of unsewered properties in each state across australia . these figures are likely to increase along with australian population growth , the continuous development of rural and unsewered urban regions , and the acceptance that owts are a cost effective and long - term option for meeting public health and environmental health goals if properly installed , operated , and maintained . assuming each person produces approximately 200 l of wastewater per day a total of approximately 31 tons of nitrogen , 8.4 tons of phosphorus , and 70 10 faecal coliform organisms will be discharged to the owts . undoubtedly , poorly performing or failing owts have economic impacts on the owners and pose environmental impacts due to nutrients that may disrupt ecosystem balance . failing owts are one of the main causes of streams , lakes , rivers , wetlands , and groundwater contamination due to the release of nutrients and pathogens into the environment . various studies worldwide and in australia have shown that poorly performing or failing owts have resulted in degradation of soil and water quality [ 3 , 6 , 7 ] . untreated effluent lying in gutters or pooling in public places provides an environment for the transmission of diseases , such as those listed below : gastroenteritis from bacteria such as salmonella or viruses such as rotavirus or protozoa ( namely giardia),bacterial dysentery , typhoid fever from salmonella , polio from enterovirus , infectious hepatitis ( hepatitis a ) causing liver disease , skin infections from viruses , bacteria , and fungi . gastroenteritis from bacteria such as salmonella or viruses such as rotavirus or protozoa ( namely giardia ) , typhoid fever from salmonella , polio from enterovirus , infectious hepatitis ( hepatitis a ) causing liver disease , skin infections from viruses , bacteria , and fungi . however the main chemical hazard of poorly performing owts is nitrate . poor performance or failures of owts are often associated with systems not situated and constructed correctly . other contributor factors to poor performance or failure of owts are poor public awareness , poor local authority guidance , continuous wastewater management issues , poor maintenance procedures , and inadequate governance . the following causes have been identified as the major reasons for owts failure : lack of area on the properties for the treatment and disposal of wastewater , undersized or inadequate wastewater treatment units or absorption trenches , poor maintenance , such as failure to desludge septic tanks , a change in occupancy from occasional to permanent , inappropriate location of absorption trenches , that is , in areas that are poorly drained , subject to flooding or high seasonal water table , poor knowledge of operation and maintenance procedures by homeowners / occupiers . lack of area on the properties for the treatment and disposal of wastewater , undersized or inadequate wastewater treatment units or absorption trenches , poor maintenance , such as failure to desludge septic tanks , a change in occupancy from occasional to permanent , inappropriate location of absorption trenches , that is , in areas that are poorly drained , subject to flooding or high seasonal water table , poor knowledge of operation and maintenance procedures by homeowners / occupiers . there is an increasing concern with the cumulative environmental impacts and local public health risk of failing or inadequately designed owts in unsewered areas . in areas where reticulated sewerage is not available , on - site disposal of wastewater is required . an application to construct or install an apparatus for the treatment of sewage in accordance with the health ( treatment of sewage and disposal of effluent and liquid waste ) regulations 1974 is required to be lodged to the local government ( lg ) in the first instance . if the wastewater volume generated is greater than 540 l / day and the building being serviced is not a single dwelling , the lg will assess the application , prepare a local government report , and forward the application to the dohwa for approval . these legislations and guidelines for owts in unsewered areas in wa are listed and briefly described below . applications for the installation of owts are only approved in the cases where sewer connections are not available . this code of practice supports the long - term reuse of greywater in both sewered and unsewered areas in wa . code of practice for the design , manufacture , installation , and operation of aerobic treatment units ( atus ) . this code of practice is published under the provisions of the health act 1911 ( wa ) and to be used simultaneously with the health ( treatment of sewage and disposal of effluent and liquid waste ) regulations 1974 . it provides guidance on the design , construction , installation , and operation of atu to serve single dwellings . the code of practice specifies the conditions necessary for on - site wastewater systems ( ows ) manufacturers who wish to sell their systems to the public or to use the systems for treating and disposing sewage . the dohwa is currently adopting standards australia and standards new zealand ( as / nzs ) 1547:2000 on - site domestic wastewater management ( as / nzs 1547.2012 ) . the dohwa is currently preparing the code of practice for on - site wastewater management with endorsement of as / nzs 1547 . the policies encourage all new developments and subdivisions in wa to connect to reticulated sewerage as the most efficient , socially equitable , and safe method of wastewater disposal . however , in situations where connection to reticulated sewerage is not possible or practicable , the policies set out the on - site wastewater disposal requirements that proposals for developments and subdivisions must meet , in order to obtain the dohwa approval [ 17 , 18 ] . sewage in the act includes sewage from faecal waste , urine from toilets , and water from shower , bath , laundry , and kitchen . it necessitates the collection , treatment , and disposal of sewage and greywater to ensure public health safety . applications for the installation of owts are only approved in the cases where sewer connections are not available . dohwa assesses and approves applications when multiple dwellings or commercial premises intend to use greywater for in - house end - uses , premises to reuse greywater via spray irrigation , or premises to reuse greywater via subsoil irrigation that produce more than 5,000 code of practice for the design , manufacture , installation , and operation of aerobic treatment units ( atus ) . this code of practice is published under the provisions of the health act 1911 ( wa ) and to be used simultaneously with the health ( treatment of sewage and disposal of effluent and liquid waste ) regulations 1974 . it provides guidance on the design , construction , installation , and operation of atu to serve single dwellings . the code of practice specifies the conditions necessary for on - site wastewater systems ( ows ) manufacturers who wish to sell their systems to the public or to use the systems for treating and disposing sewage . applicants are required to demonstrate that the ows comply with relevant australian standards and other regulations through documentations submitted to the dohwa . the dohwa is currently adopting standards australia and standards new zealand ( as / nzs ) 1547:2000 on - site domestic wastewater management ( as / nzs 1547.2012 ) . the dohwa is currently preparing the code of practice for on - site wastewater management with endorsement of as / nzs 1547 . the policies encourage all new developments and subdivisions in wa to connect to reticulated sewerage as the most efficient , socially equitable , and safe method of wastewater disposal . however , in situations where connection to reticulated sewerage is not possible or practicable , the policies set out the on - site wastewater disposal requirements that proposals for developments and subdivisions must meet , in order to obtain the dohwa approval [ 17 , 18 ] . this paper aims to review the installations and failures of owts in wa from 1997 to 2011 . recommendations to the dohwa and local government in regard to the administration of owts are also presented . a literature review related to owts legislation , standards , and approval process was undertaken . following on from this , data on the numbers of owts installed in wa was collected from two sources : centralised dohwa data management system and data provided by lg from their own data systems . as explained above , the lg is responsible for the approval of owts producing less than 540 l / day of wastewater while nondomestic and commercial owts producing more than 540 l / day are referred to the dohwa for approval . the dohwa database collected the following information : applicant 's details , date of application , dwelling address and purpose , type of apparatus installed , and date of inspection . data related to the numbers of owts approved by lg was obtained via emails that were distributed in august 2012 to 140 lgs across wa . tables 1 and 2 contain overall numbers of owts by year for periods 19972011 approved by lg and dohwa , respectively . at the end of the analysed period in 2011 , the dohwa conducted a survey to gather data from various lgs in wa on the primary causes of owts failures . the information collected in the survey included the type of owts and the reasons for system failures . overall , 28 lgs ( out of 140 ) provided data on the numbers of owts installations ( table 3 ) . the number of owts approved by the dohwa from january 1997 to december 2011 is shown in figure 2 . the proportions of owts installed in wa regional areas are larger than in urban areas . however , the risks of contamination due to failed systems are higher in urban / metro areas due to the increased density of owts . however , according to the australian bureau of statistics ( abs ) the population density of these two lgs is quite different . data collected from 1997 to 2011 have revealed that conventional septic tanks account for 76.5% and 63.7% of all wastewater systems approved by lg and dohwa , respectively . figures 4 and 5 indicate that lg and dohwa have approved more than 1,800 ( 15% ) and over 800 ( 23% ) of atu installations , respectively , during the study period of 1997 to 2011 . more than 80% of the other systems were simply described as other without any description or explanation , whereas the remaining 10% corresponds to 2% holding tanks , 4% greywater system , and 4% nutrient removal system . as mentioned in section 4 , the dohwa conducted a survey in 2011 to collect information from various lgs in wa on the major causes of owts failures . out of the 21 lgs that responded back to the survey , 6 were located within perth metro areas while the remaining 15 lgs were in the outer suburban areas . based on the data collected , there were a total of 53 owts failures , with shire of dundas and shire of derby / west kimberley reporting the highest number of failures at 11 and 6 owts failures , respectively . the most common reasons for owts failures in the survey include groundwater and surface water ingress , systems not installed properly , unsuitable soil type , and undersized systems . several lgs also mentioned other factors that contributed to owts failures , including increase in wastewater volume , root invasion , illegal installation , unauthorised tampering , cross connection to stormwater disposal , undersized systems , and unauthorised materials ( such as fats , oils , and yeasts ) . septic systems remain a preferred option for onsite wastewater management in unsewered urban and rural regions of wa . however , the use of other types of owts such as atu and composting toilets with greywater treatment has increased over the years . as expected , the number of applications was generally higher in regional areas compared to urban areas as most urban areas are connected to centralized sewerage systems . currently , national and state guidelines are available to ensure adequate construction , installation , and management of owts in order to protect public health . however , the survey conducted in 2011 indicated that a number of owts are either performing poorly or failing with about 48% of owts failures in wa , which is due to groundwater and surface water ingress . other reasons include incorrect system installation , unsuitable soil type , undersized system , increasing wastewater volume , hydrostatic pressure , high groundwater , root invasion , cross connection to stormwater disposal , and a small percentage of unauthorized tampering . although technology is improving over time and some owts are capable of producing better effluent quality , the problems identified in the dohwa survey are also reported elsewhere . therefore it is necessary to ensure that there is a minimum of 0.5 to 0.6 m of soil below the base of a trench before high permeable fissured rock , soil , or a groundwater table . identifying and comparing areas with high owts installations in wa in addition , it is essential to ensure that a fair and consistent approach to dealing with malfunctioning owts is taken by lgs under the guidance from the dohwa . inadequate management strategies and the lack of coordinate institutional arrangements in relation to owts have been previously reported in australia . for instance , in a survey of 48 septic tanks , it was found that 72% of dispersal fields were soggy and ( by inference ) ineffective , 67% required solids removal , 8% needed structural repairs , 6% had insufficient capacity , and 4% were incorrectly sited . also , a study of owts performance in south australia ( sa ) indicates that 22% of atus contains indicator bacteria at levels higher than the maximum acceptable levels specified in the sa 's regulatory guidelines . collecting data of owts installations was conducted by the dohwa via emails , relying on individual lg across wa to provide data on the number of owts installed during the study period . however , as previously mentioned , data regarding the number of approved owts applications by lg was only collected from 20% of the existing lgs in wa ( 28 out of 140 ) . a follow - up on the lg that failed to respond to the email sent out by the dohwa staff enquiring about the numbers of owts approvals indicates inadequate number of staff to review the applications or lack of adequate records to respond to the survey . several reasons for the lack of available data from lgs include the following.records were not available , due to lack of general file , lg in remote locations , and lack of full time ehos.records were not easily retrieved as data stored in offsite storage.information on owts was not recorded in the early years.for some lgs approval , inspection , and ongoing management for owts were conducted in a reactive way rather than on a proactive manner.ehos do not have the time to collate information because owts are only one part of their several duties.systems described as records were not available , due to lack of general file , lg in remote locations , and lack of full time ehos . for some lgs approval , inspection , and ongoing management for owts ehos do not have the time to collate information because owts are only one part of their several duties . lack of appropriate administration system to record owts approvals and installations is a significant drawback in the management of owts in wa . without any feasible management strategies to assess and maintain the performance of owts in each region , the resulting environmental and public health risks associated with poor system performance would increase . hence , there is a need to provide a generic approach for a standardized , common system that will allow each lg to record the number of owts applications that have been approved and to store information related to the approvals in a clear and standard record system . furthermore , there is a need for a multigovernance approach that can address the importance of an effective and robust planning and development of sites . it is important that the site assessment for on - site wastewater management be carried out in the early stage of planning phase . database should be able to record information on installation , operation , maintenance , and any other inspection requirements particularly for atus or systems achieving a secondary treated quality effluent.it should implement a soil absorption zone path length of at least 600 mm to reduce the potential for owts failure due to ground and surface water ingress.it should provide ehos with on - site wastewater management training based on as 1547 and current legislations as part of their professional development and an important measure of quality assurance for the industry , government , and general public.it should produce educational materials to owners of owts to facilitate the understanding of how owts work , how to identify signs of systems failures , and why inadequate maintenance can increase the system failures and the risk to humans and the environment . database should be able to record information on installation , operation , maintenance , and any other inspection requirements particularly for atus or systems achieving a secondary treated quality effluent . it should provide ehos with on - site wastewater management training based on as 1547 and current legislations as part of their professional development and an important measure of quality assurance for the industry , government , and general public . it should produce educational materials to owners of owts to facilitate the understanding of how owts work , how to identify signs of systems failures , and why inadequate maintenance can increase the system failures and the risk to humans and the environment . it is considered that a tolerable rate of system failure is smaller than 5% annually.they should implement a planning strategy for owts in the early stage of any development to identify the resources that are required to ensure adequate owts performance.they should implement site inspection strategies that are predominantly risk based , taking account of sensitive receptors , and should be integrated with other regulatory inspections as appropriate.they should distribute educational materials produced by the dohwa or lgs to owners of owts and provide advices to applicants on owts - related matters when required.they should update the register of installed owts in each lg jurisdiction and implement an inspection regime to identify legacy sites that will require remediation work . , monitoring of water quality ) , third party , engagement , and incentive strategies may be implemented . these strategies can involve working closely with home owners or stakeholders to ensure that those who are responsible for owts understand how to comply with the regulations and are encouraged to do so.they should embark on a series of activities to raise awareness and compliance related to the operation and maintenance of owts . information campaigns may include the communication of international best practice to water services australia ( wsa ) and specific local guidance to nongovernment organizations ( ngos ) and homeowners.they should conduct follow - up site inspections of approved owts that focus specifically on operation and maintenance . they should distribute educational materials produced by the dohwa or lgs to owners of owts and provide advices to applicants on owts - related matters when required . , monitoring of water quality ) , third party , engagement , and incentive strategies may be implemented . they should embark on a series of activities to raise awareness and compliance related to the operation and maintenance of owts . information campaigns may include the communication of international best practice to water services australia ( wsa ) and specific local guidance to nongovernment organizations ( ngos ) and homeowners .

photorefractive keratectomy ( prk ) was introduced more than 25 years ago as a corneal refractive surgical technique using the excimer laser.1,2 the advent of laser - assisted in situ keratomileusis ( lasik ) led to a decline in the popularity of prk.3 nevertheless , there is a recognition that surface ablation has several potential advantages with regard to preserving corneal biomechanical integrity and avoiding intraoperative and late flap - related complications . the main limitations of prk remain postoperative pain , delayed epithelial healing , and postoperative stromal haze . the use of alcohol as an alternative to mechanical debridement has been assessed , as has preservation of the epithelium as a flap ( laser - assisted sub - epithelial keratectomy [ lasek ] and epi - lasik).4 another alternative is transepithelial prk , where epithelial removal is generally carried out with laser phototherapeutic ( ptk ) ablation , followed by a stromal laser refractive ablation.58 transepithelial prk has several advantages , including no instrument contact with the eye , reduced intervention time , and the potential to minimize the size of an epithelial defect to that required for stromal ablation , as well as the avoidance of alcohol and , thus , potential toxicity . a number of previous studies have assessed such a two - step approach an initial ptk pre - treatment for epithelial removal , followed by prk.5,7 other studies have examined transepithelial prk ( as a two - step procedure ) versus alcohol - assisted prk.810 in this study , we evaluated a new technique for combined epithelial and stromal laser ablation . this has been termed all - surface laser ablation ( asla ) , a modified transepithelial prk technique . the technique differs from the previously described transepithelial prk technique in a number of aspects . firstly , the epithelium is not ablated using an even or broad beam ptk profile . instead a custom epithelial profile has been generated from population studies , which have demonstrated that the epithelium does not have a uniform thickness . studies using high frequency ultrasound have been utilized to form a standard epithelial model , demonstrating relatively thicker peripheral epithelium.11 the ablation profile generated from this population model thus targets 55 m centrally , and 65 m peripherally , for an 8 mm ablation zone , with further consideration to the differential ablation rate in epithelium , compared to stroma . secondly , the ablation is not carried out as two distinct steps , but rather as a single continuous profile , mitigating the risk of dehydration between steps . thirdly , the profile uniquely applies the stromal component of the ablation first , over the epithelium , prior to the additive epithelial profile . this has the potential benefit of maximising the smoothing effect of the epithelium , since it is recognized that epithelial thickening occurs in areas of stromal irregularity . this potential advantage is negated when the epithelium is removed prior to stromal ablation , thus exposing the underlying irregularity . in addition to the use of this novel surface ablation technique , we also used adjunctive mitomycin c ( mmc ) . this is recognized as a means for reducing the occurrence of postoperative stromal haze , thereby addressing the other main limitation of surface ablation techniques.12 the primary aim of this study was to assess postoperative pain , photophobia , and stromal haze , compared to a control group undergoing standard alcohol - assisted epithelial removal . additionally , we aimed to assess visual and refractive outcomes , and safety , compared to the controls . we also sought to evaluate the relative effects of asla transepithelial ablation when mmc is used . the patients of this study were recruited in emmetropia mediterranean eye institute , a private refractive practice in crete , from september 2010 to march 2011 . written informed consent was obtained prior to inclusion , and the study was performed following the mandates of the helsinki agreement . the choice of study design was based on the need for a matched control group , given the small numbers . in addition , there was the advantage of having the same patient to subjectively compare and evaluate the treatment modalities between eyes . for each patient , the choice of first eye treated and the application of intervention or control was randomized using randomization tables , with the fellow eye receiving the alternate treatment . patients and observers were masked for all features other than slit - lamp examination , which could not be masked due to differing clinical appearances . inclusion criteria were : age over 18 ; myopic refraction with astigmatism no higher than 3 diopters ( d ) ; stable refraction for 12 months preceding surgery ; maximum planned corneal stromal ablation of 110 m ; and corrected snellen distance visual acuity ( cdva ) of 0.7 snellen fraction or better . eyes with corneal epithelial pathology , keratoconus , ocular inflammation , glaucoma , and posterior segment pathology were excluded from the study . the preoperative assessment included uncorrected distance acuity ( udva ) and cdva , manifest and cycloplegic refraction , ultrasound pachymetry , corneal topography with orbscan ( bausch and lomb , rochester , ny ) and keratron scout topographer ( optikon spa , rome , italy ) . corneal aberrations were measured with the keratron scout , and ocular aberration measurements with the ork wavefront analyzer ( schwind eye - tech - solutions gmbh , kleinostheim , germany ) . the total surgical time from speculum insertion to removal was recorded in the operating theatre with a stopwatch . in both groups , surgical preparation was with iodine and topical tetracaine 0.5% instillation , prior to draping and insertion of a speculum . in the alcohol group , epithelial delamination was performed with 20% ethanol , applied using an 8 mm well , followed by epithelial removal with a spatula.8 in the asla group , treatment was preceded by standardized wet sponge application : a merocel sponge ( medtronic , minneapolis , mn ) was dipped in balanced salt solution and left to expand maximally , then applied with three slow painting movements on the corneal surface . this step avoids uneven wetting , and the subsequent risk of uneven ablation . all patients underwent treatment with the amaris 500 hz excimer laser ( schwind eye - tech - solutions ) . in the prk group , the excimer laser was administered in a single continuous session , as detailed above , with combined epithelial and stromal profile . in both groups , the postoperative regime was consistent between groups . before applying a contact lens ( frequency aspheric ; coopervision , pleasanton , ca ) , 1 drop of topical ketorolac 0.5% , and 1 drop of ofloxacin 0.3% were applied . after surgery , the patients were treated with topical instillation of ofloxacin 0.3% four times daily ( until removal of the contact lens ) , dexamethasone 0.1% drops four times daily ( reducing over 12 weeks ) , and artificial tear drops four times daily for 3 months . for all eyes in both groups , where the stromal ablation was planned to be more than 75 m , the patient received a standardized intraoperative application of 0.02% mmc , applied for 30 seconds using an mmc - impregnated sponge pledget , immediately after excimer ablation , followed by copious saline irrigation.12 twenty - eight eyes of 14 patients ( 46% ) , in both groups , received bilateral mmc after laser ablation . postoperative assessment was performed on day 1 , day 3 , at 1 week , and 1 , 3 , 6 , and 12 months after surgery . patients were masked to the assignment of eyes , while the treating and observing clinicians could not be . at each postoperative visit , udva and cdva were recorded . during the first three follow - up visits , subjective pain and photophobia postoperative corneal haze at 1 week , and 1 , 3 , 6 , and 12 months was graded at the slit lamp microscope , according to the fantes scale , by two independent ophthalmologists , and the average of the two values taken.13 contact lens replacement and early re - epithelisation were noted . subjective refraction and corneal aberrations were recorded preoperatively and at 1 , 3 , 6 , and 12 months postoperatively . all statistical analyses were performed using microsoft excel ( microsoft corporation , redmond , wa ) . intragroup variables were compared using unpaired t - tests , while temporal changes were compared using paired t - tests . sixty eyes of 30 myopic patients ( 19 male and 11 female ) undergoing excimer photorefractive keratectomy were included . the average age was 29 years ( sd : 9 ; range : 1746 ) . preoperative mean udva was 0.01 snellen fraction ( sd : 0.02 ; range : 00.1 ) , in the prk group , and 0.02 ( sd : 0.11 ; range : 00.6 ) in the asla group ( p = 0.2 ) . preoperative mean cdva was 0.98 snellen fraction ( sd : 0.06 ; range : 0.71.0 ) in the prk group , and 0.96 ( sd : 0.08 ; range : 0.71 ) in the asla group ( p = 0.2 ) . mean treated refractive error was 4.09 d ( sd : 1.87 ) in the prk group , and 4.25 d ( sd : 1.9 ) in the asla group , with a mean difference between pairs of intervention and control eyes of 0.65 d ( sd : 0.63 ) . preoperative corneal higher order aberrations , including spherical aberration , coma , and trefoil did not differ between the prk and asla groups ( p = 0.2 for all variables ) ( table 1 ) . the average stromal ablation was 72.77 m ( sd : 28.4 ) in the prk group and 71.33 m ( sd 26.3 ) in the asla group ( p = 0.4 ) . both eyes received essentially identical optical zone ( oz ) , transition zone ( tz ) , and total ablation zone ( taz ) . ( oz : 6.36 0.28 mm versus 6.35 0.27 mm ; tz : 1.28 0.48 mm versus 1.24 0.46 mm ; taz : 7.64 0.48 mm versus 7.59 0.43 mm for prk and asla , respectively ) ( p = 0.4 for all variables ) . fourteen ( 47% ) patients , with prk and asla in fellow eyes , received mmc bilaterally , immediately after ablation ; the rest did not receive mmc in either eye . a small subset of patients ( n = 5 ) had operative times recorded . approximate surgery time for the asla patients was 1 minute , compared to 3 minutes for prk . no patients were lost to follow - up . at day 3 , pain scores were statistically significantly lower in the asla group , at 0.9 ( sd : 1.6 ; range : 08 ) , compared to 2.6 ( sd : 1.7 ; range : 06 ) in the prk group ( p < 0.0005 ) . there was no significant difference in the very early pain scores at the first postoperative day : 4.5 ( sd : 2.6 ; range : 010 ) in the asla group ( p = 0.3 ) , and 4.1 ( sd : 2.6 ; range : 08 ) in the prk group ( figure 1b ) . day 1 photophobia scores were similar between groups at 6.2 ( sd : 2.4 ) in the prk group , and 6.1 ( sd : 2.6 ) in the asla group ( p = 0.4 ) . at the third postoperative day , the difference remained negligible : 1.8 ( sd : 2.5 ) in the asla group , compared to 2.2 ( sd : 2.1 ) in the prk group ( p = 0.6 ) . by day 3 , significantly more eyes in the asla group had epithelial closure ( 97% ) compared to the alcohol group , where only 17 eyes ( 57% ) had a healed epithelium ( p = 0.0002 [ chi - square test ] ) . this was also reflected in the need for a further bandage contact lens at this time ; 43% of eyes treated with conventional ( alcohol - assisted ) prk , but only 3% in the asla group required replacement of contact lens . at 1 week , all eyes in both groups had epithelial closure . there was no clear effect of mmc on epithelial healing , with 19 of 28 ( 68% ) eyes exposed to mmc epithelialising at day 3 versus 27/32 ( 84% ) in untreated eyes ( p = 0.13 ) . at 1 week , haze scores averaged 0.3 ( sd : 0.6 ; range : 03 ) in the prk group and 0.1 ( sd : 0.3 ; range : 01 ) in the asla group ( p = 0.07 ) . by 1 month , this difference was statistically significant , with a mean score of 0.4 ( sd : 0.8 ; range 03 ) in the prk group , and 0.2 ( sd : 0.5 ; range : 02 ) in the asla group ( p < 0.05 ) . significantly reduced haze in the asla group persisted at 3 and 6 months ( p < 0.05 ) , although by 1 year , all eyes in either group had a score of 0 . there was no evidence of increased corneal haze in eyes with greater ablation depths with our protocol , where all ablations > 75 m received mmc treatment ( p > 0.05 ) ( figure 1a ) . day 1 mean udva was similar at 0.42 ( sd : 0.18 ; range : 0.10.7 ) in the asla group , and 0.46 ( sd : 0.19 ; range : 0.10.9 ) in the prk group ( p = 0.2 ) . however , by day 3 , mean udva was better in the asla group at 0.63 ( sd : 0.15 ; range : 0.30.9 ) versus 0.42 ( sd : 0.15 ; range : 0.20.8 ) in the prk group ( p < 0.05 ) . at this third postoperative day , 90% ( 27 ) of eyes undergoing asla achieved 0.7 or better vision , compared to 43% of prk treated eyes . this statistically significant early visual recovery in the asla group persisted at 1 week , with mean udva of 0.85 ( sd : 0.19 ; range : 1.20.4 ) , compared to 0.66 ( sd : 0.24 ; range 1.00.1 ) in the prk group ( p < 0.0001 ) . at 1 month , mean udva was 0.94 ( sd : 0.1 ; range : 1.00.6 ) in the prk group , and 0.97 ( sd : 0.1 ; range : 1.20.6 ) in the asla group , with no remaining difference ( p = 0.1 ) . there remained no difference at 3 , 6 , and 12 months ( p = 0.5 at all time points ) ( figure 1c ) . the levels of accuracy for both techniques were high , with no statistically significant difference in attempted versus achieved final refraction ( figures 2 and 3 ) . both techniques were equivalent in safety , with one eye in each group losing one line . there were no differences between groups noted with regard to higher order aberrations , measured at any time point . there was a significant increase ( p < 0.05 ) in spherical higher order aberration postoperatively , equivalent in both groups ( table 1 ) . sixty eyes of 30 myopic patients ( 19 male and 11 female ) undergoing excimer photorefractive keratectomy were included . the average age was 29 years ( sd : 9 ; range : 1746 ) . preoperative mean udva was 0.01 snellen fraction ( sd : 0.02 ; range : 00.1 ) , in the prk group , and 0.02 ( sd : 0.11 ; range : 00.6 ) in the asla group ( p = 0.2 ) . preoperative mean cdva was 0.98 snellen fraction ( sd : 0.06 ; range : 0.71.0 ) in the prk group , and 0.96 ( sd : 0.08 ; range : 0.71 ) in the asla group ( p = 0.2 ) . mean treated refractive error was 4.09 d ( sd : 1.87 ) in the prk group , and 4.25 d ( sd : 1.9 ) in the asla group , with a mean difference between pairs of intervention and control eyes of 0.65 d ( sd : 0.63 ) . preoperative corneal higher order aberrations , including spherical aberration , coma , and trefoil did not differ between the prk and asla groups ( p = 0.2 for all variables ) ( table 1 ) . the average stromal ablation was 72.77 m ( sd : 28.4 ) in the prk group and 71.33 m ( sd 26.3 ) in the asla group ( p = 0.4 ) . both eyes received essentially identical optical zone ( oz ) , transition zone ( tz ) , and total ablation zone ( taz ) . ( oz : 6.36 0.28 mm versus 6.35 0.27 mm ; tz : 1.28 0.48 mm versus 1.24 0.46 mm ; taz : 7.64 0.48 mm versus 7.59 0.43 mm for prk and asla , respectively ) ( p = 0.4 for all variables ) . fourteen ( 47% ) patients , with prk and asla in fellow eyes , received mmc bilaterally , immediately after ablation ; the rest did not receive mmc in either eye . a small subset of patients ( n = 5 ) had operative times recorded . approximate surgery time for the asla patients was 1 minute , compared to 3 minutes for prk . at day 3 , pain scores were statistically significantly lower in the asla group , at 0.9 ( sd : 1.6 ; range : 08 ) , compared to 2.6 ( sd : 1.7 ; range : 06 ) in the prk group ( p < 0.0005 ) . there was no significant difference in the very early pain scores at the first postoperative day : 4.5 ( sd : 2.6 ; range : 010 ) in the asla group ( p = 0.3 ) , and 4.1 ( sd : 2.6 ; range : 08 ) in the prk group ( figure 1b ) . day 1 photophobia scores were similar between groups at 6.2 ( sd : 2.4 ) in the prk group , and 6.1 ( sd : 2.6 ) in the asla group ( p = 0.4 ) . at the third postoperative day , the difference remained negligible : 1.8 ( sd : 2.5 ) in the asla group , compared to 2.2 ( sd : 2.1 ) in the prk group ( p = 0.6 ) . by day 3 , significantly more eyes in the asla group had epithelial closure ( 97% ) compared to the alcohol group , where only 17 eyes ( 57% ) had a healed epithelium ( p = 0.0002 [ chi - square test ] ) . this was also reflected in the need for a further bandage contact lens at this time ; 43% of eyes treated with conventional ( alcohol - assisted ) prk , but only 3% in the asla group required replacement of contact lens . at 1 week , all eyes in both groups had epithelial closure . there was no clear effect of mmc on epithelial healing , with 19 of 28 ( 68% ) eyes exposed to mmc epithelialising at day 3 versus 27/32 ( 84% ) in untreated eyes ( p = 0.13 ) . at 1 week , haze scores averaged 0.3 ( sd : 0.6 ; range : 03 ) in the prk group and 0.1 ( sd : 0.3 ; range : 01 ) in the asla group ( p = 0.07 ) . by 1 month , this difference was statistically significant , with a mean score of 0.4 ( sd : 0.8 ; range 03 ) in the prk group , and 0.2 ( sd : 0.5 ; range : 02 ) in the asla group ( p < 0.05 ) . significantly reduced haze in the asla group persisted at 3 and 6 months ( p < 0.05 ) , although by 1 year , all eyes in either group had a score of 0 . there was no evidence of increased corneal haze in eyes with greater ablation depths with our protocol , where all ablations > 75 m received mmc treatment ( p > 0.05 ) ( figure 1a ) . day 1 mean udva was similar at 0.42 ( sd : 0.18 ; range : 0.10.7 ) in the asla group , and 0.46 ( sd : 0.19 ; range : 0.10.9 ) in the prk group ( p = 0.2 ) . however , by day 3 , mean udva was better in the asla group at 0.63 ( sd : 0.15 ; range : 0.30.9 ) versus 0.42 ( sd : 0.15 ; range : 0.20.8 ) in the prk group ( p < 0.05 ) . at this third postoperative day , 90% ( 27 ) of eyes undergoing asla achieved 0.7 or better vision , compared to 43% of prk treated eyes . this statistically significant early visual recovery in the asla group persisted at 1 week , with mean udva of 0.85 ( sd : 0.19 ; range : 1.20.4 ) , compared to 0.66 ( sd : 0.24 ; range 1.00.1 ) in the prk group ( p < 0.0001 ) . at 1 month , mean udva was 0.94 ( sd : 0.1 ; range : 1.00.6 ) in the prk group , and 0.97 ( sd : 0.1 ; range : 1.20.6 ) in the asla group , with no remaining difference ( p = 0.1 ) . there remained no difference at 3 , 6 , and 12 months ( p = 0.5 at all time points ) ( figure 1c ) . the levels of accuracy for both techniques were high , with no statistically significant difference in attempted versus achieved final refraction ( figures 2 and 3 ) . both techniques were equivalent in safety , with one eye in each group losing one line . there were no differences between groups noted with regard to higher order aberrations , measured at any time point . there was a significant increase ( p < 0.05 ) in spherical higher order aberration postoperatively , equivalent in both groups ( table 1 ) . in this study , we sought to assess a modified transepithelial prk technique , with a customized epithelial ablation profile ( asla ) , with respect to the main current limitations of surface ablation techniques , namely postoperative pain / photophobia , haze , and delayed visual recovery.4,7,12,14,15 the primary finding of this study was that the use of this technique offers patients significantly reduced early postoperative pain ( with a mean main score of 0.9 versus 2.6 at the third postoperative day ) , reduced photophobia , and more rapid epithelial closure , compared to conventional alcohol - assisted prk . a specific practical resultant benefit was that asla required bandage contact lens replacement in only one eye ( 3% ) by day 3 , whereas , in the prk group , the lens had to be replaced in 43% of eyes . with regard to early visual recovery , there is also a significant improvement , with approximately three snellen lines better vision on day 3 in the asla group ( 0.4 versus 0.2 ; p < 0.05 ) . it was one month before the prk group approached the asla group , in terms of vision ( p = 0.08 ) . this improvement on prk s limitations occurs at no cost to outcomes : the asla technique was equivalent or superior to the conventional alcohol - assisted prk in all metrics . with specific reference to safety , there was no difference , whereas the asla group appeared to have better outcomes , with 33% versus 13% having improved cdva ( p > 0.05 ) . the main further limitation of conventional prk is postoperative haze.12,14,15 a key finding of this study is that the use of asla transepithelial treatment seems to offer some further additional benefit over and above that provided by mmc , which was used equivalently in both groups . haze is a relatively rare side effect of prk , associated with high ablations , discontinuation of steroids , postoperative uv exposure , and male sex.14,15 a key turning point in the evolution of surface ablation treatments has been the introduction of mmc adjunctive therapy , which has gone some way towards ameliorating postoperative haze , and has been pivotal to the current resurgence of prk . notably , objective postoperative haze was less in the asla group at 1 week ( p = 0.07 ) , and 1 , 3 , and 6 months ( p < 0.05 ) , although absent in both groups at 1 year . in previous studies , the peak of corneal haze was observed at a mean time of 7.4 months.15 the asla cohort had consistently lower levels of haze over a 6-month period , which reached a plateau after the third month , with no attributable visual deficit . it has previously been shown to be effective in the management of keratoplasty and post - refractive surgery complex errors.5,6 a small study comparing alcohol - assisted versus ptk epithelial removal found no difference in healing time , and greater pain in the transepithelial treatment.8 another comparison of transepithelial prk , mechanical removal prk , and lasek found no difference in pain or haze.10 a large retrospective comparison of transpithelial prk with epi - lasik , lasek , and lasik found better visual outcomes with transepithelial treatment for high myopia.9 these previous studies , however , utilized the two - step technique , of ptk followed by prk.5,6,8,10,16 this is distinct from our reported asla transepithelial prk , which uses a population - based epithelium - removal profile , rather than ptk , and a single - step ablation . moreover , during asla , first the refractive component , and then the epithelial profile is ablated . so , the refractive ablation is applied over and completely or incompletely removes epithelium , whereas the subsequent epithelial profile advances the curvature change down to stroma . this reverse sequence maximizes the smoothing effect of epithelium over stromal irregularities , and utilizes the early period of expected better cooperation and fixation for the critical refractive ablation , leaving the less alignment - critical profile for the end . another advantage is the rapid treatment time ; we found that the one - step laser treatment takes considerably less time than either alcohol removal or two - step ptk laser removal . however , the follow - up period was limited to 3 months , and the use of a retrospective control group introduces potential bias during patient selection and consent , which are avoided in our fellow eye methodology.17 a key difference in the findings was early visual recovery , which was superior in the alcohol group in the above study , compared to ours . however , unlike our study , mmc was not used . it is known that keratocyte apoptosis and myofibroblast activation is a key factor in stromal recovery after surface ablation procedures , and that transepithelial ablation results in lower levels of keratocyte apoptosis.18,19 epithelial regeneration and integrity is paramount , in order to initiate cytokine induction , which in turn activates stromal wound healing.20 animal studies confirm that transepithelial epithelium removal produces a smooth uniform surface , ideal for epithelial regeneration.21 although alcohol delamination of the corneal epithelium can provide a smooth surface at the level of the bowman s membrane , the viability of the epithelial cells is reduced , with potential limbal effects.20,22 agrawal et al observed an increased inflammatory response , as well as damage to the anterior stromal keratocytes , after alcohol exposure , which could potentiate haze formation.23 we believe that the lower scores for both early- and late - onset corneal haze in asla - treated eyes are due to a smoother stromal bed , facilitating centripetal growth of new epithelium , faster treatment time , and optimal epithelial healing . the main limitation of this study relates to sample size , although , even in this relatively small sample , the study was sufficiently powered to demonstrate a statistically significant improvement in early recovery with asla transepithelial prk . all - surface laser ablation , a no - touch , single - step transepithelial prk technique , demonstrates significantly faster epithelial healing , reduced corneal haze , and reduced postoperative pain , compared with alcohol - assisted prk . the refractive and visual outcomes are at least equivalent to those of alcohol - assisted prk .

purposeto evaluate postoperative pain , corneal epithelial healing , development of corneal haze , refractive outcomes , and corneal aberrations in a novel one - step , modified transepithelial photorefractive keratectomy ( prk ) , termed all - surface laser ablation ( asla ) , compared to conventional , alcohol - assisted prk.materials and methodssixty eyes of 30 myopic patients were prospectively recruited to a randomized fellow eye study . patients underwent conventional alcohol - assisted prk in one eye ( control group ) and asla - modified transepithelial prk in the other ( 30 eyes in each treatment arm ) . primary endpoints were postoperative pain and haze scores at 1 day , 3 days , 1 week , and 1 , 3 , 6 , and 12 months . secondary endpoints included visual acuity at 1 , 3 , 6 , and 12 months , corneal aberrations at 3 , 6 , and 12 months , and early and late onset haze . refractive predictability , safety , and efficacy of the two methods were considered.resultsthe average age of the cohort was 29 years ( standard deviation [ sd ] : 9 ; range : 1846 ) , and the average spherical equivalent refractive error was 4.18 diopters ( sd : 1.9 ) . at 3 days after surgery , the average pain score was 64% lower in the asla group ( p < 0.0005 ) . at this point , 96% of asla eyes had no epithelial defect , whereas 43% in the alcohol - assisted group did not achieve complete epithelial healing , and required replacement of bandage contact lens . the haze level was consistently lower in the asla group at all time points from 1 to 6 months.conclusionthis study shows that the asla technique may have a future role in refractive surgery , due to the fact that it offers faster epithelial healing , lower pain scores , and significantly less haze formation .

Introduction Materials and methods Results Baseline demographics and control group equivalence Postoperative pain Postoperative photophobia Epithelial healing Corneal haze Uncorrected vision and refraction Higher order aberrations Discussion Conclusion

photorefractive keratectomy ( prk ) was introduced more than 25 years ago as a corneal refractive surgical technique using the excimer laser.1,2 the advent of laser - assisted in situ keratomileusis ( lasik ) led to a decline in the popularity of prk.3 nevertheless , there is a recognition that surface ablation has several potential advantages with regard to preserving corneal biomechanical integrity and avoiding intraoperative and late flap - related complications . the main limitations of prk remain postoperative pain , delayed epithelial healing , and postoperative stromal haze . the use of alcohol as an alternative to mechanical debridement has been assessed , as has preservation of the epithelium as a flap ( laser - assisted sub - epithelial keratectomy [ lasek ] and epi - lasik).4 another alternative is transepithelial prk , where epithelial removal is generally carried out with laser phototherapeutic ( ptk ) ablation , followed by a stromal laser refractive ablation.58 transepithelial prk has several advantages , including no instrument contact with the eye , reduced intervention time , and the potential to minimize the size of an epithelial defect to that required for stromal ablation , as well as the avoidance of alcohol and , thus , potential toxicity . a number of previous studies have assessed such a two - step approach an initial ptk pre - treatment for epithelial removal , followed by prk.5,7 other studies have examined transepithelial prk ( as a two - step procedure ) versus alcohol - assisted prk.810 in this study , we evaluated a new technique for combined epithelial and stromal laser ablation . this has been termed all - surface laser ablation ( asla ) , a modified transepithelial prk technique . this is recognized as a means for reducing the occurrence of postoperative stromal haze , thereby addressing the other main limitation of surface ablation techniques.12 the primary aim of this study was to assess postoperative pain , photophobia , and stromal haze , compared to a control group undergoing standard alcohol - assisted epithelial removal . additionally , we aimed to assess visual and refractive outcomes , and safety , compared to the controls . before applying a contact lens ( frequency aspheric ; coopervision , pleasanton , ca ) , 1 drop of topical ketorolac 0.5% , and 1 drop of ofloxacin 0.3% were applied . after surgery , the patients were treated with topical instillation of ofloxacin 0.3% four times daily ( until removal of the contact lens ) , dexamethasone 0.1% drops four times daily ( reducing over 12 weeks ) , and artificial tear drops four times daily for 3 months . for all eyes in both groups , where the stromal ablation was planned to be more than 75 m , the patient received a standardized intraoperative application of 0.02% mmc , applied for 30 seconds using an mmc - impregnated sponge pledget , immediately after excimer ablation , followed by copious saline irrigation.12 twenty - eight eyes of 14 patients ( 46% ) , in both groups , received bilateral mmc after laser ablation . postoperative assessment was performed on day 1 , day 3 , at 1 week , and 1 , 3 , 6 , and 12 months after surgery . during the first three follow - up visits , subjective pain and photophobia postoperative corneal haze at 1 week , and 1 , 3 , 6 , and 12 months was graded at the slit lamp microscope , according to the fantes scale , by two independent ophthalmologists , and the average of the two values taken.13 contact lens replacement and early re - epithelisation were noted . subjective refraction and corneal aberrations were recorded preoperatively and at 1 , 3 , 6 , and 12 months postoperatively . sixty eyes of 30 myopic patients ( 19 male and 11 female ) undergoing excimer photorefractive keratectomy were included . the average age was 29 years ( sd : 9 ; range : 1746 ) . preoperative mean udva was 0.01 snellen fraction ( sd : 0.02 ; range : 00.1 ) , in the prk group , and 0.02 ( sd : 0.11 ; range : 00.6 ) in the asla group ( p = 0.2 ) . preoperative mean cdva was 0.98 snellen fraction ( sd : 0.06 ; range : 0.71.0 ) in the prk group , and 0.96 ( sd : 0.08 ; range : 0.71 ) in the asla group ( p = 0.2 ) . mean treated refractive error was 4.09 d ( sd : 1.87 ) in the prk group , and 4.25 d ( sd : 1.9 ) in the asla group , with a mean difference between pairs of intervention and control eyes of 0.65 d ( sd : 0.63 ) . preoperative corneal higher order aberrations , including spherical aberration , coma , and trefoil did not differ between the prk and asla groups ( p = 0.2 for all variables ) ( table 1 ) . the average stromal ablation was 72.77 m ( sd : 28.4 ) in the prk group and 71.33 m ( sd 26.3 ) in the asla group ( p = 0.4 ) . at day 3 , pain scores were statistically significantly lower in the asla group , at 0.9 ( sd : 1.6 ; range : 08 ) , compared to 2.6 ( sd : 1.7 ; range : 06 ) in the prk group ( p < 0.0005 ) . there was no significant difference in the very early pain scores at the first postoperative day : 4.5 ( sd : 2.6 ; range : 010 ) in the asla group ( p = 0.3 ) , and 4.1 ( sd : 2.6 ; range : 08 ) in the prk group ( figure 1b ) . day 1 photophobia scores were similar between groups at 6.2 ( sd : 2.4 ) in the prk group , and 6.1 ( sd : 2.6 ) in the asla group ( p = 0.4 ) . at the third postoperative day , the difference remained negligible : 1.8 ( sd : 2.5 ) in the asla group , compared to 2.2 ( sd : 2.1 ) in the prk group ( p = 0.6 ) . by day 3 , significantly more eyes in the asla group had epithelial closure ( 97% ) compared to the alcohol group , where only 17 eyes ( 57% ) had a healed epithelium ( p = 0.0002 [ chi - square test ] ) . this was also reflected in the need for a further bandage contact lens at this time ; 43% of eyes treated with conventional ( alcohol - assisted ) prk , but only 3% in the asla group required replacement of contact lens . at 1 week , haze scores averaged 0.3 ( sd : 0.6 ; range : 03 ) in the prk group and 0.1 ( sd : 0.3 ; range : 01 ) in the asla group ( p = 0.07 ) . by 1 month , this difference was statistically significant , with a mean score of 0.4 ( sd : 0.8 ; range 03 ) in the prk group , and 0.2 ( sd : 0.5 ; range : 02 ) in the asla group ( p < 0.05 ) . significantly reduced haze in the asla group persisted at 3 and 6 months ( p < 0.05 ) , although by 1 year , all eyes in either group had a score of 0 . day 1 mean udva was similar at 0.42 ( sd : 0.18 ; range : 0.10.7 ) in the asla group , and 0.46 ( sd : 0.19 ; range : 0.10.9 ) in the prk group ( p = 0.2 ) . however , by day 3 , mean udva was better in the asla group at 0.63 ( sd : 0.15 ; range : 0.30.9 ) versus 0.42 ( sd : 0.15 ; range : 0.20.8 ) in the prk group ( p < 0.05 ) . this statistically significant early visual recovery in the asla group persisted at 1 week , with mean udva of 0.85 ( sd : 0.19 ; range : 1.20.4 ) , compared to 0.66 ( sd : 0.24 ; range 1.00.1 ) in the prk group ( p < 0.0001 ) . at 1 month , mean udva was 0.94 ( sd : 0.1 ; range : 1.00.6 ) in the prk group , and 0.97 ( sd : 0.1 ; range : 1.20.6 ) in the asla group , with no remaining difference ( p = 0.1 ) . there remained no difference at 3 , 6 , and 12 months ( p = 0.5 at all time points ) ( figure 1c ) . sixty eyes of 30 myopic patients ( 19 male and 11 female ) undergoing excimer photorefractive keratectomy were included . the average age was 29 years ( sd : 9 ; range : 1746 ) . preoperative mean udva was 0.01 snellen fraction ( sd : 0.02 ; range : 00.1 ) , in the prk group , and 0.02 ( sd : 0.11 ; range : 00.6 ) in the asla group ( p = 0.2 ) . preoperative mean cdva was 0.98 snellen fraction ( sd : 0.06 ; range : 0.71.0 ) in the prk group , and 0.96 ( sd : 0.08 ; range : 0.71 ) in the asla group ( p = 0.2 ) . mean treated refractive error was 4.09 d ( sd : 1.87 ) in the prk group , and 4.25 d ( sd : 1.9 ) in the asla group , with a mean difference between pairs of intervention and control eyes of 0.65 d ( sd : 0.63 ) . preoperative corneal higher order aberrations , including spherical aberration , coma , and trefoil did not differ between the prk and asla groups ( p = 0.2 for all variables ) ( table 1 ) . the average stromal ablation was 72.77 m ( sd : 28.4 ) in the prk group and 71.33 m ( sd 26.3 ) in the asla group ( p = 0.4 ) . at day 3 , pain scores were statistically significantly lower in the asla group , at 0.9 ( sd : 1.6 ; range : 08 ) , compared to 2.6 ( sd : 1.7 ; range : 06 ) in the prk group ( p < 0.0005 ) . there was no significant difference in the very early pain scores at the first postoperative day : 4.5 ( sd : 2.6 ; range : 010 ) in the asla group ( p = 0.3 ) , and 4.1 ( sd : 2.6 ; range : 08 ) in the prk group ( figure 1b ) . day 1 photophobia scores were similar between groups at 6.2 ( sd : 2.4 ) in the prk group , and 6.1 ( sd : 2.6 ) in the asla group ( p = 0.4 ) . at the third postoperative day , the difference remained negligible : 1.8 ( sd : 2.5 ) in the asla group , compared to 2.2 ( sd : 2.1 ) in the prk group ( p = 0.6 ) . by day 3 , significantly more eyes in the asla group had epithelial closure ( 97% ) compared to the alcohol group , where only 17 eyes ( 57% ) had a healed epithelium ( p = 0.0002 [ chi - square test ] ) . this was also reflected in the need for a further bandage contact lens at this time ; 43% of eyes treated with conventional ( alcohol - assisted ) prk , but only 3% in the asla group required replacement of contact lens . at 1 week , haze scores averaged 0.3 ( sd : 0.6 ; range : 03 ) in the prk group and 0.1 ( sd : 0.3 ; range : 01 ) in the asla group ( p = 0.07 ) . by 1 month , this difference was statistically significant , with a mean score of 0.4 ( sd : 0.8 ; range 03 ) in the prk group , and 0.2 ( sd : 0.5 ; range : 02 ) in the asla group ( p < 0.05 ) . significantly reduced haze in the asla group persisted at 3 and 6 months ( p < 0.05 ) , although by 1 year , all eyes in either group had a score of 0 . day 1 mean udva was similar at 0.42 ( sd : 0.18 ; range : 0.10.7 ) in the asla group , and 0.46 ( sd : 0.19 ; range : 0.10.9 ) in the prk group ( p = 0.2 ) . however , by day 3 , mean udva was better in the asla group at 0.63 ( sd : 0.15 ; range : 0.30.9 ) versus 0.42 ( sd : 0.15 ; range : 0.20.8 ) in the prk group ( p < 0.05 ) . at this third postoperative day , 90% ( 27 ) of eyes undergoing asla achieved 0.7 or better vision , compared to 43% of prk treated eyes . this statistically significant early visual recovery in the asla group persisted at 1 week , with mean udva of 0.85 ( sd : 0.19 ; range : 1.20.4 ) , compared to 0.66 ( sd : 0.24 ; range 1.00.1 ) in the prk group ( p < 0.0001 ) . at 1 month , mean udva was 0.94 ( sd : 0.1 ; range : 1.00.6 ) in the prk group , and 0.97 ( sd : 0.1 ; range : 1.20.6 ) in the asla group , with no remaining difference ( p = 0.1 ) . there remained no difference at 3 , 6 , and 12 months ( p = 0.5 at all time points ) ( figure 1c ) . in this study , we sought to assess a modified transepithelial prk technique , with a customized epithelial ablation profile ( asla ) , with respect to the main current limitations of surface ablation techniques , namely postoperative pain / photophobia , haze , and delayed visual recovery.4,7,12,14,15 the primary finding of this study was that the use of this technique offers patients significantly reduced early postoperative pain ( with a mean main score of 0.9 versus 2.6 at the third postoperative day ) , reduced photophobia , and more rapid epithelial closure , compared to conventional alcohol - assisted prk . a specific practical resultant benefit was that asla required bandage contact lens replacement in only one eye ( 3% ) by day 3 , whereas , in the prk group , the lens had to be replaced in 43% of eyes . with regard to early visual recovery , there is also a significant improvement , with approximately three snellen lines better vision on day 3 in the asla group ( 0.4 versus 0.2 ; p < 0.05 ) . this improvement on prk s limitations occurs at no cost to outcomes : the asla technique was equivalent or superior to the conventional alcohol - assisted prk in all metrics . with specific reference to safety , there was no difference , whereas the asla group appeared to have better outcomes , with 33% versus 13% having improved cdva ( p > 0.05 ) . notably , objective postoperative haze was less in the asla group at 1 week ( p = 0.07 ) , and 1 , 3 , and 6 months ( p < 0.05 ) , although absent in both groups at 1 year . in previous studies , the peak of corneal haze was observed at a mean time of 7.4 months.15 the asla cohort had consistently lower levels of haze over a 6-month period , which reached a plateau after the third month , with no attributable visual deficit . it has previously been shown to be effective in the management of keratoplasty and post - refractive surgery complex errors.5,6 a small study comparing alcohol - assisted versus ptk epithelial removal found no difference in healing time , and greater pain in the transepithelial treatment.8 another comparison of transepithelial prk , mechanical removal prk , and lasek found no difference in pain or haze.10 a large retrospective comparison of transpithelial prk with epi - lasik , lasek , and lasik found better visual outcomes with transepithelial treatment for high myopia.9 these previous studies , however , utilized the two - step technique , of ptk followed by prk.5,6,8,10,16 this is distinct from our reported asla transepithelial prk , which uses a population - based epithelium - removal profile , rather than ptk , and a single - step ablation . however , the follow - up period was limited to 3 months , and the use of a retrospective control group introduces potential bias during patient selection and consent , which are avoided in our fellow eye methodology.17 a key difference in the findings was early visual recovery , which was superior in the alcohol group in the above study , compared to ours . it is known that keratocyte apoptosis and myofibroblast activation is a key factor in stromal recovery after surface ablation procedures , and that transepithelial ablation results in lower levels of keratocyte apoptosis.18,19 epithelial regeneration and integrity is paramount , in order to initiate cytokine induction , which in turn activates stromal wound healing.20 animal studies confirm that transepithelial epithelium removal produces a smooth uniform surface , ideal for epithelial regeneration.21 although alcohol delamination of the corneal epithelium can provide a smooth surface at the level of the bowman s membrane , the viability of the epithelial cells is reduced , with potential limbal effects.20,22 agrawal et al observed an increased inflammatory response , as well as damage to the anterior stromal keratocytes , after alcohol exposure , which could potentiate haze formation.23 we believe that the lower scores for both early- and late - onset corneal haze in asla - treated eyes are due to a smoother stromal bed , facilitating centripetal growth of new epithelium , faster treatment time , and optimal epithelial healing . all - surface laser ablation , a no - touch , single - step transepithelial prk technique , demonstrates significantly faster epithelial healing , reduced corneal haze , and reduced postoperative pain , compared with alcohol - assisted prk .

obesity is the second cause of death in the world and has reached epidemic proportions in recent years . the world health organization estimates that there are over 1 billion overweight adults globally , 300 million of whom are obese . obesity is a chronic disease and is associated with numerous comorbidities , including type 2 diabetes mellitus ( t2 dm ) , cardiovascular disease , hypertension , and dyslipidemia . moreover , obesity is a risk factor for major causes of morbidity and mortality and increasing evidence shows that a binge eating disorder ( bed ) affects a subset of obese patients . binge eating disorder is a psychiatric disorder characterized by recurrent episodes of binge eating ( ie , eating large amounts of food ) and is associated with a loss of control and significant distress in the absence of the regular compensatory weight - reducing behaviors commonly observed among patients with bulimia nervosa . the prevalence of bed in the normal adult population varies from 2% to 5% , but this proportion rises up to 50% among obese adults seeking weight reduction . european data indicate that the general prevalence of bed in italy is approximately 1.12% ( 0.26% among men and 1.92% among women ) . binge eating disorder is strongly linked with obesity and binge eating per se is linked with a high burden of metabolic risk factors in the general population . studies on obese patients with bed suggest an increased risk for the metabolic syndrome both in adults and adolescents . several studies showed a higher prevalence of bed in young and overweight patients with t2 dm . furthermore , bed is associated with poorer glycemic control and higher rates of diabetic complications in adolescents and adults with type 1 diabetes . binge eating disorder and binging behaviors are associated with poorer response to weight loss therapy . a few studies , however , have assessed the metabolic profile of bed obese without t2 dm and no data are available on the relation between different eating behaviors and the metabolic profile of these patients . indeed , a body of studies suggests the presence of an overall , low - grade inflammation in obesity , with increased levels of several circulating factors , such as high - sensitive c - reactive protein ( hs - crp ) , tumor necrosis factor- , interleukin-6 , and other biologic markers of inflammation . conversely , a reduction in body weight is accompanied by a decrease or even a normalization of these inflammatory markers , which are known to have a causal relationship with obesity and its comorbidities , such as insulin resistance , t2 dm , and cardiovascular risk . low - grade inflammation is strongly linked with obesity but , to our best knowledge , no data are available on the possible differences regarding the inflammatory profile between obese patients with and without bed . the aim of this study was to evaluate whether obese patients with bed and without t2 dm have a different metabolic and inflammatory profile related to their eating behaviors compared with non - bed obese . we also wished to test the hypothesis that the characteristic eating patterns of bed obese lead to an altered metabolic and inflammatory profile when compared with non - bed obese . a total of 115 white obese patients without t2 dm were consecutively recruited in this cross - sectional study at the department of medical and surgical sciences of the university magna graecia of catanzaro ( italy ) from december 2013 to december 2014 . patients were enrolled according to the following eligibility criteria : aged between 20 and 65 years , body mass index ( bmi ) > 30 exclusion criteria were as follows : pregnancy or having recently given birth , previous diagnosis of diabetes mellitus , known inflammatory disease , a history of malignant disease or pathologies , or drugs able to modify glucose metabolism . after 12-hour fasting , a venous blood sample was drawn for laboratory determinations and a 75 g oral glucose tolerance test was performed with 0 , 30 , 60 , 90 , and 120 minutes of sampling for plasma glucose and insulin for each patient . glucose tolerance status was defined on the basis of body mass index according to the american diabetes association criteria . patients underwent anthropometrical evaluation , wearing light indoor clothing , and no shoes , with a standing height to the nearest 0.1 cm and a body weight to the nearest 0.1 kg at 8.00 am . height and weight were measured using a portable stadiometer ( seca 220 , gmbh & co. , hamburg , germany ) and a balance scale ( seca 761 , gmbh & co. , hamburg , germany ) ; then , their bmi ( kg / m ) was calculated . in addition , waist and hip circumference were measured , and body composition was estimated by bioelectrical impedance . blood pressure was measured using a calibrated manual sphygmomanometer and a stethoscope on the brachial artery at the antecubital area of the left elbow with patients supine after 5 minutes of rest . the hospital ethical committee ( comitato etico azienda ospedaliera mater domini ) approved protocol in september 2013 , and written informed consent was obtained from all patients . all the investigations were performed in accordance with the principles of the declaration of helsinki . binge eating scale is an easy to administer test with adequate internal consistency and validity . it has been widely used in research either to measure binge eating severity in the nonpurge binge eating population or to determine whether potential research patients meet the inclusion criteria for binge eating . binge eating scale is made up of 16 items describing the behavioral manifestations , feelings , and cognitions associated with binge eating . each item consists of 4 statements that reflect a range of severity from which patients choose the 1 that best describes their perceptions and feelings about their eating behavior . a total bes score < 17 indicates unlikely bed , a 17 to 27 score possible bed and values > 27 probable bed . psychiatric researchers with adequate training in the field of eating disorders interviewed each patient a week later . the interviewers examined each patient by means of the structured clinical interview for diagnostic and statistic manual of mental disorders iv ( dsm - iv ) axis i disorders and the binge eating disorder - clinical interview to respectively assess / confirm the diagnosis of bed and to deepen understanding of their eating behaviors ( ie , night eating , postdinner eating , social eating , sweet eating , emotional eating , grazing , craving for carbohydrates , and hyperphagia ) and exercise habits . the clinical interview through the structured clinical interview for dsm - iv axis i disorders confirmed the diagnosis of bed for all the obese patients with bes scores 17 . no bed diagnosis was confirmed among patients with bes < 17 . according to the diagnosis of the bed , the patients were divided into 2 groups : non - bed obese patients ( n = 85 ) and bed - obese patients ( n = 30 ) . plasma glucose , total and high - density lipoprotein ( hdl ) cholesterol , triglycerides , and uric acid concentrations were measured by enzymatic methods ( roche diagnostics , mannheim , germany ) . glycated hemoglobin ( hba1c ) was measured with high - performance liquid chromatography using a national glycohemoglobin standardization program certified automated analyzer and ifcc ( adams ha-8160 hba1c analyzer , menarini , italy ; normal reference range , 4.3%5.9% ) . plasma insulin concentration was determined by a chemiluminescence - based assay ( immulite , siemens healthcare , italy ) . high - sensitivity c reactive protein levels were measured by an automated instrument ( cardiophase hscrp , milan , italy ) . the erythrocyte sedimentation rate ( esr ) was measured automatically by the stopped - flow technique in a capillary microphotometer ( alifax test 1 system polverara , italy ) . insulin resistance was estimated by the homeostasis model assessment ( homa - ir ) index , calculated from the fasting glucose , and insulin concentrations according to the following formula ( fasting insulin fasting glucose)/22.5 . the visceral adiposity index ( vai ) was calculated using the formulas proposed by amato et al for men , vai = [ white blood cell ( wc)/36.58 + ( 1.89 bmi ) ] ( tryglicerides ( tg)/0.81 ) ( 1.52/hdl ) and for women , vai = [ wc/39.68 + ( 1.88 bmi ) ] ( tg/1.03 ) ( 1.31/hdl ) . data were analyzed using the statistical package for the social science , version 21.0 ( spss inc . , variables with skewed distribution , including triglyceride , hscrp , esr , wbc , and fasting insulin were natural log transformed for statistical analyses . anthropometric and metabolic differences between groups were tested after adjusting for age , sex , and bmi using a general linear model . cohen 's effect sizes ( es ) were calculated for all significant findings , with values ( negative or positive ) of 0.2 , 0.6 , 1.2 , and > 1.2 indicating trivial , small , moderate , and large es , respectively . the structural equation modeling using maximum likelihood estimation was conducted by means of spss / amos 21.0 to test for the validity of the hypothesized model . structural equation modeling is a method that allows the simultaneous estimation of all relationships between observed ( manifest or unmeasured ) and unobserved ( or latent ) variables of a model . it is graphically represented with observed variables enclosed by rectangles and latent variables by circles . an assumed causal path between 2 variables is shown by a directed edge ( single - headed arrow ) . path coefficients on the edges are partial standardized regression coefficients , which measure the effect of 1 variable on another , while controlling for all other variables prior in the model ; all coefficients show a positive association , but those with a minus sign indicate a negative association . model fit was evaluated using the comparative fit index ( cfi ) , the tucker lewis incremental fit index ( tli ) , and root mean square error of approximation ( rmsea ) . the magnitudes of these indices were evaluated according to the recommendations of hu and bentler . for cfi and tli , values of 0.90 and above were considered adequate , whereas values of 0.95 or above were considered very good ; for rmsea values of 0.08 and below were considered adequate and 0.05 or less very good . the ratio of and degree of freedom was also evaluated , considering that values below 3.0 are good and those below 2.0 are very good . a total of 115 white obese patients without t2 dm were consecutively recruited in this cross - sectional study at the department of medical and surgical sciences of the university magna graecia of catanzaro ( italy ) from december 2013 to december 2014 . patients were enrolled according to the following eligibility criteria : aged between 20 and 65 years , body mass index ( bmi ) > 30 exclusion criteria were as follows : pregnancy or having recently given birth , previous diagnosis of diabetes mellitus , known inflammatory disease , a history of malignant disease or pathologies , or drugs able to modify glucose metabolism . after 12-hour fasting , a venous blood sample was drawn for laboratory determinations and a 75 g oral glucose tolerance test was performed with 0 , 30 , 60 , 90 , and 120 minutes of sampling for plasma glucose and insulin for each patient . glucose tolerance status was defined on the basis of body mass index according to the american diabetes association criteria . patients underwent anthropometrical evaluation , wearing light indoor clothing , and no shoes , with a standing height to the nearest 0.1 cm and a body weight to the nearest 0.1 kg at 8.00 am . height and weight were measured using a portable stadiometer ( seca 220 , gmbh & co. , hamburg , germany ) and a balance scale ( seca 761 , gmbh & co. , hamburg , germany ) ; then , their bmi ( kg / m ) was calculated . in addition , waist and hip circumference were measured , and body composition was estimated by bioelectrical impedance . blood pressure was measured using a calibrated manual sphygmomanometer and a stethoscope on the brachial artery at the antecubital area of the left elbow with patients supine after 5 minutes of rest . the hospital ethical committee ( comitato etico azienda ospedaliera mater domini ) approved protocol in september 2013 , and written informed consent was obtained from all patients . all the investigations were performed in accordance with the principles of the declaration of helsinki . binge eating scale is an easy to administer test with adequate internal consistency and validity . it has been widely used in research either to measure binge eating severity in the nonpurge binge eating population or to determine whether potential research patients meet the inclusion criteria for binge eating . binge eating scale is made up of 16 items describing the behavioral manifestations , feelings , and cognitions associated with binge eating . each item consists of 4 statements that reflect a range of severity from which patients choose the 1 that best describes their perceptions and feelings about their eating behavior . a total bes score < 17 indicates unlikely bed , a 17 to 27 score possible bed and values > 27 probable bed . psychiatric researchers with adequate training in the field of eating disorders interviewed each patient a week later . the interviewers examined each patient by means of the structured clinical interview for diagnostic and statistic manual of mental disorders iv ( dsm - iv ) axis i disorders and the binge eating disorder - clinical interview to respectively assess / confirm the diagnosis of bed and to deepen understanding of their eating behaviors ( ie , night eating , postdinner eating , social eating , sweet eating , emotional eating , grazing , craving for carbohydrates , and hyperphagia ) and exercise habits . the clinical interview through the structured clinical interview for dsm - iv axis i disorders confirmed the diagnosis of bed for all the obese patients with bes scores 17 . no bed diagnosis was confirmed among patients with bes < 17 . according to the diagnosis of the bed , the patients were divided into 2 groups : non - bed obese patients ( n = 85 ) and bed - obese patients ( n = 30 ) . plasma glucose , total and high - density lipoprotein ( hdl ) cholesterol , triglycerides , and uric acid concentrations were measured by enzymatic methods ( roche diagnostics , mannheim , germany ) . glycated hemoglobin ( hba1c ) was measured with high - performance liquid chromatography using a national glycohemoglobin standardization program certified automated analyzer and ifcc ( adams ha-8160 hba1c analyzer , menarini , italy ; normal reference range , 4.3%5.9% ) . plasma insulin concentration was determined by a chemiluminescence - based assay ( immulite , siemens healthcare , italy ) . high - sensitivity c reactive protein levels were measured by an automated instrument ( cardiophase hscrp , milan , italy ) . the erythrocyte sedimentation rate ( esr ) was measured automatically by the stopped - flow technique in a capillary microphotometer ( alifax test 1 system polverara , italy ) . insulin resistance was estimated by the homeostasis model assessment ( homa - ir ) index , calculated from the fasting glucose , and insulin concentrations according to the following formula ( fasting insulin fasting glucose)/22.5 . the visceral adiposity index ( vai ) was calculated using the formulas proposed by amato et al for men , vai = [ white blood cell ( wc)/36.58 + ( 1.89 bmi ) ] ( tryglicerides ( tg)/0.81 ) ( 1.52/hdl ) and for women , vai = [ wc/39.68 + ( 1.88 bmi ) ] ( tg/1.03 ) ( 1.31/hdl ) . data were analyzed using the statistical package for the social science , version 21.0 ( spss inc . , chicago , il ) . variables with skewed distribution , including triglyceride , hscrp , esr , wbc , and fasting insulin anthropometric and metabolic differences between groups were tested after adjusting for age , sex , and bmi using a general linear model . cohen 's effect sizes ( es ) were calculated for all significant findings , with values ( negative or positive ) of 0.2 , 0.6 , 1.2 , and > 1.2 indicating trivial , small , moderate , and large es , respectively . the structural equation modeling using maximum likelihood estimation was conducted by means of spss / amos 21.0 to test for the validity of the hypothesized model . structural equation modeling is a method that allows the simultaneous estimation of all relationships between observed ( manifest or unmeasured ) and unobserved ( or latent ) variables of a model . it is graphically represented with observed variables enclosed by rectangles and latent variables by circles . an assumed causal path between 2 variables is shown by a directed edge ( single - headed arrow ) . path coefficients on the edges are partial standardized regression coefficients , which measure the effect of 1 variable on another , while controlling for all other variables prior in the model ; all coefficients show a positive association , but those with a minus sign indicate a negative association . model fit was evaluated using the comparative fit index ( cfi ) , the tucker lewis incremental fit index ( tli ) , and root mean square error of approximation ( rmsea ) . the magnitudes of these indices were evaluated according to the recommendations of hu and bentler . for cfi and tli , values of 0.90 and above were considered adequate , whereas values of 0.95 or above were considered very good ; for rmsea values of 0.08 and below were considered adequate and 0.05 or less very good . the ratio of and degree of freedom was also evaluated , considering that values below 3.0 are good and those below 2.0 are very good . table 1 shows the anthropometrical characteristics and laboratory findings for the 2 study groups . there were no differences in age and sex between bed and non - bed obese . binge eating disorder obese exhibited significantly higher bmi , waist circumference , hip circumference , waist / hip ratio , and fat mass , and a lower lean mass as compared with non - bed obese ; these differences remained significant after adjusting for bmi ( table 1 ) . there were no differences between the groups in blood pressure , total cholesterol , low - density lipoprotein cholesterol , triglycerides . binge eating disorder obese had a worse metabolic and inflammatory profile , exhibiting significantly lower hdl cholesterol and higher levels of hba1c , uric acid , esr , hs - crp , and wbc and all these differences remained significant after adjusting for bmi ( table 1 ) . binge eating disorder obese also exhibited significantly higher levels of fasting plasma insulin and higher degree of insulin resistance as assessed by homa - ir index and vai compared with non - bed obese ; similarly , differences remained significant after adjusting for bmi . blood pressure , low - density lipoprotein cholesterol , triglycerides , fasting glucose , and 2-hour postload plasma glucose were statistically significant after adjusting for bmi . comparison of anthropometrical and laboratory characteristics between groups regarding eating habits , bed obese revealed significantly higher percentages of grazing , emotional eating , night eating , sweet eating , and craving for carbohydrates than non - bed obese ( figure 1 ) . eating behaviors in binge eating disorder and non - binge eating disorder obese patients . structural equation modeling was conducted to test the hypothesis that altered eating behaviors affect anthropometrical , metabolic , and inflammatory variables . the model achieved goodness - of - fit : = 69.755 ; p = 0.261 ; ratio of and degree of freedom = 1.107 ; cfi = 0.980 ; tli = 0.970 ; and rmsea = 0.032 . in our model , altered eating behaviors ( ie , night eating , sweet eating , craving for carbohydrates , emotional eating , and grazing ) showed an indirect effect on waist circumference mediated by the diagnosis of bed and body weight ; waist circumference had an indirect effect on hba1c mediated by insulin resistance assessed either by homa - ir or vai ; and vai had a direct effect on the inflammatory indexes ( ie , esr , hs - crp , and uric acid ; figure 2 ) . craving for bed = binge eating disorder , cfi = comparative fit index , ch = craving for carbohydrates , cmin = , df = degree of freedom , esr = erythrocyte sedimentation rate ; rmsea = root mean square error of approximation , tli = tucker in this study , we showed an unfavorable metabolic and inflammatory profile in obese patients with bed as compared with non - bed obese . notably , we reported , for the first time that bed obese , exhibited a worse anthropometric and metabolic profile in relation to their different eating behaviors , as compared with non - bed obese . there are several potential mechanisms by which binge eating may cause or contribute to obesity . the characteristic eating patterns of bed individuals seem to be associated with specific metabolic abnormalities of obesity . for example , the ingestion of fewer but larger meals has adverse metabolic consequences than the ingestion of frequent , small meals , including increase in fasting glucose , insulin secretion , serum lipids , and deterioration of glucose tolerance . eating rapidly results in elevated serum lipids , higher waist - hip circumference ratio , and liver steatosis . the mechanism(s ) underlying the relationship between eating rate and fatty liver are not known . as reported by kral et al , it is hypothesized that the enhance and rapid glucose absorption , mediated by a brisk insulin response via cephalic phase release , an incretin effect and rapid intestinal handling , causes fatty infiltration of the liver via glucose toxicity . alone or in conjunction with rapid absorption of lipid , glucose and insulin increases may lead to insulin resistance and metabolic syndrome with central / visceral fat distribution and dyslipidemia . besides , increased insulin levels induced by binging may contribute to increased hunger . in fact , bed patients eat significantly more than do non - bed obese during the stimulation of a binge eating episode . despite the relation between specific metabolic alterations and these eating features of bed individuals ( ie , speed and amount ) , little is known about the metabolic consequences of the other eating habits typically observed in bed individuals ( eg , craving for carbohydrates , sweet eating , grazing , or emotional eating ) . herein , we provide evidence that bed obese showed a higher frequency of altered eating behaviors ( ie , binging , grazing , emotional eating , sweet eating , and craving for carbohydrates ) than non - bed obese . we did not observe a significant difference in binge eating frequency between bed and non - bed obese patients . the symptom binge eating is a behavior marked by consumption of a large amount of food within a short period of time with the sense of loss of control . but to fulfill the diagnosis of bed , the patient needs to have other symptoms , such as eating quickly , eating until feeling full , eating even if not hungry , eating alone for embarrassment and/or feeling disgusted , depressed , or guilty for overeating . so binge is a characteristic symptom but not an exclusive symptom of patients with bed . on the contrary , dsm - iv requires that binges occur at least 2 days a week for 6 months , so many obese patients could not satisfy the criteria of frequency while still binging . as binging becomes a distraction related to aversive emotional states , bed obese our results are in agreement with previous data showing that emotional eating is associated with the preference for sweet foods and that bed obese crave carbohydrates more than fats , compared with non - bed obese . as previously reported , grazing , another dysfunctional eating behavior that consists of smaller , subjective episodes of overeating , was significantly more evident among bed obese as compared with non - bed obese . we postulate that the compensatory pattern , demonstrated in healthy lean patients , could be impaired in patients who are already obese and have chronic binge habits . these habits could be responsible , in association with obesity , for the worse metabolic profile observed in bed obese . accordingly , we found that bed obese exhibited a significantly higher bmi , fat mass , fasting insulin levels , homa - ir index , and hba1c when compared with non - bed obese . moreover , among bed obese , we found an increased abdominal circumference , an indirect index of visceral adiposity and an increased vai , an indicator of a dysfunction of adipose tissue . in obese patients , visceral adipose tissue accumulation has been associated with an increased production of free fatty acids , interleukin-6 , tumor necrosis factor- , hs - crp , and a decreased production of adiponectin , each of which may contribute to insulin resistance . it is known that these cytokines and chemokines activate intracellular pathways that lead to the development of insulin resistance , increasing the risk of t2 dm . a previously study demonstrated that women with bed had significantly reduced plasma levels of adiponectin . accordingly , it is conceivable that increased visceral adiposity observed in bed obese may be responsible for increased insulin resistance , estimated by the homa - ir index . importantly , we observed that the bed obese showed elevated inflammatory markers , such as hs - crp , esr , and wbc counts . several studies showed that chronic subclinical inflammation is associated with t2 dm , cardiovascular disease , and patients at high risk of developing t2 dm . elevation in hs - crp is considered a marker of cardiovascular risk that has also been correlated with insulin resistance . increasing evidence has shown that the accumulation of lipids in adipose tissue and the expansion of fat mass determine the initiation of the obesity - induced inflammatory process through the production of proinflammatory cytokines and chemokines by the fat tissue . our results , in line with hudson et al , disagree with abraham et al . a possible explanation is that the research by hudson et al was drawn among obese patients , as in our case , whereas abraham et al used a large population - based cohort comparing obese ( most bingers ) with overweight ( most nonbingers ) . moreover , we found , that bed obese showed higher uric acid levels than non - bed obese did . elevation in uric acid has been associated with obesity and insulin resistance , all risk factors for atherosclerosis , and t2 dm . the prooxidant and proinflammatory effects of uric acid that interfere with glucose uptake may explain this association . furthermore , hyperuricemia is frequently documented in patients with cardiovascular diseases and with subclinical organ damage . structural equation modeling analysis helps to explain the relation between eating behaviors and metabolic impairment . night eating , sweet eating , grazing , emotional eating , and craving for carbohydrates may lead to increased waist circumference , an index of visceral adiposity , and insulin resistance , which , in turn , may favor production of inflammatory molecules , and alteration in glucose metabolism . the mechanism(s ) by which the eating behaviors are able to induce an alteration of metabolic profile and/or the inflammatory profile is not known . it might represent a direct effect of binge eating , perhaps because of the large amount of food ingested in typical eating binges . also , rapid consumption of large amounts of food can increase oxidative and inflammatory stress , and inflammatory changes could represent an important causal pathway for developing metabolic alterations . alternatively , because bed appears to be partially caused by genetic factors independent of obesity , it is possible that these or other underlying nongenetic factors might be responsible for these alterations . the novelty of this article is that an eating disorder , namely bed , and the related altered eating behaviors , can help to explain an impaired metabolic and inflammatory profile in a group of obese patients that could have an increased cardiometabolic risk . strengths of this study are the exclusion of patients with t2 dm or confounding disorders characterized by elevation in inflammatory molecules , inclusion of both sexes , the accurate anthropometric , metabolic , inflammatory and psychiatric characterization , and the evaluation of eating behaviors . the findings of this study , however , need to be interpreted in light of some limitations . first , the current results derive from a cross - sectional research with a small sample of obese patients . nevertheless , the sample included all obese patients that consecutively asked for weight reduction therapy in our department . furthermore , es and fit indexes of structural equation modeling demonstrated that the results were not influenced by the sample size . a second limitation of the current study is that we have evaluated insulin sensitivity by the homa - ir index . although the euglycaemic hyperinsulinaemic clamp , which is considered the gold standard method to measure insulin sensitivity , may provide a more accurate estimate of insulin sensitivity , it is time consuming and expensive , and is not feasible in large - scale studies . despite the limitations the current study confirms and expands the actual knowledge about the effects of specific dysfunctional eating behaviors commonly observed among obese patients on important metabolic and inflammatory alterations . all obese patients should be assessed for bed and it can be done through an easy tool , the bes . this could represent an important clinical target to identify patients potentially at high risk for obesity , t2 dm , and/or cardiometabolic disease . further research and prospective studies , however , are needed to assess and evaluate whether early detection and intervention for this specific disorder could have a positive prognostic impact on the long - term outcome ( eg , t2 dm and/or cardiovascular risk ) of this specific group of obese patients seeking weight reduction .

abstractto evaluate whether obese patients with a binge eating disorder ( bed ) have an altered metabolic and inflammatory profile related to their eating behaviors compared with non - bed obese.a total of 115 white obese patients consecutively recruited underwent biochemical , anthropometrical evaluation , and a 75-g oral glucose tolerance test . patients answered the binge eating scale and were interviewed by a psychiatrist . the patients were subsequently divided into 2 groups according to diagnosis : non - bed obese ( n = 85 ) and bed obese ( n = 30 ) . structural equation modeling analysis was performed to elucidate the relation between eating behaviors and metabolic and inflammatory profile.bed obese exhibited significantly higher percentages of altered eating behaviors , body mass index ( p < 0.001 ) , waist circumference ( p < 0.01 ) , fat mass ( p < 0.001 ) , and a lower lean mass ( p < 0.001 ) when compared with non - bed obese . binge eating disorder obese also had a worse metabolic and inflammatory profile , exhibiting significantly lower high - density lipoprotein cholesterol levels ( p < 0.05 ) , and higher levels of glycated hemoglobin ( p < 0.01 ) , uric acid ( p < 0.05 ) , erythrocyte sedimentation rate ( p < 0.001 ) , high - sensitive c - reactive protein ( p < 0.01 ) , and white blood cell counts ( p < 0.01 ) . higher fasting insulin ( p < 0.01 ) and higher insulin resistance ( p < 0.01 ) , assessed by homeostasis model assessment index and visceral adiposity index ( p < 0.001 ) , were observed among bed obese . all differences remained significant after adjusting for body mass index . no significant differences in fasting plasma glucose or 2-hour postchallenge plasma glucose were found . structural equation modeling analysis confirmed the relation between the altered eating behaviors of bed and the metabolic and inflammatory profile.binge eating disorder obese exhibited an unfavorable metabolic and inflammatory profile , which is related to their characteristic eating habits .

INTRODUCTION METHODS Study Participants Psychiatric Evaluation Laboratory Determinations Statistical Analysis RESULTS DISCUSSION

obesity is a chronic disease and is associated with numerous comorbidities , including type 2 diabetes mellitus ( t2 dm ) , cardiovascular disease , hypertension , and dyslipidemia . moreover , obesity is a risk factor for major causes of morbidity and mortality and increasing evidence shows that a binge eating disorder ( bed ) affects a subset of obese patients . binge eating disorder is a psychiatric disorder characterized by recurrent episodes of binge eating ( ie , eating large amounts of food ) and is associated with a loss of control and significant distress in the absence of the regular compensatory weight - reducing behaviors commonly observed among patients with bulimia nervosa . binge eating disorder is strongly linked with obesity and binge eating per se is linked with a high burden of metabolic risk factors in the general population . studies on obese patients with bed suggest an increased risk for the metabolic syndrome both in adults and adolescents . binge eating disorder and binging behaviors are associated with poorer response to weight loss therapy . a few studies , however , have assessed the metabolic profile of bed obese without t2 dm and no data are available on the relation between different eating behaviors and the metabolic profile of these patients . indeed , a body of studies suggests the presence of an overall , low - grade inflammation in obesity , with increased levels of several circulating factors , such as high - sensitive c - reactive protein ( hs - crp ) , tumor necrosis factor- , interleukin-6 , and other biologic markers of inflammation . conversely , a reduction in body weight is accompanied by a decrease or even a normalization of these inflammatory markers , which are known to have a causal relationship with obesity and its comorbidities , such as insulin resistance , t2 dm , and cardiovascular risk . low - grade inflammation is strongly linked with obesity but , to our best knowledge , no data are available on the possible differences regarding the inflammatory profile between obese patients with and without bed . the aim of this study was to evaluate whether obese patients with bed and without t2 dm have a different metabolic and inflammatory profile related to their eating behaviors compared with non - bed obese . we also wished to test the hypothesis that the characteristic eating patterns of bed obese lead to an altered metabolic and inflammatory profile when compared with non - bed obese . a total of 115 white obese patients without t2 dm were consecutively recruited in this cross - sectional study at the department of medical and surgical sciences of the university magna graecia of catanzaro ( italy ) from december 2013 to december 2014 . patients were enrolled according to the following eligibility criteria : aged between 20 and 65 years , body mass index ( bmi ) > 30 exclusion criteria were as follows : pregnancy or having recently given birth , previous diagnosis of diabetes mellitus , known inflammatory disease , a history of malignant disease or pathologies , or drugs able to modify glucose metabolism . after 12-hour fasting , a venous blood sample was drawn for laboratory determinations and a 75 g oral glucose tolerance test was performed with 0 , 30 , 60 , 90 , and 120 minutes of sampling for plasma glucose and insulin for each patient . glucose tolerance status was defined on the basis of body mass index according to the american diabetes association criteria . patients underwent anthropometrical evaluation , wearing light indoor clothing , and no shoes , with a standing height to the nearest 0.1 cm and a body weight to the nearest 0.1 kg at 8.00 am . height and weight were measured using a portable stadiometer ( seca 220 , gmbh & co. , hamburg , germany ) and a balance scale ( seca 761 , gmbh & co. , hamburg , germany ) ; then , their bmi ( kg / m ) was calculated . in addition , waist and hip circumference were measured , and body composition was estimated by bioelectrical impedance . binge eating scale is an easy to administer test with adequate internal consistency and validity . binge eating scale is made up of 16 items describing the behavioral manifestations , feelings , and cognitions associated with binge eating . the interviewers examined each patient by means of the structured clinical interview for diagnostic and statistic manual of mental disorders iv ( dsm - iv ) axis i disorders and the binge eating disorder - clinical interview to respectively assess / confirm the diagnosis of bed and to deepen understanding of their eating behaviors ( ie , night eating , postdinner eating , social eating , sweet eating , emotional eating , grazing , craving for carbohydrates , and hyperphagia ) and exercise habits . the clinical interview through the structured clinical interview for dsm - iv axis i disorders confirmed the diagnosis of bed for all the obese patients with bes scores 17 . according to the diagnosis of the bed , the patients were divided into 2 groups : non - bed obese patients ( n = 85 ) and bed - obese patients ( n = 30 ) . plasma glucose , total and high - density lipoprotein ( hdl ) cholesterol , triglycerides , and uric acid concentrations were measured by enzymatic methods ( roche diagnostics , mannheim , germany ) . glycated hemoglobin ( hba1c ) was measured with high - performance liquid chromatography using a national glycohemoglobin standardization program certified automated analyzer and ifcc ( adams ha-8160 hba1c analyzer , menarini , italy ; normal reference range , 4.3%5.9% ) . high - sensitivity c reactive protein levels were measured by an automated instrument ( cardiophase hscrp , milan , italy ) . the erythrocyte sedimentation rate ( esr ) was measured automatically by the stopped - flow technique in a capillary microphotometer ( alifax test 1 system polverara , italy ) . insulin resistance was estimated by the homeostasis model assessment ( homa - ir ) index , calculated from the fasting glucose , and insulin concentrations according to the following formula ( fasting insulin fasting glucose)/22.5 . the visceral adiposity index ( vai ) was calculated using the formulas proposed by amato et al for men , vai = [ white blood cell ( wc)/36.58 + ( 1.89 bmi ) ] ( tryglicerides ( tg)/0.81 ) ( 1.52/hdl ) and for women , vai = [ wc/39.68 + ( 1.88 bmi ) ] ( tg/1.03 ) ( 1.31/hdl ) . anthropometric and metabolic differences between groups were tested after adjusting for age , sex , and bmi using a general linear model . the structural equation modeling using maximum likelihood estimation was conducted by means of spss / amos 21.0 to test for the validity of the hypothesized model . structural equation modeling is a method that allows the simultaneous estimation of all relationships between observed ( manifest or unmeasured ) and unobserved ( or latent ) variables of a model . model fit was evaluated using the comparative fit index ( cfi ) , the tucker lewis incremental fit index ( tli ) , and root mean square error of approximation ( rmsea ) . a total of 115 white obese patients without t2 dm were consecutively recruited in this cross - sectional study at the department of medical and surgical sciences of the university magna graecia of catanzaro ( italy ) from december 2013 to december 2014 . patients were enrolled according to the following eligibility criteria : aged between 20 and 65 years , body mass index ( bmi ) > 30 exclusion criteria were as follows : pregnancy or having recently given birth , previous diagnosis of diabetes mellitus , known inflammatory disease , a history of malignant disease or pathologies , or drugs able to modify glucose metabolism . after 12-hour fasting , a venous blood sample was drawn for laboratory determinations and a 75 g oral glucose tolerance test was performed with 0 , 30 , 60 , 90 , and 120 minutes of sampling for plasma glucose and insulin for each patient . glucose tolerance status was defined on the basis of body mass index according to the american diabetes association criteria . patients underwent anthropometrical evaluation , wearing light indoor clothing , and no shoes , with a standing height to the nearest 0.1 cm and a body weight to the nearest 0.1 kg at 8.00 am . height and weight were measured using a portable stadiometer ( seca 220 , gmbh & co. , hamburg , germany ) and a balance scale ( seca 761 , gmbh & co. , hamburg , germany ) ; then , their bmi ( kg / m ) was calculated . in addition , waist and hip circumference were measured , and body composition was estimated by bioelectrical impedance . binge eating scale is an easy to administer test with adequate internal consistency and validity . binge eating scale is made up of 16 items describing the behavioral manifestations , feelings , and cognitions associated with binge eating . a total bes score < 17 indicates unlikely bed , a 17 to 27 score possible bed and values > 27 probable bed . the interviewers examined each patient by means of the structured clinical interview for diagnostic and statistic manual of mental disorders iv ( dsm - iv ) axis i disorders and the binge eating disorder - clinical interview to respectively assess / confirm the diagnosis of bed and to deepen understanding of their eating behaviors ( ie , night eating , postdinner eating , social eating , sweet eating , emotional eating , grazing , craving for carbohydrates , and hyperphagia ) and exercise habits . the clinical interview through the structured clinical interview for dsm - iv axis i disorders confirmed the diagnosis of bed for all the obese patients with bes scores 17 . according to the diagnosis of the bed , the patients were divided into 2 groups : non - bed obese patients ( n = 85 ) and bed - obese patients ( n = 30 ) . plasma glucose , total and high - density lipoprotein ( hdl ) cholesterol , triglycerides , and uric acid concentrations were measured by enzymatic methods ( roche diagnostics , mannheim , germany ) . glycated hemoglobin ( hba1c ) was measured with high - performance liquid chromatography using a national glycohemoglobin standardization program certified automated analyzer and ifcc ( adams ha-8160 hba1c analyzer , menarini , italy ; normal reference range , 4.3%5.9% ) . the erythrocyte sedimentation rate ( esr ) was measured automatically by the stopped - flow technique in a capillary microphotometer ( alifax test 1 system polverara , italy ) . insulin resistance was estimated by the homeostasis model assessment ( homa - ir ) index , calculated from the fasting glucose , and insulin concentrations according to the following formula ( fasting insulin fasting glucose)/22.5 . the visceral adiposity index ( vai ) was calculated using the formulas proposed by amato et al for men , vai = [ white blood cell ( wc)/36.58 + ( 1.89 bmi ) ] ( tryglicerides ( tg)/0.81 ) ( 1.52/hdl ) and for women , vai = [ wc/39.68 + ( 1.88 bmi ) ] ( tg/1.03 ) ( 1.31/hdl ) . variables with skewed distribution , including triglyceride , hscrp , esr , wbc , and fasting insulin anthropometric and metabolic differences between groups were tested after adjusting for age , sex , and bmi using a general linear model . structural equation modeling is a method that allows the simultaneous estimation of all relationships between observed ( manifest or unmeasured ) and unobserved ( or latent ) variables of a model . path coefficients on the edges are partial standardized regression coefficients , which measure the effect of 1 variable on another , while controlling for all other variables prior in the model ; all coefficients show a positive association , but those with a minus sign indicate a negative association . model fit was evaluated using the comparative fit index ( cfi ) , the tucker lewis incremental fit index ( tli ) , and root mean square error of approximation ( rmsea ) . there were no differences in age and sex between bed and non - bed obese . binge eating disorder obese exhibited significantly higher bmi , waist circumference , hip circumference , waist / hip ratio , and fat mass , and a lower lean mass as compared with non - bed obese ; these differences remained significant after adjusting for bmi ( table 1 ) . there were no differences between the groups in blood pressure , total cholesterol , low - density lipoprotein cholesterol , triglycerides . binge eating disorder obese had a worse metabolic and inflammatory profile , exhibiting significantly lower hdl cholesterol and higher levels of hba1c , uric acid , esr , hs - crp , and wbc and all these differences remained significant after adjusting for bmi ( table 1 ) . binge eating disorder obese also exhibited significantly higher levels of fasting plasma insulin and higher degree of insulin resistance as assessed by homa - ir index and vai compared with non - bed obese ; similarly , differences remained significant after adjusting for bmi . blood pressure , low - density lipoprotein cholesterol , triglycerides , fasting glucose , and 2-hour postload plasma glucose were statistically significant after adjusting for bmi . comparison of anthropometrical and laboratory characteristics between groups regarding eating habits , bed obese revealed significantly higher percentages of grazing , emotional eating , night eating , sweet eating , and craving for carbohydrates than non - bed obese ( figure 1 ) . eating behaviors in binge eating disorder and non - binge eating disorder obese patients . structural equation modeling was conducted to test the hypothesis that altered eating behaviors affect anthropometrical , metabolic , and inflammatory variables . in our model , altered eating behaviors ( ie , night eating , sweet eating , craving for carbohydrates , emotional eating , and grazing ) showed an indirect effect on waist circumference mediated by the diagnosis of bed and body weight ; waist circumference had an indirect effect on hba1c mediated by insulin resistance assessed either by homa - ir or vai ; and vai had a direct effect on the inflammatory indexes ( ie , esr , hs - crp , and uric acid ; figure 2 ) . craving for bed = binge eating disorder , cfi = comparative fit index , ch = craving for carbohydrates , cmin = , df = degree of freedom , esr = erythrocyte sedimentation rate ; rmsea = root mean square error of approximation , tli = tucker in this study , we showed an unfavorable metabolic and inflammatory profile in obese patients with bed as compared with non - bed obese . notably , we reported , for the first time that bed obese , exhibited a worse anthropometric and metabolic profile in relation to their different eating behaviors , as compared with non - bed obese . the characteristic eating patterns of bed individuals seem to be associated with specific metabolic abnormalities of obesity . for example , the ingestion of fewer but larger meals has adverse metabolic consequences than the ingestion of frequent , small meals , including increase in fasting glucose , insulin secretion , serum lipids , and deterioration of glucose tolerance . alone or in conjunction with rapid absorption of lipid , glucose and insulin increases may lead to insulin resistance and metabolic syndrome with central / visceral fat distribution and dyslipidemia . in fact , bed patients eat significantly more than do non - bed obese during the stimulation of a binge eating episode . despite the relation between specific metabolic alterations and these eating features of bed individuals ( ie , speed and amount ) , little is known about the metabolic consequences of the other eating habits typically observed in bed individuals ( eg , craving for carbohydrates , sweet eating , grazing , or emotional eating ) . herein , we provide evidence that bed obese showed a higher frequency of altered eating behaviors ( ie , binging , grazing , emotional eating , sweet eating , and craving for carbohydrates ) than non - bed obese . we did not observe a significant difference in binge eating frequency between bed and non - bed obese patients . as binging becomes a distraction related to aversive emotional states , bed obese our results are in agreement with previous data showing that emotional eating is associated with the preference for sweet foods and that bed obese crave carbohydrates more than fats , compared with non - bed obese . as previously reported , grazing , another dysfunctional eating behavior that consists of smaller , subjective episodes of overeating , was significantly more evident among bed obese as compared with non - bed obese . these habits could be responsible , in association with obesity , for the worse metabolic profile observed in bed obese . accordingly , we found that bed obese exhibited a significantly higher bmi , fat mass , fasting insulin levels , homa - ir index , and hba1c when compared with non - bed obese . moreover , among bed obese , we found an increased abdominal circumference , an indirect index of visceral adiposity and an increased vai , an indicator of a dysfunction of adipose tissue . in obese patients , visceral adipose tissue accumulation has been associated with an increased production of free fatty acids , interleukin-6 , tumor necrosis factor- , hs - crp , and a decreased production of adiponectin , each of which may contribute to insulin resistance . accordingly , it is conceivable that increased visceral adiposity observed in bed obese may be responsible for increased insulin resistance , estimated by the homa - ir index . increasing evidence has shown that the accumulation of lipids in adipose tissue and the expansion of fat mass determine the initiation of the obesity - induced inflammatory process through the production of proinflammatory cytokines and chemokines by the fat tissue . moreover , we found , that bed obese showed higher uric acid levels than non - bed obese did . elevation in uric acid has been associated with obesity and insulin resistance , all risk factors for atherosclerosis , and t2 dm . structural equation modeling analysis helps to explain the relation between eating behaviors and metabolic impairment . night eating , sweet eating , grazing , emotional eating , and craving for carbohydrates may lead to increased waist circumference , an index of visceral adiposity , and insulin resistance , which , in turn , may favor production of inflammatory molecules , and alteration in glucose metabolism . the novelty of this article is that an eating disorder , namely bed , and the related altered eating behaviors , can help to explain an impaired metabolic and inflammatory profile in a group of obese patients that could have an increased cardiometabolic risk . strengths of this study are the exclusion of patients with t2 dm or confounding disorders characterized by elevation in inflammatory molecules , inclusion of both sexes , the accurate anthropometric , metabolic , inflammatory and psychiatric characterization , and the evaluation of eating behaviors . first , the current results derive from a cross - sectional research with a small sample of obese patients . although the euglycaemic hyperinsulinaemic clamp , which is considered the gold standard method to measure insulin sensitivity , may provide a more accurate estimate of insulin sensitivity , it is time consuming and expensive , and is not feasible in large - scale studies . despite the limitations the current study confirms and expands the actual knowledge about the effects of specific dysfunctional eating behaviors commonly observed among obese patients on important metabolic and inflammatory alterations . all obese patients should be assessed for bed and it can be done through an easy tool , the bes . further research and prospective studies , however , are needed to assess and evaluate whether early detection and intervention for this specific disorder could have a positive prognostic impact on the long - term outcome ( eg , t2 dm and/or cardiovascular risk ) of this specific group of obese patients seeking weight reduction .

menopause is associated with increased cardiovascular disease and once women develop acute coronary symptoms , they have worse short and longterm outcomes than men.1 many different factors contribute , including marked hormonal changes,2 changes in metabolic profile associated with increased risk of the metabolic syndrome,3 and relative increase in intraabdominal fat with age.4 accumulation of intraabdominal fat is associated with increased waist circumference and liver fat,5 overproduction of very lowdensity lipoprotein ( vldl ) , and decreased catabolism of apolipoprotein ( apo)bcontaining particles in men.6 the catabolism of apobcontaining particles is partly determined by plasma apociii concentrations , and higher plasma apociii has been associated with dyslipidemia in obese men.7 although abdominal obesity tends to be associated with obesity in men , data from the united states have been used to estimate that 40% of women have an abdominal fat distribution pattern as defined by waist : hip ratio.8 normally , fasting plasma triglycerides ( tg ) are determined by 2 distinct subclasses of vldl9 ; vldl1 is larger and more tgrich than vldl2 , the latter can either be secreted directly from the liver , or formed by the peripheral hydrolysis of vldl1 . hypertriglyceridemia is associated with atherogenic dyslipidemia including the production of small dense ldl , lower hdl cholesterol , and accumulation of postprandial tgrich lipoproteins.10 in men with type 2 diabetes , the secretion of vldl1 is associated with liver fat , hypertriglyceridemia , and increased atherogenic risk.11 impaired hepatic fatty acid oxidation has been reported to be related to obesity and insulin resistance by some12 , 13 but not all.14 few detailed studies have investigated vldl1 and vldl2 kinetics in women , and none have compared the kinetics of vldl or apociii in pre and postmenopausal women . we hypothesized that vldl1tg and apob secretion would be higher in abdominally obese compared with abdominally lean women and aimed to investigate the effect of menopause status on this relationship by measuring hepatic de novo fatty acid synthesis ( dnl ) , oxidation , and desaturation in relation to vldl1 and vldl2 kinetics in pre and postmenopausal women . we recruited 60 healthy white women from local advertising and the oxford biobank as previously reported15 equally into pre and postmenopausal groups aged 35 to 45 and 55 to 65 , respectively . the age groups ensured that perimenopausal women were not included and postmenopausal status was defined as absence of menses for at least 12 months and folliclestimulating hormone > 30 iu / l . since we also wished to investigate the effect of android fat ( abdominal obesity ) , we used waist circumference , a marker of android fat , to facilitate recruitment of women into groups with low or high android fat . for simplicity , we have referred to the group with low android fat as lean . a waist circumference of 80 cm was selected as the proxy measure of high android fat , with increased risk of cardiovascular disease in europid women defined by the international diabetes federation16 and additionally , we recruited women into small waist ( < 80 cm , n=30 ) , or large waist ( 80 to 84 cm , n=5 ; 85 to 91 cm , n=5 ; and 92 to 110 cm , n=5 ) categories in both menopausal groups . this was to ensure a good range of android fat in our cohort , and ensure exact matching of abdominal obesity between menopausal groups . other inclusion and exclusion criteria have been previously described in a study relating to energy intake in a subset of the participants15 but briefly , women were excluded if they had any condition or treatment that would affect metabolic or hormonal status ( including smoking , diabetes , or hormone replacement therapy ) , or had body mass index ( bmi ) < 18.5 or > 34.9 . smokers or women exceeding alcohol consumption guidelines of 2 to 3 units per day were also excluded.17 all participants gave informed , written consent and the study was approved by the oxfordshire clinical research ethics committee . participants attended the clinical research unit prior to the metabolic day in order to be given deuterated water for consumption the evening before the study day , and to give a blood sample for background isotopic enrichment measurements relating to the measurement of dnl ( see below ) . intrahepatic fat was measured by magnetic resonance spectroscopy , visceral and subcutaneous fat were measured by magnetic resonance imaging after an overnight fast and within 2 weeks of the study day,18 and whole body composition and fat distribution ( eg , android and gynoid fat ) were measured using dexa.15 participants arrived after an overnight fast and after consuming deuterated water ( h2o , in order to measure de novo lipogenesis , dnl ) ( 3 g / kg body water ) at 8 and 10 pm the evening before the study day and then continued to consume enriched water ( 2.5 g per 500 ml water ) , in order to achieve and maintain a plasma water enrichment of 0.3%.19 a cannula was placed in an antecubital vein in order to take blood samples for the estimation of dnl in vldl1 and vldl2palmitate , and background isotopic enrichments for the kinetic studies . another cannula was placed in the contralateral arm to administer intravenous boluses of [ h3]leucine ( 7 mg / kg ) and [ h5]glycerol ( 500 mg ) , while an intravenous infusion of [ uc]palmitic acid , potassium salt complexed with albumin20 at 0.03 mol / kg per minute , was started . blood samples were taken for a further 8 hours and vldl1 and vldl2 were isolated from plasma using density gradient ultracentrifugation.20 due to technical problems , 1 participant did not receive the palmitate infusion , 1 participant 's infusion was stopped early , and 1 participant did not complete the metabolic study day . blood samples were drawn into heparinized syringes ( sarstedt , leicester , uk ) and plasma was rapidly separated at 4c . plasma metabolites were analyzed enzymatically,20 insulin was measured by radioimmunoassay ( millipore [ uk ] ltd , watford , uk ) , and homeostatic model assessment of insulin resistance was calculated as an index of insulin resistance.21 a timeaveraged areaunderthecurve for plasma 3hydroxybutyrate ( 3ohb ) and nonesterified fatty acids ( nefa ) was calculated from hourly values taken during the study . serum steroids ( cortisol , dehydroepiandrosterone , and androstenedione ) were measured by liquid chromatography / tandem mass spectrometry using a waters xevo mass spectrometer with acquity uplc system as described previously.22 [ h5]glycerol in plasma , vldl1 and vldl2tg ( to trace tg ) and [ h3]leucine in plasma , and vldl1and vldl2apob ( to trace whole particles ) were measured by gas chromatography mass spectrometry.23 [ uc]palmitic acid was measured in plasma nefa and vldl1 and vldl2tg by gas chromatography mass spectrometry20 and the proportion of fatty acids ( fas ) in vldltg that were derived from nonsystemic sources was calculated,20 assuming that 16:0 is representative of all fas . mathematical modeling of vldl kinetics ( vldl1tg , vldl2tg , vldl1apob , vldl2apob production and clearance ) was calculated from [ h5]glycerol and [ h3]leucine enrichments in plasma and lipoprotein fractions.11 see figures s1 and s2 for examples of raw data used for modeling . vldltg production rates were corrected for lean mass in order to consider delivery of tg to muscle as previously described24 but not corrected when considering hepatic fa trafficking . total plasma apociii and apociii in plasma devoid of apobcontaining particles were measured using a hydragel lp ciii electroimmunodiffusion kit ( sebia , france ) with appropriate standards and quality controls according to the manufacturer 's instructions . by difference , we calculated apociii concentrations in apobcontaining particles ( apociii lpb ) . apociii kinetic modeling was carried out as previously described25 and assumes ( consistent with previous studies , and earlier radiotracer studies ) that apociii exchanges between vldl and hdl particles , and therefore that measuring apociii kinetics in plasma is valid . the ratio of [ uc]16:1n7/[uc]16:0 in vldl1 and vldl2tg was determined as a shortterm index of hepatic stearoylcoa desaturase ( scd ) activity ( the isotopic desaturation index ) and also the scd16 and scd18 fa ratios.26 fa methyl esters prepared from vldl1 and vldl2tg fas20 were analyzed by gc13 to quantify 16:0 and 16:1n7 , and by gcisotope ratio mass spectrometer to measure isotopic enrichment.26 hepatic dnl was measured on the study day , based on the incorporation of h in plasma water ( finnigan gasbenchii ; thermofisher scientific , uk ) and into vldl1 and vldl2tg palmitate using gas chromatography % dnl and represents synthesis of fas from precursors such as sugars and amino acids.28 fa rate of appearance ( ranefa ) was calculated from the [ uc]16:0 infusion rate and enrichment in the plasma nefa fraction and rdnefa was assumed to equal ranefa.24 statistical analysis was performed using spss statistics 19 ( ibm , spss products , chertsey , uk ) . twoway anova was used to determine the effect of abdominal obesity and menopausal status ( fixed factors ) on each dependent variable , and interaction between the fixed factors . a significant interaction term indicated that the relationship between the dependent variable and abdominal obesity was significantly different in pre and postmenopausal women . associations between variables were carried out using spearman 's rank correlation coefficient ( univariate analysis ) . in order to visualize relationships between metabolic variables , we plotted significant correlations between metabolic and anthropometric variables related to hepatic fa partitioning using hive plots.29 each variable is represented by a node and the nodes are joined by blue ( significant positive correlations ) or red ( significant negative correlations ) lines . the nodes are placed on 3 duplicated radial axes , which represent grouped variables ( anthropometric and metabolic variables / vldl1 or vldl2 ) . the axes are duplicated in order to allow for representation of correlations within the variable group ( eg , there are lines joining the isotopic desaturation index and % dnl in vldl1 for pre and postmenopausal women , representing significant positive correlations ) . using data from a study of the reproducibility of relevant kinetic parameters ( vldl tg and apob100 secretion rates , vldltg clearance rate , rate of appearance nefa,30 and dnl),19 separate power calculations were carried out and the numbers in pre and postmenopausal groups to detect a 40% difference with power of 0.80 at of 0.05 were 4 , 8 , 9 , 10 , and 15 , respectively ( in each group ) . a difference of 40% was considered to be clinically significant and was within the range of differences previously reported in other studies.11 we recruited 60 healthy white women from local advertising and the oxford biobank as previously reported15 equally into pre and postmenopausal groups aged 35 to 45 and 55 to 65 , respectively . the age groups ensured that perimenopausal women were not included and postmenopausal status was defined as absence of menses for at least 12 months and folliclestimulating hormone > 30 iu / l . since we also wished to investigate the effect of android fat ( abdominal obesity ) , we used waist circumference , a marker of android fat , to facilitate recruitment of women into groups with low or high android fat . for simplicity , we have referred to the group with low android fat as lean . a waist circumference of 80 cm was selected as the proxy measure of high android fat , with increased risk of cardiovascular disease in europid women defined by the international diabetes federation16 and additionally , we recruited women into small waist ( < 80 cm , n=30 ) , or large waist ( 80 to 84 cm , n=5 ; 85 to 91 cm , n=5 ; and 92 to 110 cm , n=5 ) categories in both menopausal groups . this was to ensure a good range of android fat in our cohort , and ensure exact matching of abdominal obesity between menopausal groups . other inclusion and exclusion criteria have been previously described in a study relating to energy intake in a subset of the participants15 but briefly , women were excluded if they had any condition or treatment that would affect metabolic or hormonal status ( including smoking , diabetes , or hormone replacement therapy ) , or had body mass index ( bmi ) < 18.5 or > 34.9 . smokers or women exceeding alcohol consumption guidelines of 2 to 3 units per day were also excluded.17 all participants gave informed , written consent and the study was approved by the oxfordshire clinical research ethics committee . participants attended the clinical research unit prior to the metabolic day in order to be given deuterated water for consumption the evening before the study day , and to give a blood sample for background isotopic enrichment measurements relating to the measurement of dnl ( see below ) . intrahepatic fat was measured by magnetic resonance spectroscopy , visceral and subcutaneous fat were measured by magnetic resonance imaging after an overnight fast and within 2 weeks of the study day,18 and whole body composition and fat distribution ( eg , android and gynoid fat ) were measured using dexa.15 participants arrived after an overnight fast and after consuming deuterated water ( h2o , in order to measure de novo lipogenesis , dnl ) ( 3 g / kg body water ) at 8 and 10 pm the evening before the study day and then continued to consume enriched water ( 2.5 g per 500 ml water ) , in order to achieve and maintain a plasma water enrichment of 0.3%.19 a cannula was placed in an antecubital vein in order to take blood samples for the estimation of dnl in vldl1 and vldl2palmitate , and background isotopic enrichments for the kinetic studies . another cannula was placed in the contralateral arm to administer intravenous boluses of [ h3]leucine ( 7 mg / kg ) and [ h5]glycerol ( 500 mg ) , while an intravenous infusion of [ uc]palmitic acid , potassium salt complexed with albumin20 at 0.03 mol / kg per minute , was started . blood samples were taken for a further 8 hours and vldl1 and vldl2 were isolated from plasma using density gradient ultracentrifugation.20 due to technical problems , 1 participant did not receive the palmitate infusion , 1 participant 's infusion was stopped early , and 1 participant did not complete the metabolic study day . blood samples were drawn into heparinized syringes ( sarstedt , leicester , uk ) and plasma was rapidly separated at 4c . plasma metabolites were analyzed enzymatically,20 insulin was measured by radioimmunoassay ( millipore [ uk ] ltd , watford , uk ) , and homeostatic model assessment of insulin resistance was calculated as an index of insulin resistance.21 a timeaveraged areaunderthecurve for plasma 3hydroxybutyrate ( 3ohb ) and nonesterified fatty acids ( nefa ) was calculated from hourly values taken during the study . serum steroids ( cortisol , dehydroepiandrosterone , and androstenedione ) were measured by liquid chromatography / tandem mass spectrometry using a waters xevo mass spectrometer with acquity uplc system as described previously.22 [ h5]glycerol in plasma , vldl1 and vldl2tg ( to trace tg ) and [ h3]leucine in plasma , and vldl1and vldl2apob ( to trace whole particles ) were measured by gas chromatography mass spectrometry.23 [ uc]palmitic acid was measured in plasma nefa and vldl1 and vldl2tg by gas chromatography mass spectrometry20 and the proportion of fatty acids ( fas ) in vldltg that were derived from nonsystemic sources was calculated,20 assuming that 16:0 is representative of all fas . mathematical modeling of vldl kinetics ( vldl1tg , vldl2tg , vldl1apob , vldl2apob production and clearance ) was calculated from [ h5]glycerol and [ h3]leucine enrichments in plasma and lipoprotein fractions.11 see figures s1 and s2 for examples of raw data used for modeling . vldltg production rates were corrected for lean mass in order to consider delivery of tg to muscle as previously described24 but not corrected when considering hepatic fa trafficking . total plasma apociii and apociii in plasma devoid of apobcontaining particles were measured using a hydragel lp ciii electroimmunodiffusion kit ( sebia , france ) with appropriate standards and quality controls according to the manufacturer 's instructions . by difference , we calculated apociii concentrations in apobcontaining particles ( apociii lpb ) . apociii kinetic modeling was carried out as previously described25 and assumes ( consistent with previous studies , and earlier radiotracer studies ) that apociii exchanges between vldl and hdl particles , and therefore that measuring apociii kinetics in plasma is valid . the ratio of [ uc]16:1n7/[uc]16:0 in vldl1 and vldl2tg was determined as a shortterm index of hepatic stearoylcoa desaturase ( scd ) activity ( the isotopic desaturation index ) and also the scd16 and scd18 fa ratios.26 fa methyl esters prepared from vldl1 and vldl2tg fas20 were analyzed by gc13 to quantify 16:0 and 16:1n7 , and by gcisotope ratio mass spectrometer to measure isotopic enrichment.26 hepatic dnl was measured on the study day , based on the incorporation of h in plasma water ( finnigan gasbenchii ; thermofisher scientific , uk ) and into vldl1 and vldl2tg palmitate using gas chromatography mass spectrometry.27 for simplicity , this is referred to as % dnl and represents synthesis of fas from precursors such as sugars and amino acids.28 fa rate of appearance ( ranefa ) was calculated from the [ uc]16:0 infusion rate and enrichment in the plasma nefa fraction and rdnefa was assumed to equal ranefa.24 statistical analysis was performed using spss statistics 19 ( ibm , spss products , chertsey , uk ) . twoway anova was used to determine the effect of abdominal obesity and menopausal status ( fixed factors ) on each dependent variable , and interaction between the fixed factors . a significant interaction term indicated that the relationship between the dependent variable and abdominal obesity was significantly different in pre and postmenopausal women . associations between variables were carried out using spearman 's rank correlation coefficient ( univariate analysis ) . in order to visualize relationships between metabolic variables , we plotted significant correlations between metabolic and anthropometric variables related to hepatic fa partitioning using hive plots.29 each variable is represented by a node and the nodes are joined by blue ( significant positive correlations ) or red ( significant negative correlations ) lines . the nodes are placed on 3 duplicated radial axes , which represent grouped variables ( anthropometric and metabolic variables / vldl1 or vldl2 ) . the axes are duplicated in order to allow for representation of correlations within the variable group ( eg , there are lines joining the isotopic desaturation index and % dnl in vldl1 for pre and postmenopausal women , representing significant positive correlations ) . using data from a study of the reproducibility of relevant kinetic parameters ( vldl tg and apob100 secretion rates , vldltg clearance rate , rate of appearance nefa,30 and dnl),19 separate power calculations were carried out and the numbers in pre and postmenopausal groups to detect a 40% difference with power of 0.80 at of 0.05 were 4 , 8 , 9 , 10 , and 15 , respectively ( in each group ) . a difference of 40% was considered to be clinically significant and was within the range of differences previously reported in other studies.11 sixty women were recruited : mean age was 41.0 years ( range 35 to 45 ) for premenopausal and 58.1 years ( 55 to 64 ) for postmenopausal women . mean age when divided according to abdominal obesity was 49.3 ( 35 to 64 ) and 49.3 ( 35 to 63 ) for lean and abdominally obese women , respectively . plasma folliclestimulating hormone concentrations ranged from 3.0 to 21.3 and 46.5 to 125 iu / l in pre and postmenopausal women , respectively ( confirming menopausal status ) . fiftyeight women from whom dexa scans were available are included in this study , divided according to menopause status and android fat measurement ( corrected for total fat ) , for statistical analyses . lean women had a mean android fat of 0.065 ( sd 0.013 ) and abdominally obese women had a mean value of 0.094 ( sd 0.008 ) . gynoid fat was significantly lower , and intraabdominal fat was significantly higher in postmenopausal women , despite being matched for abdominal obesity ( table 1 ) . liver fat was generally low , although 6 women , all abdominally obese , had values of > 5% . bmi was not significantly different between menopausal groups and ranged from 21.5 to 33.0 kg / m in abdominally obese and 19.5 to 27.6 thus , some abdominally lean women would be classified as overweight by bmi , and some abdominally obese women would be classified as lean by bmi . body composition in women according to menopausal status and abdominal obesity data presented as mean ( sem ) or median ( range ) . statistical significance based on 2way anova : p meno , statistical significance for an effect of menopausal status ; p abd obesity , statistical significance for an effect of abdominal obesity . bmi indicates body mass index ; intraab fat , intraabdominal fat ; ns , not significant ; subcut fat , subcutaneous fat ; whr , waisttohip ratio . n=30 for postmenopausal women apart from data derived from dexa measurements , which were n=28 ( gynoid fat , android : gynoid ratio , fat mass , lean mass and fat : lean mass ) . corrected for total fat mass in order to investigate differences in body fat distribution . postmenopausal women had significantly higher concentrations of plasma total , ldl , nonhdl cholesterol , apociiilpb , plasma apob , and systolic bp than premenopausal women ( table 2 ) . women with abdominal obesity were more insulin resistant with a more adverse lipid profile ( higher nonhdl cholesterol , plasma tg , vldl1 and vldl2tg concentrations ) . biochemical and metabolic variables in women according to menopausal status and abdominal obesity data presented as mean ( sem ) or median ( range ) . statistical significance based on 2way anova : p meno , statistical significance for an effect of menopausal status ; p abd obesity , statistical significance for an effect of abdominal obesity ; ns , not significant ; no significant interaction between abdominal obesity and menopausal status was found . 3ohb indicates plasma 3hydroxybutyrate ; apob , apolipoprotein b ; apociii lpb , apociii associated with lipoprotein b containing particles ; auc , area under the curve ; bp , blood pressure ; chol , cholesterol ; hdl , highdensity lipoprotein ; homair , homeostatic model assessment of insulin resistance ; ldl , lowdensity lipoprotein ; nefa , nonesterified fatty acids ; tg , triglyceride ; vldl , very lowdensity lipoprotein . n=30 for postmenopausal women apart from auc measurements for nefa and 3ohb which are n=29 . the rate of disappearance of fas ( rdnefa ) , expressed per kg lean mass was significantly higher in post compared to premenopausal women ( table 3 ) . release of fas into plasma ( ranefa ) per unit weight of adipose tissue was lower in women with abdominal obesity , but vldl1tg and vldl1apob production were significantly higher . the ratio of vldl2tg direct production : vldl2apob production was significantly higher in post compared to premenopausal women , indicating production of larger particles . kinetic estimates relating to nefa , vldl , and apociii metabolism in women according to menopausal status and abdominal obesity data presented as mean ( sem ) . statistical significance based on 2way anova : p meno , statistical significance for an effect of menopausal status ; p abd obesity , statistical significance for an effect of abdominal obesity . apob indicates apolipoprotein b100 ; dirprod , direct production ; fcr , fractional clearance rate ; fdc , fractional direct clearance ; ftr , fractional transfer rate ; indirprod , indirect production ; lean , lean tissue ; nefa , nonesterified fatty acids ; ns , not significant ; prod , production ; pr , production rate ; ra , rate of appearance ; rd , rate of disappearance ; tg , triacylglycerol ; vldl , very lowdensity lipoprotein . there were no significant positive correlations between age and liver fat , vldl1tg and vldl2tg direct production , vldl1 and vldl2 direct apob production , or vldl1 and vldl2tg : apob production ratios within menopausal groups . plasma apociii concentrations positively correlated with apociii production rate ( r s=0.59 , p=9.010 ) but not clearance rate , indicating that plasma apociii concentrations were determined by production rate . plasma , hdl and apociii associated withlipoprotein bcontaining particle ( apociii lpb ) concentrations were not affected by abdominal obesity , but plasma apociii lpb concentrations were higher in postmenopausal women . overall , mean % dnl was less than 10% in vldl1 and vldl2palmitate ( data not shown ) and when corrected for flux from the liver , was not significantly different between menopausal groups , but was higher with abdominal obesity . menopause status affected the relationship between abdominal obesity and 3ohb : nefa , and abdominal obesity per se had a strong influence on factors related to fa partitioning ( table 4 ) . of note , the systemic fa contribution to vldl1tg production was significantly higher in abdominally obese women , in line with higher vldl1tg secretion . variables relating to fa metabolism in women according to menopausal status and abdominal obesity data presented as mean ( sem ) or median ( range ) . statistical significance based on 2way anova : p meno , statistical significance for an effect of menopausal status ; p abd obesity , statistical significance for an effect of abdominal obesity . 3ohb indicates 3hydroxybutyrate ; fa , fatty acid ; nefa , nonesterified fatty acids ; ns , not significant ; tg , triglyceride ; vldl , very lowdensity lipoprotein . * p<0.05 , * * p<0.01 for a statistically significant interaction between abdominal obesity and menopausal status . to explore fa partitioning in relation to metabolic and anthropometric measurements , we tabulated univariate correlations between relevant variables in pre and postmenopausal women ( selected data in tables 5 through 8 and complete analysis in tables s1 through s4 ) . the data are illustrated in hive plots ( figure 1 ) , which clearly show that the patterns of correlations are quite different in pre and postmenopausal women . in particular , the density of correlations in the top left of the figure for premenopausal women ( figure 1a , anthropometric and metabolic variables with vldl2 metabolism ) is markedly less for postmenopausal women ( figure 1b ) . correlations relating to % dnl and vldl production are shown in figure 2a and 2b and between % dnl and plasma 3ohb in figure 2c and 2d . the most marked univariate correlations relating to hepatic fa partitioning were between the isotopic desaturation index in vldl1 and vldl2tg and plasma 3ohb area under the curve ( figure 2e and 2f ) . vldlapob and tg production were highly correlated for vldl1 and less so for vldl2 ( figure 3 ) . serum cortisol concentrations were negatively correlated with waisttohip ratio in pre ( r s=0.38 , p=0.04 ) but not postmenopausal women . there was a significant correlation between abdominal fat and liver fat ( r s=0.50 , p<0.001 , n=60 ) . the importance of menopausal status in this relationship is shown in the hive plots and tables 5 through 8 which showed , remarkably , that a significant correlation between liver fat and abdominal obesity was observed only in premenopausal women . correlation coefficients ( r s ) for premenopausal women between selected variables relating to vldl1 metabolism , liver fat , and intraabdominal fat see supplemental material for full statistical analysis . 3ohb indicates 3hydroxybutyrate ; apob , apolipoprotein b ; auc , area under the curve ; d , day ; dnl , de novo lipogenesis ; homair , homeostatic model assessment of insulin resistance ; iso , isotopic ; nefa , nonesterified fatty acids ; prod , production ; scd , stearoylcoa desaturase 1 ; scd16 , ratio of 16:1n7/16:0 ; scd18 , ratio 18:1n9/18:0 ; subcut , subcutaneous ; tg , triglyceride ; tot , total ; vldl , very lowdensity lipoprotein . correlation coefficients ( r s ) for premenopausal women between selected variables relating to vldl2 metabolism , liver fat , and intraabdominal fat see supplementary material for full statistical analysis . 3ohb indicates 3hydroxybutyrate ; apob , apolipoprotein b ; auc , area under the curve ; d , day ; dnl , de novo lipogenesis ; homair , homeostatic model assessment of insulin resistance ; iso , isotopic ; nefa , nonesterified fatty acids ; prod , production ; scd , stearoylcoa desaturase 1 ; scd16 , ratio of 16:1n7/16:0 ; scd18 , ratio 18:1n9/18:0 ; subcut , subcutaneous ; tg , triglyceride ; tot , total ; vldl , very lowdensity lipoprotein . correlation coefficients ( r s ) for postmenopausal women between selected variables relating to vldl1 metabolism , liver fat and intraabdominal fat see supplementary material for full statistical analysis . 3ohb indicates 3hydroxybutyrate ; apob , apolipoprotein b ; auc , area under the curve ; d , day ; dnl , de novo lipogenesis ; homair , homeostatic model assessment of insulin resistance ; iso , isotopic ; nefa , nonesterified fatty acids ; prod , production ; scd , stearoylcoa desaturase 1 ; scd16 , ratio of 16:1n7/16:0 ; scd18 , ratio 18:1n9/18:0 ; subcut , subcutaneous ; tg , triglyceride ; tot , total ; vldl , very lowdensity lipoprotein . correlation coefficients ( r s ) for postmenopausal women between selected variables relating to vldl2 metabolism , liver fat , and intraabdominal fat see supplementary material for full statistical analysis . 3ohb indicates 3hydroxybutyrate ; apob , apolipoprotein b ; auc , area under the curve ; d , day ; dnl , de novo lipogenesis ; homair , homeostatic model assessment of insulin resistance ; iso , isotopic ; nefa , nonesterified fatty acids ; prod , production ; scd , stearoylcoa desaturase 1 ; scd16 , ratio of 16:1n7/16:0 ; scd18 , ratio 18:1n9/18:0 ; subcut , subcutaneous ; tg , triglyceride ; tot , total ; vldl , very lowdensity lipoprotein . significant correlations between variables relating to vldltg metabolism represented as hive plots for premenopausal ( a ) and postmenopausal ( b ) women . each variable is represented by a node and the nodes are joined by blue ( significant positive correlations ) or red ( significant negative correlations ) lines . the thickness of the line represents the strength of the correlation . the nodes are placed on 3 duplicated radial axes that represent grouped variables ( anthropometric and metabolic variables / vldl 1 or vldl 2 ) . individual nodes are coded as indicated and specific correlation coefficients are given in supplemental material . node codes : a , liver fat ( % ) ; b , total body fat ( kg ) ; c , android fat / total fat ; d , gynoid fat / total fat ; e , visceral fat ( cm ) ; f , subcut fat ( cm ) ; g , nefa ( mol / l ) ; h , 3ohb ( auc ) ; i , homair ; k , vldltg scd isotopic index ; m , vldltg scd18 index ; o , vldltg 18:2n6 ( % ) ; q , vldltg production per day ; s , vldltg scd16 ; u , vldltg dnl ( % ) ; w , vldltg production / apob production . 3ohb indicates plasma 3hydroxybutyrate ; apob , apolipoprotein b ; auc , area under the curve ; dnl , hepatic de novo lipogenesis ; homair , homeostatic model assessment of insulin resistance ; nefa , plasma nonesterified fatty acids ; scd , stearoylcoa desaturase ; scd16 , 16:1 n7/16:0 ratio in vldltg ; scd18 , 18:1 n9/18:0 ratio in vldltg ; subcut , subcutaneous ; tg , triglyceride ; vldl , very lowdensity lipoprotein . correlations between vldl 1tg production ( mg / day ) and the proportion ( % ) of dnl fatty acids vldl 1tg ( a ) , vldl 2tg direct production ( mg / day ) and the proportion ( % ) of dnl fatty acids vldl 2tg ( b ) , the proportion ( % ) of dnl fatty acids vldl 1tg and the auc for plasma 3hydroxybutyrate ( mol / l ) ( c ) , the proportion ( % ) of dnl fatty acids vldl 2tg and the auc for plasma 3hydroxybutyrate ( mol / l ) ( d ) , and the association between plasma 3hydroxybutyrate concentrations ( mol / l ) and the isotopic desaturation index ( [ uc16:1n7/uc16:0]*1000 ) in vldl 1tg ( e ) and vldl 2tg ( f ) in pre ( ) and post ( ) menopausal women . auc indicates area under the curve ; dnl , hepatic de novo lipogenesis ; ns , not significant ; tg , triglyceride ; vldl , very lowdensity lipoprotein . correlations between vldl 1apob production ( mg / day ) and vldl 1tg production ( mg / day ) ( a ) , vldl 2apob production ( mg / day ) and vldl 2tg direct production ( mg / day ) ( b ) in pre ( ) and post ( ) menopausal women . apob , apolipoprotein b ; ns , not significant ; tg , triglyceride ; vldl , very lowdensity lipoprotein . we also took the opportunity to examine metabolic variables according to liver fat content because of the importance of liver fat with respect to the metabolic complications of obesity.32 the median value in the cohort of 60 women was 0.85% . of the 50% of women with lower liver fat , 17 were premenopausal and 13 were postmenopausal . in general , significant effects reflected those found by considering women according to abdominal obesity ( tables s5 through s7 ) . however , the effect of liver fat on ldl , hdl , and nonhdl cholesterol as well as vldltg production was less than for abdominal obesity . using a combination of stable isotope tracer techniques , we investigated kinetic parameters of apob , apociii , and tg metabolism in pre and postmenopausal women . we report for the first time that menopausal status is a determinant of hepatic tg flux through enhancement of adipose tissue nefa flux , altered intrahepatic fa partitioning , and secretion of larger vldl2 . vldltg secretion is normally dependent on vldlapob100 secretion,33 but we found that vldl2tg secretion after the menopause was dissociated from vldl2apob production . systemic fas were the major source of vldl2tg in all women , but both systemic and nonsystemic fas contributed to greater vldl2tg secretion in postmenopausal women . we also report for the first time that vldl1tg secretion is higher in abdominally obese women . vldl1 and vldl2 metabolism have not previously been measured in relation to menopausal and abdominal obesity status in women . we measured 2 aspects of vldl secretion : vldlapob secretion rate , which measures whole particle secretion ; and vldltg secretion , which tracks the lipid component . using these 2 parameters we were also able to estimate the relative sizes of vldl1 and vldl2 at the point of hepatic secretion . vldl1 and vldl2 secretion rates were correlated but in agreement with previous findings , their metabolism was independent34 as shown in hive plots . vldl1tg and vldl1apob production rates were significantly higher in there are no previous comparable studies , but in lean and obese premenopausal women there were no differences in total vldlapob or vldltg secretion.35 , 36 another study in premenopausal women found higher total vldltg production in upperbody obese compared with lean women,37 although production was not corrected for any measure of body mass . vldl2tg direct production was higher in post compared to premenopausal women , due to production of tgenriched vldl2 . this has not been reported previously and the relevance is not clear as yet , but small rather than large vldl is implicated in atherosclerosis progression.38 vldl2tg production was higher in postmenopausal women than men matched for plasma tg concentrations24 and as discussed by the authors , vldl2 is more efficiently converted to ldl than vldl1 . moderate hypercholesterolemia arises principally from overproduction of vldl2 particles in men39 ; our study was not designed to measure ldl , kinetics but higher plasma and ldl cholesterol concentrations after the menopause were not accompanied by an increase in vldl2apob production . menopausal status did not affect vldl1 or vldl2 clearance , in contrast to the work of mittendorfer.40 vldl kinetics are sensitive to intraabdominal and liver fat content,32 and discrepancies between published studies may be due to liver fat content , and/or kinetic parameters not corrected for adiposity or lean mass . although estrogen per se may affect vldl kinetics,41 other factors such as the changes in body composition accompany estrogen deficiency . we found increased concentrations of plasma total and ldl cholesterol and apob , which are typical features of dyslipidemia in postmenopausal women.42 additionally , we found significantly higher apociii lpb in postmenopausal women . since apociii plays a pivotal role in the development of hypertriglyceridemia,7 apociii may play a role in the development of dyslipidemia in older , postmenopausal women . higher rdnefa in postmenopausal women is consistent with the finding that exogenous estrogen reduced nefa flux in postmenopausal women.43 this suggests that higher nefa flux in postmenopausal women is a result of estrogen depletion . ranefa , when corrected for fat mass , was significantly lower with increasing abdominal obesity , in agreement with the concept of downregulated adipose tissue fa trafficking in obesity with reduced expression of lipolytic genes such as hormonesensitive lipase and adipose triglyceride lipase.44 the relationship between obesity , insulin resistance , and lipolysis is not clear in the literature and has been elegantly reviewed.45 at a whole body level , lipolysis was similar in lean and abdominally obese groups , but the contribution of systemic fa to increased vldl1tg was significantly higher in abdominally obese women , as was the contribution of nonsystemic fa , with a tendency toward increased de novo hepatic fa secretion indicating an upregulation of secretion of fa from all sources . higher secretion of de novo palmitate has previously been found in obese hypertriglyceridemic men compared with lean normolipidemic men and women.46 our findings of inverse correlations between plasma 3ohb and % dnl in vldl1tg are in agreement with studies in healthy men and women.47 moreover , we found strong inverse correlations between the isotopic desaturation index in vldl1 and vldl2tg and plasma 3ohb in the whole cohort . these 2 variables are not obviously related but provide the first evidence of a clear divergence of fa partitioning in humans in vivo such that hepatic desaturation of fas was low when fa oxidation was high ( and vice versa ) . serum cortisol concentrations were negatively correlated with waisttohip ratio in pre but not postmenopausal women . cortisol status has previously been inversely related to waisttohip ratio in women , although menopause status was not defined ; this has been explained by a higher local clearance rate of cortisol in visceral fat , which has more glucocorticoid receptors than subcutaneous fat.48 however , we found no correlation between serum cortisol concentrations and intraabdominal fat area . in agreement with previous studies of aging,49 serum cortisol concentrations were higher in postmenopausal women . strengths of our study include the unique combination of kinetic and anthropometric measurements in large groups of wellmatched women , but a limitation is the crosssectional design . do not reflect a difference in hormone concentrations , but rather the natural course of events ( menopause plus aging with accompanying changes in body fat distribution ) . indeed , postmenopausal women had equal abdominal obesity but higher intraabdominal fat and less gynoid fat , demonstrating a change in body fat distribution.4 given the importance of estrogen in determining body fat distribution and direct effects on lipid metabolism , it may have been insightful to measure serum estrogen concentrations , although it is clear that many factors beyond sex hormones contribute to lipid and lipoprotein metabolism.50 the study design meant that we were able to look at correlations within each menopausal group . we also analyzed our data according to liver fat and we found that overall , the results were similar to when we divided according to abdominal obesity . this is in contrast to findings in individuals with a large range of liver fat,32 where liver fat was found to be more discriminatory . however , we found that abdominal obesity in women was more related to impaired vldltg secretion than liver fat . we did not include a comparator group of men , but other groups have compared lipoprotein metabolism in men and women.50 one study found that vldltg secretion rate was significantly higher in premenopausal women than men,31 whereas another found that vldl2tg but not vldl1tg secretion rate was higher in postmenopausal women than men.24 we have previously reported no difference in the postprandial contribution of dietary and nonsystemic fa to vldltg between insulinsensitive men and women.13 however , lipoprotein metabolism is dependent on a many factors , and accumulation of excess body fat seems to affect lipid kinetics differently in men and women as recently discussed.50 total body fat and body fat distribution are obvious differences between men and women , and this study has highlighted the importance of body fat distribution in women . vldl1 and vldl2 metabolism is complex in women , and hive plots illustrate that the patterns of associations with metabolic variables are different between menopausal groups . a lack of significant correlation between hepatic vldl2tg and vldl2apob production in postmenopausal women is intriguing and requires further study . abdominal obesity was characterized by increased cardiovascular disease risk factors such as vldl1tg and apob production , liver fat , and nonhdl cholesterol . interestingly , this was observed despite a considerable overlap in bmi between abdominally lean and abdominally obese groups . our study is the first to report that vldl1tg secretion is significantly higher in abdominally obese women and accounts for increased plasma vldl1tg and plasma tg concentrations . this is important because there is increasing evidence that there is a causal relationship between tgmediated pathways and coronary heart disease.51 weight gain in postmenopausal women is likely to impact on both vldl1tg and vldl2tg secretory pathways with consequent implications for cardiovascular disease risk . the study was funded by the british heart foundation ( project grant pg/09/003 ) and hodson is a british heart foundation intermediate fellow in basic science ( fs/11/18/28633 ) . the british heart foundation had no role in the design , analysis , or writing of this article . marinou was funded by the european commission under the marie curie programme ( fp7people2011ief ) . the contents reflect only the author 's views and not the views of the european commission . apociii kinetics were funded by a grant from the national heart foundation of australia ( g 11p 5739 ) . phrb is a senior research fellow of the national health and medical research council ( nhmrc ) of australia . table s1 . correlation coefficients ( r s ) for premenopausal women correlations between variables relating to vldl1 metabolism table s2 . correlation coefficients ( r s ) for premenopausal women correlations between variables relating to vldl2 metabolism table s3 . correlation coefficients ( r s ) for postmenopausal women correlations between variables relating to vldl1 metabolism table s4 . correlation coefficients ( r s ) for postmenopausal women correlations between variables relating to vldl2 metabolism table s5 . biochemical and metabolic variables in women according to abdominal obesity and liver fat table s6 . kinetic estimates relating to nefa , vldl , and apociii metabolism in women according to abdominal obesity and liver fat table s7 . variables relating to fatty acid metabolism in women according to abdominal obesity and liver fat figure s1 . mean tracer : trace ratio for glycerol stable isotope enrichment used to model vldltg kinetics ( n=58 , meansem , filled circles represent vldl1tg , open circles represent vldl2tg ) . figure s2 . mean percent enrichment for leucine stable isotope enrichment used to model vldlapob100 kinetics ( n=59 , meansem , filled circles represent vldl1tg , open circles represent vldl2tg ) . overview of hepatic fatty acid trafficking according to abdominal obesity and menopausal status in healthy women .

backgroundandroid fat distribution ( abdominal obesity ) is associated with insulin resistance , hepatic steatosis , and greater secretion of large very lowdensity lipoprotein ( vldl ) particles in men . since abdominal obesity is becoming increasingly prevalent in women , we aimed to investigate the relationship between android fat and hepatic lipid metabolism in pre and postmenopausal women.methods and resultswe used a combination of stable isotope tracer techniques to investigate intrahepatic fatty acid synthesis and partitioning in 29 lean and 29 abdominally obese women ( android fat / total fat 0.065 [ 0.02 to 0.08 ] and 0.095 [ 0.08 to 0.11 ] , respectively ) . thirty women were premenopausal aged 35 to 45 and they were matched for abdominal obesity with 28 postmenopausal women aged 55 to 65 . as anticipated , abdominal obese women were more insulin resistant with enhanced hepatic secretion of large ( 40430 versus 26826 mg / kg lean mass , p<0.001 ) but not small vldl ( 16011 versus 14213 ) . however , postmenopausal status had a pronounced effect on the characteristics of small vldl particles , which were considerably triglycerideenriched ( production ratio of vldl 2 triglyceride : apolipoprotein b 305.3 versus 191.6 , p<0.05 ) . in contrast to postmenopausal women , there was a tight control of hepatic fatty acid metabolism and triglyceride production in premenopausal women , whereby oxidation ( r s=0.49 , p=0.006 ) , de novo lipogenesis ( r s=0.55 , p=0.003 ) , and desaturation ( r s=0.48 , p=0.012 ) were closely correlated with abdominal obesitydriven large vldltriglyceride secretion rate.conclusionsin women , abdominal obesity is a major driver of hepatic large vldl particle secretion , whereas postmenopausal status was characterized by increased small vldl particle size . these data provide a mechanistic basis for the hyperlipidemia observed in postmenopausal obesity .

Introduction Materials and Methods Subjects Measurement of Liver, Subcutaneous, and Visceral Fat and Body Composition Metabolic Study Day Biochemical Analyses Statistical Analysis Power Calculation Results Discussion Conclusions Sources of Funding Disclosures Supporting information

menopause is associated with increased cardiovascular disease and once women develop acute coronary symptoms , they have worse short and longterm outcomes than men.1 many different factors contribute , including marked hormonal changes,2 changes in metabolic profile associated with increased risk of the metabolic syndrome,3 and relative increase in intraabdominal fat with age.4 accumulation of intraabdominal fat is associated with increased waist circumference and liver fat,5 overproduction of very lowdensity lipoprotein ( vldl ) , and decreased catabolism of apolipoprotein ( apo)bcontaining particles in men.6 the catabolism of apobcontaining particles is partly determined by plasma apociii concentrations , and higher plasma apociii has been associated with dyslipidemia in obese men.7 although abdominal obesity tends to be associated with obesity in men , data from the united states have been used to estimate that 40% of women have an abdominal fat distribution pattern as defined by waist : hip ratio.8 normally , fasting plasma triglycerides ( tg ) are determined by 2 distinct subclasses of vldl9 ; vldl1 is larger and more tgrich than vldl2 , the latter can either be secreted directly from the liver , or formed by the peripheral hydrolysis of vldl1 . hypertriglyceridemia is associated with atherogenic dyslipidemia including the production of small dense ldl , lower hdl cholesterol , and accumulation of postprandial tgrich lipoproteins.10 in men with type 2 diabetes , the secretion of vldl1 is associated with liver fat , hypertriglyceridemia , and increased atherogenic risk.11 impaired hepatic fatty acid oxidation has been reported to be related to obesity and insulin resistance by some12 , 13 but not all.14 few detailed studies have investigated vldl1 and vldl2 kinetics in women , and none have compared the kinetics of vldl or apociii in pre and postmenopausal women . we hypothesized that vldl1tg and apob secretion would be higher in abdominally obese compared with abdominally lean women and aimed to investigate the effect of menopause status on this relationship by measuring hepatic de novo fatty acid synthesis ( dnl ) , oxidation , and desaturation in relation to vldl1 and vldl2 kinetics in pre and postmenopausal women . we recruited 60 healthy white women from local advertising and the oxford biobank as previously reported15 equally into pre and postmenopausal groups aged 35 to 45 and 55 to 65 , respectively . since we also wished to investigate the effect of android fat ( abdominal obesity ) , we used waist circumference , a marker of android fat , to facilitate recruitment of women into groups with low or high android fat . intrahepatic fat was measured by magnetic resonance spectroscopy , visceral and subcutaneous fat were measured by magnetic resonance imaging after an overnight fast and within 2 weeks of the study day,18 and whole body composition and fat distribution ( eg , android and gynoid fat ) were measured using dexa.15 participants arrived after an overnight fast and after consuming deuterated water ( h2o , in order to measure de novo lipogenesis , dnl ) ( 3 g / kg body water ) at 8 and 10 pm the evening before the study day and then continued to consume enriched water ( 2.5 g per 500 ml water ) , in order to achieve and maintain a plasma water enrichment of 0.3%.19 a cannula was placed in an antecubital vein in order to take blood samples for the estimation of dnl in vldl1 and vldl2palmitate , and background isotopic enrichments for the kinetic studies . the ratio of [ uc]16:1n7/[uc]16:0 in vldl1 and vldl2tg was determined as a shortterm index of hepatic stearoylcoa desaturase ( scd ) activity ( the isotopic desaturation index ) and also the scd16 and scd18 fa ratios.26 fa methyl esters prepared from vldl1 and vldl2tg fas20 were analyzed by gc13 to quantify 16:0 and 16:1n7 , and by gcisotope ratio mass spectrometer to measure isotopic enrichment.26 hepatic dnl was measured on the study day , based on the incorporation of h in plasma water ( finnigan gasbenchii ; thermofisher scientific , uk ) and into vldl1 and vldl2tg palmitate using gas chromatography % dnl and represents synthesis of fas from precursors such as sugars and amino acids.28 fa rate of appearance ( ranefa ) was calculated from the [ uc]16:0 infusion rate and enrichment in the plasma nefa fraction and rdnefa was assumed to equal ranefa.24 statistical analysis was performed using spss statistics 19 ( ibm , spss products , chertsey , uk ) . a significant interaction term indicated that the relationship between the dependent variable and abdominal obesity was significantly different in pre and postmenopausal women . the axes are duplicated in order to allow for representation of correlations within the variable group ( eg , there are lines joining the isotopic desaturation index and % dnl in vldl1 for pre and postmenopausal women , representing significant positive correlations ) . using data from a study of the reproducibility of relevant kinetic parameters ( vldl tg and apob100 secretion rates , vldltg clearance rate , rate of appearance nefa,30 and dnl),19 separate power calculations were carried out and the numbers in pre and postmenopausal groups to detect a 40% difference with power of 0.80 at of 0.05 were 4 , 8 , 9 , 10 , and 15 , respectively ( in each group ) . a difference of 40% was considered to be clinically significant and was within the range of differences previously reported in other studies.11 we recruited 60 healthy white women from local advertising and the oxford biobank as previously reported15 equally into pre and postmenopausal groups aged 35 to 45 and 55 to 65 , respectively . since we also wished to investigate the effect of android fat ( abdominal obesity ) , we used waist circumference , a marker of android fat , to facilitate recruitment of women into groups with low or high android fat . intrahepatic fat was measured by magnetic resonance spectroscopy , visceral and subcutaneous fat were measured by magnetic resonance imaging after an overnight fast and within 2 weeks of the study day,18 and whole body composition and fat distribution ( eg , android and gynoid fat ) were measured using dexa.15 participants arrived after an overnight fast and after consuming deuterated water ( h2o , in order to measure de novo lipogenesis , dnl ) ( 3 g / kg body water ) at 8 and 10 pm the evening before the study day and then continued to consume enriched water ( 2.5 g per 500 ml water ) , in order to achieve and maintain a plasma water enrichment of 0.3%.19 a cannula was placed in an antecubital vein in order to take blood samples for the estimation of dnl in vldl1 and vldl2palmitate , and background isotopic enrichments for the kinetic studies . a significant interaction term indicated that the relationship between the dependent variable and abdominal obesity was significantly different in pre and postmenopausal women . the axes are duplicated in order to allow for representation of correlations within the variable group ( eg , there are lines joining the isotopic desaturation index and % dnl in vldl1 for pre and postmenopausal women , representing significant positive correlations ) . using data from a study of the reproducibility of relevant kinetic parameters ( vldl tg and apob100 secretion rates , vldltg clearance rate , rate of appearance nefa,30 and dnl),19 separate power calculations were carried out and the numbers in pre and postmenopausal groups to detect a 40% difference with power of 0.80 at of 0.05 were 4 , 8 , 9 , 10 , and 15 , respectively ( in each group ) . a difference of 40% was considered to be clinically significant and was within the range of differences previously reported in other studies.11 sixty women were recruited : mean age was 41.0 years ( range 35 to 45 ) for premenopausal and 58.1 years ( 55 to 64 ) for postmenopausal women . mean age when divided according to abdominal obesity was 49.3 ( 35 to 64 ) and 49.3 ( 35 to 63 ) for lean and abdominally obese women , respectively . plasma folliclestimulating hormone concentrations ranged from 3.0 to 21.3 and 46.5 to 125 iu / l in pre and postmenopausal women , respectively ( confirming menopausal status ) . lean women had a mean android fat of 0.065 ( sd 0.013 ) and abdominally obese women had a mean value of 0.094 ( sd 0.008 ) . gynoid fat was significantly lower , and intraabdominal fat was significantly higher in postmenopausal women , despite being matched for abdominal obesity ( table 1 ) . n=30 for postmenopausal women apart from data derived from dexa measurements , which were n=28 ( gynoid fat , android : gynoid ratio , fat mass , lean mass and fat : lean mass ) . women with abdominal obesity were more insulin resistant with a more adverse lipid profile ( higher nonhdl cholesterol , plasma tg , vldl1 and vldl2tg concentrations ) . 3ohb indicates plasma 3hydroxybutyrate ; apob , apolipoprotein b ; apociii lpb , apociii associated with lipoprotein b containing particles ; auc , area under the curve ; bp , blood pressure ; chol , cholesterol ; hdl , highdensity lipoprotein ; homair , homeostatic model assessment of insulin resistance ; ldl , lowdensity lipoprotein ; nefa , nonesterified fatty acids ; tg , triglyceride ; vldl , very lowdensity lipoprotein . the rate of disappearance of fas ( rdnefa ) , expressed per kg lean mass was significantly higher in post compared to premenopausal women ( table 3 ) . plasma apociii concentrations positively correlated with apociii production rate ( r s=0.59 , p=9.010 ) but not clearance rate , indicating that plasma apociii concentrations were determined by production rate . menopause status affected the relationship between abdominal obesity and 3ohb : nefa , and abdominal obesity per se had a strong influence on factors related to fa partitioning ( table 4 ) . to explore fa partitioning in relation to metabolic and anthropometric measurements , we tabulated univariate correlations between relevant variables in pre and postmenopausal women ( selected data in tables 5 through 8 and complete analysis in tables s1 through s4 ) . the data are illustrated in hive plots ( figure 1 ) , which clearly show that the patterns of correlations are quite different in pre and postmenopausal women . serum cortisol concentrations were negatively correlated with waisttohip ratio in pre ( r s=0.38 , p=0.04 ) but not postmenopausal women . there was a significant correlation between abdominal fat and liver fat ( r s=0.50 , p<0.001 , n=60 ) . the importance of menopausal status in this relationship is shown in the hive plots and tables 5 through 8 which showed , remarkably , that a significant correlation between liver fat and abdominal obesity was observed only in premenopausal women . 3ohb indicates 3hydroxybutyrate ; apob , apolipoprotein b ; auc , area under the curve ; d , day ; dnl , de novo lipogenesis ; homair , homeostatic model assessment of insulin resistance ; iso , isotopic ; nefa , nonesterified fatty acids ; prod , production ; scd , stearoylcoa desaturase 1 ; scd16 , ratio of 16:1n7/16:0 ; scd18 , ratio 18:1n9/18:0 ; subcut , subcutaneous ; tg , triglyceride ; tot , total ; vldl , very lowdensity lipoprotein . 3ohb indicates 3hydroxybutyrate ; apob , apolipoprotein b ; auc , area under the curve ; d , day ; dnl , de novo lipogenesis ; homair , homeostatic model assessment of insulin resistance ; iso , isotopic ; nefa , nonesterified fatty acids ; prod , production ; scd , stearoylcoa desaturase 1 ; scd16 , ratio of 16:1n7/16:0 ; scd18 , ratio 18:1n9/18:0 ; subcut , subcutaneous ; tg , triglyceride ; tot , total ; vldl , very lowdensity lipoprotein . 3ohb indicates 3hydroxybutyrate ; apob , apolipoprotein b ; auc , area under the curve ; d , day ; dnl , de novo lipogenesis ; homair , homeostatic model assessment of insulin resistance ; iso , isotopic ; nefa , nonesterified fatty acids ; prod , production ; scd , stearoylcoa desaturase 1 ; scd16 , ratio of 16:1n7/16:0 ; scd18 , ratio 18:1n9/18:0 ; subcut , subcutaneous ; tg , triglyceride ; tot , total ; vldl , very lowdensity lipoprotein . 3ohb indicates 3hydroxybutyrate ; apob , apolipoprotein b ; auc , area under the curve ; d , day ; dnl , de novo lipogenesis ; homair , homeostatic model assessment of insulin resistance ; iso , isotopic ; nefa , nonesterified fatty acids ; prod , production ; scd , stearoylcoa desaturase 1 ; scd16 , ratio of 16:1n7/16:0 ; scd18 , ratio 18:1n9/18:0 ; subcut , subcutaneous ; tg , triglyceride ; tot , total ; vldl , very lowdensity lipoprotein . 3ohb indicates plasma 3hydroxybutyrate ; apob , apolipoprotein b ; auc , area under the curve ; dnl , hepatic de novo lipogenesis ; homair , homeostatic model assessment of insulin resistance ; nefa , plasma nonesterified fatty acids ; scd , stearoylcoa desaturase ; scd16 , 16:1 n7/16:0 ratio in vldltg ; scd18 , 18:1 n9/18:0 ratio in vldltg ; subcut , subcutaneous ; tg , triglyceride ; vldl , very lowdensity lipoprotein . correlations between vldl 1tg production ( mg / day ) and the proportion ( % ) of dnl fatty acids vldl 1tg ( a ) , vldl 2tg direct production ( mg / day ) and the proportion ( % ) of dnl fatty acids vldl 2tg ( b ) , the proportion ( % ) of dnl fatty acids vldl 1tg and the auc for plasma 3hydroxybutyrate ( mol / l ) ( c ) , the proportion ( % ) of dnl fatty acids vldl 2tg and the auc for plasma 3hydroxybutyrate ( mol / l ) ( d ) , and the association between plasma 3hydroxybutyrate concentrations ( mol / l ) and the isotopic desaturation index ( [ uc16:1n7/uc16:0]*1000 ) in vldl 1tg ( e ) and vldl 2tg ( f ) in pre ( ) and post ( ) menopausal women . auc indicates area under the curve ; dnl , hepatic de novo lipogenesis ; ns , not significant ; tg , triglyceride ; vldl , very lowdensity lipoprotein . using a combination of stable isotope tracer techniques , we investigated kinetic parameters of apob , apociii , and tg metabolism in pre and postmenopausal women . we report for the first time that menopausal status is a determinant of hepatic tg flux through enhancement of adipose tissue nefa flux , altered intrahepatic fa partitioning , and secretion of larger vldl2 . we measured 2 aspects of vldl secretion : vldlapob secretion rate , which measures whole particle secretion ; and vldltg secretion , which tracks the lipid component . vldl1tg and vldl1apob production rates were significantly higher in there are no previous comparable studies , but in lean and obese premenopausal women there were no differences in total vldlapob or vldltg secretion.35 , 36 another study in premenopausal women found higher total vldltg production in upperbody obese compared with lean women,37 although production was not corrected for any measure of body mass . ranefa , when corrected for fat mass , was significantly lower with increasing abdominal obesity , in agreement with the concept of downregulated adipose tissue fa trafficking in obesity with reduced expression of lipolytic genes such as hormonesensitive lipase and adipose triglyceride lipase.44 the relationship between obesity , insulin resistance , and lipolysis is not clear in the literature and has been elegantly reviewed.45 at a whole body level , lipolysis was similar in lean and abdominally obese groups , but the contribution of systemic fa to increased vldl1tg was significantly higher in abdominally obese women , as was the contribution of nonsystemic fa , with a tendency toward increased de novo hepatic fa secretion indicating an upregulation of secretion of fa from all sources . cortisol status has previously been inversely related to waisttohip ratio in women , although menopause status was not defined ; this has been explained by a higher local clearance rate of cortisol in visceral fat , which has more glucocorticoid receptors than subcutaneous fat.48 however , we found no correlation between serum cortisol concentrations and intraabdominal fat area . indeed , postmenopausal women had equal abdominal obesity but higher intraabdominal fat and less gynoid fat , demonstrating a change in body fat distribution.4 given the importance of estrogen in determining body fat distribution and direct effects on lipid metabolism , it may have been insightful to measure serum estrogen concentrations , although it is clear that many factors beyond sex hormones contribute to lipid and lipoprotein metabolism.50 the study design meant that we were able to look at correlations within each menopausal group . we did not include a comparator group of men , but other groups have compared lipoprotein metabolism in men and women.50 one study found that vldltg secretion rate was significantly higher in premenopausal women than men,31 whereas another found that vldl2tg but not vldl1tg secretion rate was higher in postmenopausal women than men.24 we have previously reported no difference in the postprandial contribution of dietary and nonsystemic fa to vldltg between insulinsensitive men and women.13 however , lipoprotein metabolism is dependent on a many factors , and accumulation of excess body fat seems to affect lipid kinetics differently in men and women as recently discussed.50 total body fat and body fat distribution are obvious differences between men and women , and this study has highlighted the importance of body fat distribution in women . abdominal obesity was characterized by increased cardiovascular disease risk factors such as vldl1tg and apob production , liver fat , and nonhdl cholesterol . variables relating to fatty acid metabolism in women according to abdominal obesity and liver fat figure s1 .

falls in older adults are a global health issue , resulting in injury , disability , restriction in physical activities , fear of falls , poor quality of life , hospitalization , and institutionalization . falls are a major cause of mortality and morbidity among older adults , put huge burden on the health - care system , and cost a large amount of health resources1,2,3 . in the united kingdom , fall - related costs among older adults reached $ 1.6 billion in 20083 . in the united states , the total direct medical cost of fall injuries for people 65 and over was $ 30 billion in 20104 . these figures do not include the indirect and intangible costs for family and society . therefore , a study on fall incidence and related factors would contribute to fall prevention and policy development . fall incidence varied widely across different ages , races , living residences and so on1 , 5 . previous studies have reported that approximately 2835% , 3242% , and 50% of adults over 65 , adults 70 or over , and adults 80 and over have one or more falls annually , respectively6,7,8,9,10 . fall incidence varied from 28 to 42% per year in older western adults , while it ranged from 14.7 to 34% in older chinese adults , about half that found in the caucasian older adults population11,12,13,14 . about 3050% of older adults living in long - term care institutions fell at least once , which was higher than the rate for those living in the community15 , 16 . a series of studies have reported that demographics ( e.g. , age , gender , ethnicity ) , chronic diseases ( e.g. , dementia , parkinsonism , stroke , hypertension , depression , and diabetes mellitus ) , medications ( e.g. , antidepressants , sedatives , antihypertensives , antipsychotics ) , and other factors were correlated with falls in older adults , but there were also some areas of divergence in these research results1 , 5 , 17,18,19 . the discrepancy may come from differences in race , living residence , geographical variation , cultural background , lifestyle , or other factors . china , the world s most populated country , has up to 160 million older adults aged 60 and over , and 99% of older chinese adults live in china19 . epidemiologic studies of falling in older chinese adults have predominantly adopted retrospective methods causing recall bias , which has reduced their accuracy . a few prospective studies aimed at the fall status of the chinese elderly were carried out in hong kong , taiwan , and australia . in addition , no study has sufficiently focused on very old chinese adults ( aged 85 or over)14,19,20,21 . the characteristics of this group are advanced age , predominantly male , clustered living in military communities , possession of excellent health - care insurance , and a higher socioeconomic level . until now , the fall status of older chinese veterans in military communities has been unknown . the objective of the present study was to fill the gap in data concerning fall incidence and related clinical factors of older chinese veterans in military communities . the prevention of falls network europe ( profane ) group defined a fall as an unexpected event in which the participant comes to rest on the ground , floor , or lower level22 . in the present study , we adopted this definition and exclude external force ( e.g. , forcefully pushed down ) . this was a prospective population - based cohort study performed among older chinese veterans living in 13 military communities of the air force from may to july 2012 . the inclusion criteria were aged 60 or over , living in a military community , and ability to walk with or without an assistive device . the exclusion criteria were unavailability for interviews because of residing in a hospital or being out , anticipated survival of less than 6 months , and being unwilling to cooperate in this study . a total of 466 eligible subjects , accounting for 77.5% older veterans in 13 military communities , were interviewed . all procedures were approved by the institutional ethical review committee , and all subjects provided written informed consent to participate in this study . in our study , fall was the target event , and we combined two ways to record fall data during the 12-month follow - up . first , if the veterans fell , they and/or their caregivers reported to military community liaisons who recorded the details of the fall in a fall diary for the subjects . the liaisons in our study were the medical staff in charge of health care in the chinese military communities . fall data from community liaisons and researchers were compiled at the end of the 12-month follow - up . if there were discrepancies , we contacted the subjects and liaisons to confirm the facts . we collected demographic data and information about health status , assistive device use , and fall history in the past 6 months . health status included physical measurements , chronic disease history , medication use , chemical measurements of blood , and physical function assessment . physical measurements included vision and hearing acuity assessment , body mass index ( bmi ) , systolic blood pressure ( sbp ) , and diastolic blood pressure ( dbp ) . visual acuity assessment was performed with an international standard visual acuity chart for both eyes separately and recorded in decimal values from 0.00 to 1.50 , and the better eye s measurement was chosen as the subject s visual acuity . hearing acuity assessment was divided into 4 groups : normal , slight decrease ( can not hear a whisper ) , modest decrease ( can not hear a normal voice ) , and severe decrease ( can only hear loud talking ) . bmi was calculated as mass ( kg)/height ( m ) and was classified into three groups : underweight ( < 18.5 ) , normal ( 18.5 but 25 ) , and overweight ( > 25 ) . sbp and dbp were measured in the seated position by a standard mercury sphygmomanometer . chronic disease history included information about dementia , parkinsonism , stroke , cervical diseases , lumbar diseases , knee diseases , coronary diseases , sinus bradycardia , atrial fibrillation , atrioventricular block , chronic heart failure ( chf ) , hypertension , diabetes mellitus , chronic obstructive pulmonary disease ( copd ) , sleep apnea syndrome ( sas ) , chronic renal failure ( crf ) , hyperlipemia , hyperuricemia , cancer , and anemia . medication use included information about sedatives , antihypertensives , antiarrhythmics , antidepressants , oral hypoglycemics , and the total number of oral medications ( including prescription drugs and over - the - counter drugs ) . the chronic diseases and medications chosen to be investigated in the present study were the most common chronic diseases in our subjects or had been reported in the literatures as pertinent diseases or medications for falls in older adults . the total number of selected chronic diseases was counted and divided into a low count ( 1 ) , medium count ( 2 to 4 ) , and high count ( 5 ) . the total number of oral medications was also counted and divided into a low count ( 5 ) , medium count ( 6 to 9 ) , and high count ( 10 ) . chemical measurements of blood included measurement of hemoglobin , albumin , total protein , blood urea nitrogen , urinary creatinine , uric acid , fasting blood sugar , total cholesterol , triglyceride , low density lipoproteins ( ldl ) , and high density lipoproteins ( hdl ) . physical function assessment was performed with the five - repetition sit - to - stand test ( frsts ) . the frsts test recorded the time to transfer from a sitting position to standing position five times successively with the arms folded across the chest . the frsts test score was divided into 5 groups : a score of 1 was > 0 s but 9 s , a score of 2 was > 9 s but 12 s , a score of 3 was > 12 s but 15 s , a score of 4 was > 15 s , and a score of 5 was unable to complete the test23 . face - to - face interviews were carried out at the beginning of the study to collect the demographic data and information about medication use ( including prescription drugs and over - the - counter drugs ) , assistive device use , and fall history in the past 6 months . physical and chemical measurements were compiled from the annual health examination reports of 2012 . descriptive statistics were reported as mean and standard deviations for continuous data and as percentages for categorical data . the incidence of falls was calculated as the number of falls divided by 1,000 person - years ( py ) , and the incidence of fallers was calculated as the number of fallers divided by 1,000 py . to compare fall incidence with other studies , we calculated the standardized incidence . the clinical related factors for falls were explored by cox regression models with time to the first fall for the information collected about demographics , physical measurements , physical function assessment , chronic diseases , medication use , chemical measurements of blood , assistive device use , and fall history . in model 1 , only demographic data were included . in model 2 , after adjustment for demographics , all variables of physical measurements , chronic diseases , medication use , chemical measurements of blood , assistive device use , and fall history were examined , respectively . in model 3 , after adjustment for demographics , all significant variables in model 2 were simultaneously entered into a summary we explored the association of multiple diseases and multiple medications with falls after adjustment for demographics , assistive device use , and history of falls by cox regression analysis . we explored the relationship of physical function with falls after adjustment for demographics , assistive device use , and history of falls with another cox regression analysis . statistical significance was accepted at p<0.05 , and all statistical analyses were carried out with spss 16.0 version . thirteen subjects died ; 3 of these subjects experienced 1 fall and 1 experienced 2 falls before they died . thirteen subjects died ; three of these subjects experienced 1 fall and 1 experienced . there was no significant difference in age or sex between the study subjects and the removed subjects . the demographic , health status , assistive device use , and fall history data of the 447 older veterans are shown in table 1table 1.demographic characteristics and health status for all subjects , non - fallers and fallersvariablestotal ( n=447)non - fallers ( n= 336)fallers ( n=111 ) meansd(range)number(%)meansd(range)number(%)meansd(range)number(%)demographic characteristics age ( y)82.24.7 ( 6095)81.84.8 ( 6094)83.64.0 ( 7495 ) gender ( male)417 ( 93.3)315 ( 93.8)102 ( 91.9)physical measurementssbp ( mmhg)136.718.2 ( 90210)137.918.8 ( 90210)131.016.5 ( 83170)dbp ( mmhg)71.59.9 ( 48100)72.59.8 ( 48100)67.28.3 ( 5090)bmi ( kg / m)<18.510 ( 2.2)9 ( 2.7)1 ( 0.9)18.525216 ( 48.3)165 ( 49.1)51 ( 45.9)>25221 ( 49.4)162 ( 48.2)59 ( 53.2)visual acuity0.80.3 ( 0.01.5)0.80.3 ( 0.01.5)0.72.3 ( 0.01.2)hearing acuitynormal362 ( 81.0)281 ( 83.6)81 ( 73.0)slight impairment23 ( 5.1)15 ( 4.5)8 ( 7.2)modest impairment28 ( 6.3)22 ( 6.5)6 ( 5.4)severe impairment34 ( 7.6)18 ( 5.4)16 ( 14.4 ) frsts test score190 ( 20.1)77 ( 22.9)13 ( 11.7)2160 ( 35.8)134 ( 39.9)26 ( 23.4)380 ( 17.9)63 ( 18.8)17 ( 15.3)479 ( 17.7)45 ( 13.4)34 ( 30.6)538 ( 8.5)17 ( 5.1)21 ( 18.9)chronic diseases dementia29 ( 6.5)14 ( 4.2)15 ( 13.5)parkinsonism26 ( 5.8)14 ( 4.2)12 ( 10.8)stroke44 ( 9.8)14 ( 4.2)30 ( 27.0)cervical diseases103 ( 23.0)73 ( 21.7)30 ( 27.0)lumbar diseases97 ( 21.7)65 ( 19.3)32 ( 28.8)knee diseases96 ( 21.5)67 ( 19.9)29 ( 26.1)coronary disease312 ( 69.8)224 ( 66.7)88 ( 79.3)sinus bradycardia70 ( 15.7)52 ( 15.5)18 ( 16.2)atrial fibrillation48 ( 10.7)36 ( 10.7)12 ( 10.8)atrioventricular block79 ( 17.7)58 ( 17.3)21 ( 18.9)chf30 ( 6.7)23 ( 6.8)7 ( 6.3)hypertension276 ( 61.7)207 ( 61.6)69 ( 62.2)diabetes mellitus123 ( 27.9)88 ( 26.2)35 ( 31.5)copd19 ( 4.3)15 ( 4.5)4 ( 3.6)sas11 ( 2.5)7 ( 2.1)4 ( 3.6)crf40 ( 8.9)25 ( 7.4)15 ( 13.5)hyperlipemia119 ( 26.6)86 ( 25.6)33 ( 29.7)hyperuricemia106 ( 23.7)85 ( 25.3)21 ( 18.9)cancer74 ( 16.6)53 ( 15.8)21 ( 18.9)anemia22 ( 4.9)14 ( 4.2)8 ( 7.2)multiple diseases162 ( 13.9)50 ( 14.9)12 ( 10.8)24282 ( 63.1)227 ( 67.6)55 ( 49.5)5103 ( 23.0)59 ( 17.6)44 ( 39.6)medication use sedatives 42 ( 9.4)22 ( 6.5)20 ( 18.0)antihypertensives 209 ( 46.8)149 ( 44.3)60 ( 54.1)antiarrhythmics 67 ( 15.0)51 ( 15.2)16 ( 14.4)antidepressants6 ( 1.3)5 ( 1.5)1 ( 0.9)oral hypoglycemics 79 ( 17.7)53 ( 15.8)26 ( 23.4)multiple medications5279 ( 62.4)226 ( 67.3)53 ( 47.7)69135 ( 30.2)93 ( 27.7)42 ( 37.8)1033 ( 7.4)17 ( 5.1)16 ( 14.4)chemical measurementshemoglobin ( g / l)132.214.2 ( 76194)133.014.2 ( 76194)130.013.9 ( 95164)albumin ( g / l)42.82.6 ( 16.350.0)42.92.7 ( 16.350.0)42.52.3 ( 36.048.0)total protein ( g / l)68.94.4 ( 55.-80.0)69.04.4 ( 55.080.0)68.84.6 ( 59.679.0)blood urea nitrogen ( mmol / l)6.42.2 ( 2.625.4)6.42.2 ( 2.925.4)6.42.3 ( 2.617.3)urinary creatine ( mmol / l)85.528.9 ( 33382)85.228.4 ( 33382)86.230.4 ( 39252)uric acid ( mmol / l)347.380.7 ( 154675)347.282.1 ( 158675)347.876.6 ( 154670)fasting blood sugar ( mmol / l)5.41.3 ( 3.215.1)5.41.2 ( 3.215.1)5.51.5 ( 4.013.1)total cholesterol ( mmol / l)4.50.8 ( 1.17.5)4.50.8 ( 1.17.6)4.50.8 ( 2.26.9)triglyceride ( mmol / l)1.40.9 ( 0.39.5)1.50.9 ( 0.39.5)1.40.8 ( 0.45.2)ldl ( mmol / l)2.80.7 ( 1.15.4)2.80.7 ( 1.15.4)2.70.7 ( 1.14.7)hdl ( mmol / l)1.20.4 ( 0.74.3)1.20.4 ( 0.74.3)1.20.3 ( 0.72.4)othersassistive device use122 ( 27.3)74 ( 22.0)48 ( 43.2)falls history ( the past 6 months)67 ( 15.0)30 ( 8.9)37 ( 33.3)sbp : systolic blood pressure ; dbp : diastolic blood pressure ; frsts test : five repetition sit - to - stand test ; chf : chronic heart failure ; copd : chronic obstructive pulmonary disease ; sas : sleep apnea syndrome ; crf : chronic renal failure ; ldl : low density lipoproteins ; hdl : high density lipoproteins . * p<0.05 ; * * p<0.01 ; * * * p<0.001 . there were 429 subjects over 75 years old , which accounted for 96% of the subjects in the final samples ( table 2table 2.comparison of the sample distribution and the incidence of falls and fallers between hong kong and the present studyage ( y)sample distributionfall incidencefaller incidencehong kong % ( n)present study % ( n)hong kong ( /1,000 py)present study ( /1,000 py)hong kong ( /1,000 py)present study ( /1,000 py)6064/1.1 ( 5)/0/0656933.9 ( 514)1.1 ( 5)16701320707429.1 ( 441)1.8 ( 8)248125173125757919.2 ( 291)14.5 ( 65)363200254154808412.5 ( 189)55.3 ( 247)387328285239855.4 ( 82)26.2 ( 117)4684913513578589/20.8 ( 93)/510/34790/5.4 ( 24)/421/379all100 ( 1517)100 ( 447)270342198249standardized incidence 19641964300187219146the incidence of falls was calculated as the number of falls divided per 1,000 person - years . the incidence of fallers was calculated as the number of fallers divided per 1,000 person - years . there were 417 male subjects , which accounted for 93.3% of the subjects in the final samples ( table 1 ) . thirteen subjects died ; three of these subjects experienced 1 fall and 1 experienced . sbp : systolic blood pressure ; dbp : diastolic blood pressure ; frsts test : five repetition sit - to - stand test ; chf : chronic heart failure ; copd : chronic obstructive pulmonary disease ; sas : sleep apnea syndrome ; crf : chronic renal failure ; ldl : low density lipoproteins ; hdl : high density lipoproteins . * p<0.05 ; * * p<0.01 ; * * * p<0.001 the incidence of falls was calculated as the number of falls divided per 1,000 person - years . the incidence of fallers was calculated as the number of fallers divided per 1,000 person - years . , 86 fell once , 25 older veterans fell two or more times , and a total of 152 falls occurred . the incidence of falls and fallers were 342/1,000 py and 249/1,000 py , respectively ( table 2 ) . the incidence of falls increased substantially when the elderly were under 90 years old , but decreased when the elderly were over 90 years old . the incidence of fallers rose sharply when the elderly were younger than 85 years old , while the incidence became stable when the elderly were older than 85 years old ( table 2 ) . in model 1 of the cox regression analyses , age was correlated with falls ( table 3table 3.cox regression analyses of clinical independent risk factors for fallsvariables model 1model 2model 3rr95% cirr95% cirr95% cidemographic characteristicsage1.081.041.131.030.981.08sex1.370.702.721.200.582.47physical measurementssbp ( mmhg)0.990.981.000.990.981.00dbp ( mmhg)0.980.961.000.980.951.08visual acuity0.360.180.710.470.230.96hearing acuity0.830.700.990.850.711.02bmi ( kg / m)<18.5 vs.18.5253.940.5229.64>25.0 vs. 18.5251.160.791.70chronic diseasesdementia2.201.253.871.550.832.90parkinsonism 2.211.214.031.410.722.75stroke4.392.876.702.431.513.93cervical diseases1.290.851.97lumbar diseases1.601.062.411.731.122.70knee diseases1.300.851.99coronary diseases1.550.972.49sinus bradycardia1.040.631.72atrial fibrillation1.020.561.86atrioventricular block1.010.621.63chf0.750.311.64hypertension0.940.611.38diabetes mellitus1.220.821.83copd0.700.261.90sas1.990.735.43crf1.440.822.52hyperlipemia1.160.771.75hyperuricemia0.770.481.24cancer1.160.721.86anemia1.320.642.74medication usesedatives2.191.343.591.801.053.08antihypertensives 1.270.871.85antiarrhythmics 0.900.491.66antidepressants0.920.541.56oral hypoglycemics 1.490.962.31chemical measurements hemoglobin ( g / l)0.990.981.01albumin ( g / l)0.970.911.04total protein ( g / l)0.990.951.03blood urea nitrogen ( g / l)0.980.891.07urinary creatinine ( mmol / l)1.000.991.01uric acid ( mmol / l)1.000.991.00fasting blood sugar ( mmol / l)1.080.951.22total cholesterol ( mmol / l)1.000.801.24triglyceride ( mmol / l)1.000.801.24ldl ( mmol / l)0.970.751.26hdl ( mmol / l)1.000.591.68assistive device use0.510.340.771.280.822.01history of falls ( past 6 months)3.282.194.912.771.784.32rr : relative risk ; ci : confidence interval ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ; frsts test : five - repetition sit - to - stand test ; chf : chronic heart failure ; copd : chronic obstructive pulmonary disease ; sas : sleep apnea syndrome ; crf : chronic renal failure . in model 1 , only demographic data were included . in model 2 , after adjustment for demographics , all variables of physical measurements , chronic diseases , medication use , chemical measurements of blood , assistive device use , and fall history were examined , respectively . in model 3 , after adjustment for demographics , all significant variables in model 2 were simultaneously entered into a summary cox regression model . * p<0.05 ; * * p<0.01 ; * * * p<0.001 ) . in model 2 , after adjustment for demographics , falls were associated with sbp , dbp , visual acuity , hearing acuity , dementia , parkinsonism , stroke , lumbar diseases , sedatives , assistive device use , and fall history in the past 6 months , respectively ( table 3 ) . in model 3 , after adjustment for demographics , the significant clinical factors associated with falls were only visual acuity ( rr=0.47 , p=0.039 ) , stroke ( rr=2.43 , p=0.000 ) , lumbar diseases ( rr=1.73 , p=0.015 ) , sedatives ( rr=1.80 , p=0.032 ) , and fall history in the past 6 months ( rr=2.77 , p=0.000 ) . after adjustment for demographics , assistive device use , and fall history by cox regression analysis , multiple diseases ( rr=1.53 , p=0.013 ) and multiple medications ( rr=1.34 , p=0.041 ) were the independent risk factors for falls . after adjustment for demographics , assistive device use , and history of falls by cox regression analysis , the fpsts test score ( rr=1.41 , p=0.000 ) was an independent predictor for falls . confidence interval ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ; frsts test : five - repetition sit - to - stand test ; chf : chronic heart failure ; copd : chronic obstructive pulmonary disease ; sas : sleep apnea syndrome ; crf : chronic renal failure . in model 1 , only demographic data were included . in model 2 , after adjustment for demographics , all variables of physical measurements , chronic diseases , medication use , chemical measurements of blood , assistive device use , and fall history were examined , respectively . in model 3 , after adjustment for demographics , all significant variables in model 2 were simultaneously entered into a summary cox regression model . * p<0.05 ; * * p<0.01 ; * * * p<0.001 this was the first prospective cohort study of falls among older chinese veterans in military communities . the incidence of falls and fallers were 342/1,000 py and 249/1,000 py , respectively . the clinical factors associated with falls among older chinese veterans in military communities were poor senses , stroke , lumbar diseases , taking sedatives , fall history in the past 6 months , having multiple diseases , taking multiple medications , and poor performance in the fpsts test . in our study , the incidence of falls and fallers among older chinese veterans was 342/1,000 py and 249/1,000 py , respectively . compared with the results of fall studies in older western adults , our fall incidence and faller incidence were lower than those found in western countries ( ranges of 300 to 809/1,000 py and 190 to 321/1,000 py)13 , 24,25,26 . the divergent incidences were derived from differences in attitudes toward falls , behaviors , lifestyle , cultural background , and races . compared with the study of chu , which investigated the fall incidence among older chinese adults in the hong kong communities , adjusted for an unbalanced age distribution , the standardized incidences of falls and faller were 187/1,000 py and 146/1,000 py in the present study , while they were 300/1,000 py and 219/1,000 py in hong kong ( table 2)20 . the reason for our low incidence may result from the following : first , the veterans had access to an excellent comprehensive health - care system ( e.g. , the professional medical staff in charge of older veteran health care , provision of a variety of health - related information and so on ) . most studies have shown that females have a higher incidence of falls than males . almost all the earlier studies showed that age was a risk factor for falls , but the relationship between age and falls in our study was not as significant as others1 , 16 , 20 . the mean age of our subjects was 82 ( 82.244.68 ) years old , which was older than those in other studies . the distribution of age showed that most subjects age were over 75 years old . old - old adults ( 7584 years old ) accounted for 69.8% of the subjects , and very - old adults ( over 85 aged ) accounted for 26.2% of the subjects ( table 2 ) . so , our study was the first time to provide sufficient fall information for very - old chinese adults14 , 19,20,21 . the incidence of falls increased substantially when the elderly were younger than 90 , but it decreased when the elderly were 90 or over ( table 2 ) . the incidence of fallers went up sharply when the older veterans were under 85 years old , but became stable when the subjects were over 85 years old . therefore , we hypothesized that the number of fallers was increased at under 85 years old , reached a peak at 85 years old , and remained stable or decreased at over 85 years old . the underlying explanations for this hypothesis are as follows : firstly , very - old people pay more attention to falls on their own compared with young - old adults , who were reluctant to admit to aging and were indifferent to falls . secondly , the caregivers who take care of very - old adults and the medical staff in charge of health care in military communities were more careful and utilized many protective strategies to prevent falls . although the fall rate in females was higher than males in accordance with other studies , we did not find a correlation between sex and falls in older chinese veterans27 , 28 . the number of female subjects in our sample was too small ( accounting for just 6.7% ) , which may explain this difference . vision and hearing are the most basic senses of human beings . this prospective study showed that poor visual acuity was a significant risk factor for falls , which was consistent with other studies1 , 29 , 30 . visual input is thought to be important in allowing individuals to coordinate and plan movement , as well as in helping maintaining balance31 . our study revealed that poor hearing acuity was close to being statistically significant as an independent risk factor . we divided hearing based on the ability to hear someone talking , which may have more application value but less statistical power . hearing loss interfered with the elderly people s awareness of their surroundings in a timely and exact manner and delayed their avoidance of environmental hazards . lastly , poor senses limited the elderly people s daily activities , reducing their muscle strength and endurance33 . a series of studies showed that cardiovascular diseases ( e.g. , hypertension , atrial fibrillation ) , neurological diseases ( e.g. , dementia , parkinsonism , stroke ) , orthopedic diseases ( e.g. , cervical , lumbar , knee ) , and diabetes mellitus were common diseases related to falls1 , 17 , 20 , 34 . on the other hand , among the chronic diseases in our study , the difference between the present and other studies may be due to the following reasons : first , chronic diseases exerted an effect on falls proportionate to their severity , which was not recorded in our study . second , the number of subjects for several chronic diseases was small ( e.g. , dementia and parkinsonism ) . in addition , our study results indicated that having multiple diseases was a strong risk contributor to falls . the effect of a single chronic disease is lower , but the accumulated effect of multiple diseases may be more significant . multiple diseases overlapped and interfered with physical function , exacerbated frailness of the body , and worsened postural control . earlier studies documented that antihypertensives , antiarrhythmics , and oral hypoglycemic drugs were common drugs that caused falls35,36,37 , but we did not find these results among older chinese veterans . it may be that medical staff have paid more attention to the adverse effects of these drugs in chinese military communities and avoided the falls associated with these drugs . antidepressants were reported to be highly related with falls36 , 37 , but were not predictors in our sample . this discrepancy may be due to the fact that there was only a small number of subjects taking this medication in our study . however , taking sedatives was a significant independent risk factor for falls in our study . sleeping disorder was a common disease in older persons , and use of sedatives was increasing ; however , awareness of adverse reactions to sedatives and monitoring strategies form them have not been considered important in military communities . therefore , sedatives became the main drugs leading to the occurrence of falls in our study . the mechanism of falls caused by sedatives was sedation , dizziness , decrease in neuromuscular function , and cognitive impairment15 . in our study , taking multiple medications ( no matter whether they were prescription or over - the - counter drugs ) was strongly associated with falls , which was similar to the findings of the study of friedman and campbell25 , 38 . adverse reactions of drugs related to falls are correlated with the number of medications in addition to drug dosage , because the interaction and cooperation of multiple medications aggravate the adverse reactions5 . in western countries , taking multiple medications may be a marker of frailty , but this is more of a behavioral factor in china . for the older chinese veterans , the abundant health resources and high socioeconomic level made them inclined to take more drugs ( the number of oral medications in our sample vs. in hong kong was 4.423.36 vs. 1.642.0120 ) to maintain their health . therefore , in order to reduce the risk of falls , medical staff in chinese military communities should pay more attention to control of the number of medications . in our study , the results of the frsts were strongly associated with falls , which was similar to the findings of the study of zhang39 . the frsts is widely used as a simple tool for assessing muscle strength , particularly lower limb strength40 . poor performance in the frsts was an indicator of frailty , general muscle weakness , and balance deterioration in the lower limbs , and also an important indicator of decreasing physical function . furthermore , poor muscle strength and low physical performance may cause the impairment of postural reflexes and loss of independence and increase the risk of falls . some studies reported that use of an assistive device was a protective strategy , while other studies found that use of an assistive device was a risk factor for falls13 , 25 . in our study , although use of an assistive device was a risk factor for falls in our univariate analysis ( table 1 , cox model 2 ) , it was not in our multivariate analysis . this suggested that use of an assistive device was not an independent risk for falls . furthermore , analysis showed that use of an assistive device was positively correlated with physical function ( , table 4table 4.relationship between assistive device use and physical function scorevariablephysical function score12345without assistive devicecount84137573611% within physical function93.3%85.6%71.3%45.6%28.9%with assistive devicecount623234327% within physical function6.7%14.4%28.8%54.4%71.1%a score of 1 was>0 s but 9 s in the five - repetition sit - to - stand test , a score of 2 was > 9 s but 12 s in the five - repetition sit - to - stand test , a score of 3 was>12 s but 15 s in the five - repetition sit - to - stand test , a score of 4 was>15 s in the five - repetition sit - to - stand test , and a score of 5 was unable to complete the five - repetition sit - to - stand test . ) . we then analyzed the association of using an assistive device with falls by stratifying physical function . ultimately , there was no association between use of an assistive device and falls by physical function stratification . so we hypothesize that use of an assistive device has an indirect association with falls though physical function . since physical function was an independent risk factor for falls in our study , use of an assistive device represented only a means of compensating for loss of physical function and improving activities of daily living , and it did not have a protective or risk - enhancing effect with respect to falls41,42,43 . having a history of falls was a strong risk factor for falls , as shown by the outcomes of other studies1 , 19 . a score of 1 was>0 s but 9 s in the five - repetition sit - to - stand test , a score of 2 was > 9 s but 12 s in the five - repetition sit - to - stand test , a score of 3 was>12 s but 15 s in the five - repetition sit - to - stand test , a score of 4 was>15 s in the five - repetition sit - to - stand test , and a score of 5 was unable to complete the five - repetition sit - to - stand test . the strengths of our study were as follows : first , it was the first prospective cohort study about the incidence of falls and related clinical factors for older chinese veterans in military communities . second , most subjects were very - old adults , which was different from other studies . it was able to provide some information about fall status in very - old adults to some extent . fourth , the integrity and exactness of the clinical information were high , because data were collected from electronic records , reports of health examinations , and professional staff assessments . fifth , older veterans living in military communities were administrated by a special department , so the stability and cooperation of subjects were very reliable . there were a few limitations in our study : first , the sample size was not large , but the number of subjects accounted for 77.5% of the populations of 13 air force military communities in beijing , which provided a good representation of older chinese veterans . second , most of the subjects were male , as an unbalanced ratio of the genders is a characteristic feature of older veterans in china . there were only 30 female subjects ( 6.7% ) in our study , which made the results less powerful and limited the generalizability of our results . but our study still offered important information about very - old adult chinese males . third , the prevalence of dementia in our subjects was 6.5% , which was lower than that among older adults . dementia was underrepresented in our samples as a result of subject exclusion : subjects unwilling to participate in this study were excluded . in chinese culture , individuals with dementia and relatives of individuals with dementia would not be willing to take part in many investigations . in summary , the present study revealed the incidence of falls and fallers to be 342/1,000 py and 249/1,000 py , which were lower than those in hong kong and in western countries . the clinical risk factors for falls among older chinese veterans in military communities were poor senses , stroke , lumbar diseases , taking sedatives , fall history in the past 6 months , having multiple diseases , taking multiple drugs , and poor physical function . however , advanced age and use of an assistive device were not independent risk factors for falls among older chinese veterans . the preventive strategies targeting the above risk factors are very significant for reducing falls in older chinese veterans .

[ purpose ] the aim of this study was to determine fall incidence and explore clinical factors of falls among older chinese veterans in military communities . [ subjects and methods ] we carried out a 12-month prospective study among 13 military communities in beijing , china . fall events were obtained by self - report to military community liaisons and monthly telephone interviews by researchers . [ results ] among the final sample of 447 older veterans , 86 fell once , 25 fell twice or more , and 152 falls occurred altogether . the incidence of falls and fallers were 342/1,000 person - years and 249/1,000 person - years . in cox regression models , independent clinical factors associated with falls were visual acuity ( rr=0.47 ) , stroke ( rr=2.43 ) , lumbar diseases ( rr=1.73 ) , sedatives ( rr=1.80 ) , fall history in the past 6 months ( rr=2.77 ) , multiple chronic diseases ( rr=1.53 ) , multiple medications ( rr=1.34 ) , and five - repetition sit - to - stand test score ( rr=1.41 ) . hearing acuity was close to being statistically significant . [ conclusion ] the incidences of falls and fallers among older chinese veterans were lower than those of hong kong and western countries . the clinical risk factors of falls were poor senses , stroke , lumbar diseases , taking sedatives , fall history in the past 6 months , having multiple chronic diseases , taking multiple medications , and poor physical function . the preventive strategies targeting the above risk factors are very significant for reducing falls .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

the objective of the present study was to fill the gap in data concerning fall incidence and related clinical factors of older chinese veterans in military communities . physical function assessment was performed with the five - repetition sit - to - stand test ( frsts ) . face - to - face interviews were carried out at the beginning of the study to collect the demographic data and information about medication use ( including prescription drugs and over - the - counter drugs ) , assistive device use , and fall history in the past 6 months . the clinical related factors for falls were explored by cox regression models with time to the first fall for the information collected about demographics , physical measurements , physical function assessment , chronic diseases , medication use , chemical measurements of blood , assistive device use , and fall history . the demographic , health status , assistive device use , and fall history data of the 447 older veterans are shown in table 1table 1.demographic characteristics and health status for all subjects , non - fallers and fallersvariablestotal ( n=447)non - fallers ( n= 336)fallers ( n=111 ) meansd(range)number(%)meansd(range)number(%)meansd(range)number(%)demographic characteristics age ( y)82.24.7 ( 6095)81.84.8 ( 6094)83.64.0 ( 7495 ) gender ( male)417 ( 93.3)315 ( 93.8)102 ( 91.9)physical measurementssbp ( mmhg)136.718.2 ( 90210)137.918.8 ( 90210)131.016.5 ( 83170)dbp ( mmhg)71.59.9 ( 48100)72.59.8 ( 48100)67.28.3 ( 5090)bmi ( kg / m)<18.510 ( 2.2)9 ( 2.7)1 ( 0.9)18.525216 ( 48.3)165 ( 49.1)51 ( 45.9)>25221 ( 49.4)162 ( 48.2)59 ( 53.2)visual acuity0.80.3 ( 0.01.5)0.80.3 ( 0.01.5)0.72.3 ( 0.01.2)hearing acuitynormal362 ( 81.0)281 ( 83.6)81 ( 73.0)slight impairment23 ( 5.1)15 ( 4.5)8 ( 7.2)modest impairment28 ( 6.3)22 ( 6.5)6 ( 5.4)severe impairment34 ( 7.6)18 ( 5.4)16 ( 14.4 ) frsts test score190 ( 20.1)77 ( 22.9)13 ( 11.7)2160 ( 35.8)134 ( 39.9)26 ( 23.4)380 ( 17.9)63 ( 18.8)17 ( 15.3)479 ( 17.7)45 ( 13.4)34 ( 30.6)538 ( 8.5)17 ( 5.1)21 ( 18.9)chronic diseases dementia29 ( 6.5)14 ( 4.2)15 ( 13.5)parkinsonism26 ( 5.8)14 ( 4.2)12 ( 10.8)stroke44 ( 9.8)14 ( 4.2)30 ( 27.0)cervical diseases103 ( 23.0)73 ( 21.7)30 ( 27.0)lumbar diseases97 ( 21.7)65 ( 19.3)32 ( 28.8)knee diseases96 ( 21.5)67 ( 19.9)29 ( 26.1)coronary disease312 ( 69.8)224 ( 66.7)88 ( 79.3)sinus bradycardia70 ( 15.7)52 ( 15.5)18 ( 16.2)atrial fibrillation48 ( 10.7)36 ( 10.7)12 ( 10.8)atrioventricular block79 ( 17.7)58 ( 17.3)21 ( 18.9)chf30 ( 6.7)23 ( 6.8)7 ( 6.3)hypertension276 ( 61.7)207 ( 61.6)69 ( 62.2)diabetes mellitus123 ( 27.9)88 ( 26.2)35 ( 31.5)copd19 ( 4.3)15 ( 4.5)4 ( 3.6)sas11 ( 2.5)7 ( 2.1)4 ( 3.6)crf40 ( 8.9)25 ( 7.4)15 ( 13.5)hyperlipemia119 ( 26.6)86 ( 25.6)33 ( 29.7)hyperuricemia106 ( 23.7)85 ( 25.3)21 ( 18.9)cancer74 ( 16.6)53 ( 15.8)21 ( 18.9)anemia22 ( 4.9)14 ( 4.2)8 ( 7.2)multiple diseases162 ( 13.9)50 ( 14.9)12 ( 10.8)24282 ( 63.1)227 ( 67.6)55 ( 49.5)5103 ( 23.0)59 ( 17.6)44 ( 39.6)medication use sedatives 42 ( 9.4)22 ( 6.5)20 ( 18.0)antihypertensives 209 ( 46.8)149 ( 44.3)60 ( 54.1)antiarrhythmics 67 ( 15.0)51 ( 15.2)16 ( 14.4)antidepressants6 ( 1.3)5 ( 1.5)1 ( 0.9)oral hypoglycemics 79 ( 17.7)53 ( 15.8)26 ( 23.4)multiple medications5279 ( 62.4)226 ( 67.3)53 ( 47.7)69135 ( 30.2)93 ( 27.7)42 ( 37.8)1033 ( 7.4)17 ( 5.1)16 ( 14.4)chemical measurementshemoglobin ( g / l)132.214.2 ( 76194)133.014.2 ( 76194)130.013.9 ( 95164)albumin ( g / l)42.82.6 ( 16.350.0)42.92.7 ( 16.350.0)42.52.3 ( 36.048.0)total protein ( g / l)68.94.4 ( 55.-80.0)69.04.4 ( 55.080.0)68.84.6 ( 59.679.0)blood urea nitrogen ( mmol / l)6.42.2 ( 2.625.4)6.42.2 ( 2.925.4)6.42.3 ( 2.617.3)urinary creatine ( mmol / l)85.528.9 ( 33382)85.228.4 ( 33382)86.230.4 ( 39252)uric acid ( mmol / l)347.380.7 ( 154675)347.282.1 ( 158675)347.876.6 ( 154670)fasting blood sugar ( mmol / l)5.41.3 ( 3.215.1)5.41.2 ( 3.215.1)5.51.5 ( 4.013.1)total cholesterol ( mmol / l)4.50.8 ( 1.17.5)4.50.8 ( 1.17.6)4.50.8 ( 2.26.9)triglyceride ( mmol / l)1.40.9 ( 0.39.5)1.50.9 ( 0.39.5)1.40.8 ( 0.45.2)ldl ( mmol / l)2.80.7 ( 1.15.4)2.80.7 ( 1.15.4)2.70.7 ( 1.14.7)hdl ( mmol / l)1.20.4 ( 0.74.3)1.20.4 ( 0.74.3)1.20.3 ( 0.72.4)othersassistive device use122 ( 27.3)74 ( 22.0)48 ( 43.2)falls history ( the past 6 months)67 ( 15.0)30 ( 8.9)37 ( 33.3)sbp : systolic blood pressure ; dbp : diastolic blood pressure ; frsts test : five repetition sit - to - stand test ; chf : chronic heart failure ; copd : chronic obstructive pulmonary disease ; sas : sleep apnea syndrome ; crf : chronic renal failure ; ldl : low density lipoproteins ; hdl : high density lipoproteins . there were 429 subjects over 75 years old , which accounted for 96% of the subjects in the final samples ( table 2table 2.comparison of the sample distribution and the incidence of falls and fallers between hong kong and the present studyage ( y)sample distributionfall incidencefaller incidencehong kong % ( n)present study % ( n)hong kong ( /1,000 py)present study ( /1,000 py)hong kong ( /1,000 py)present study ( /1,000 py)6064/1.1 ( 5)/0/0656933.9 ( 514)1.1 ( 5)16701320707429.1 ( 441)1.8 ( 8)248125173125757919.2 ( 291)14.5 ( 65)363200254154808412.5 ( 189)55.3 ( 247)387328285239855.4 ( 82)26.2 ( 117)4684913513578589/20.8 ( 93)/510/34790/5.4 ( 24)/421/379all100 ( 1517)100 ( 447)270342198249standardized incidence 19641964300187219146the incidence of falls was calculated as the number of falls divided per 1,000 person - years . , 86 fell once , 25 older veterans fell two or more times , and a total of 152 falls occurred . in model 1 of the cox regression analyses , age was correlated with falls ( table 3table 3.cox regression analyses of clinical independent risk factors for fallsvariables model 1model 2model 3rr95% cirr95% cirr95% cidemographic characteristicsage1.081.041.131.030.981.08sex1.370.702.721.200.582.47physical measurementssbp ( mmhg)0.990.981.000.990.981.00dbp ( mmhg)0.980.961.000.980.951.08visual acuity0.360.180.710.470.230.96hearing acuity0.830.700.990.850.711.02bmi ( kg / m)<18.5 vs.18.5253.940.5229.64>25.0 vs. 18.5251.160.791.70chronic diseasesdementia2.201.253.871.550.832.90parkinsonism 2.211.214.031.410.722.75stroke4.392.876.702.431.513.93cervical diseases1.290.851.97lumbar diseases1.601.062.411.731.122.70knee diseases1.300.851.99coronary diseases1.550.972.49sinus bradycardia1.040.631.72atrial fibrillation1.020.561.86atrioventricular block1.010.621.63chf0.750.311.64hypertension0.940.611.38diabetes mellitus1.220.821.83copd0.700.261.90sas1.990.735.43crf1.440.822.52hyperlipemia1.160.771.75hyperuricemia0.770.481.24cancer1.160.721.86anemia1.320.642.74medication usesedatives2.191.343.591.801.053.08antihypertensives 1.270.871.85antiarrhythmics 0.900.491.66antidepressants0.920.541.56oral hypoglycemics 1.490.962.31chemical measurements hemoglobin ( g / l)0.990.981.01albumin ( g / l)0.970.911.04total protein ( g / l)0.990.951.03blood urea nitrogen ( g / l)0.980.891.07urinary creatinine ( mmol / l)1.000.991.01uric acid ( mmol / l)1.000.991.00fasting blood sugar ( mmol / l)1.080.951.22total cholesterol ( mmol / l)1.000.801.24triglyceride ( mmol / l)1.000.801.24ldl ( mmol / l)0.970.751.26hdl ( mmol / l)1.000.591.68assistive device use0.510.340.771.280.822.01history of falls ( past 6 months)3.282.194.912.771.784.32rr : relative risk ; ci : confidence interval ; sbp : systolic blood pressure ; dbp : diastolic blood pressure ; frsts test : five - repetition sit - to - stand test ; chf : chronic heart failure ; copd : chronic obstructive pulmonary disease ; sas : sleep apnea syndrome ; crf : chronic renal failure . in model 2 , after adjustment for demographics , falls were associated with sbp , dbp , visual acuity , hearing acuity , dementia , parkinsonism , stroke , lumbar diseases , sedatives , assistive device use , and fall history in the past 6 months , respectively ( table 3 ) . in model 3 , after adjustment for demographics , the significant clinical factors associated with falls were only visual acuity ( rr=0.47 , p=0.039 ) , stroke ( rr=2.43 , p=0.000 ) , lumbar diseases ( rr=1.73 , p=0.015 ) , sedatives ( rr=1.80 , p=0.032 ) , and fall history in the past 6 months ( rr=2.77 , p=0.000 ) . after adjustment for demographics , assistive device use , and fall history by cox regression analysis , multiple diseases ( rr=1.53 , p=0.013 ) and multiple medications ( rr=1.34 , p=0.041 ) were the independent risk factors for falls . * p<0.05 ; * * p<0.01 ; * * * p<0.001 this was the first prospective cohort study of falls among older chinese veterans in military communities . the clinical factors associated with falls among older chinese veterans in military communities were poor senses , stroke , lumbar diseases , taking sedatives , fall history in the past 6 months , having multiple diseases , taking multiple medications , and poor performance in the fpsts test . in our study , the incidence of falls and fallers among older chinese veterans was 342/1,000 py and 249/1,000 py , respectively . compared with the study of chu , which investigated the fall incidence among older chinese adults in the hong kong communities , adjusted for an unbalanced age distribution , the standardized incidences of falls and faller were 187/1,000 py and 146/1,000 py in the present study , while they were 300/1,000 py and 219/1,000 py in hong kong ( table 2)20 . furthermore , analysis showed that use of an assistive device was positively correlated with physical function ( , table 4table 4.relationship between assistive device use and physical function scorevariablephysical function score12345without assistive devicecount84137573611% within physical function93.3%85.6%71.3%45.6%28.9%with assistive devicecount623234327% within physical function6.7%14.4%28.8%54.4%71.1%a score of 1 was>0 s but 9 s in the five - repetition sit - to - stand test , a score of 2 was > 9 s but 12 s in the five - repetition sit - to - stand test , a score of 3 was>12 s but 15 s in the five - repetition sit - to - stand test , a score of 4 was>15 s in the five - repetition sit - to - stand test , and a score of 5 was unable to complete the five - repetition sit - to - stand test . ) a score of 1 was>0 s but 9 s in the five - repetition sit - to - stand test , a score of 2 was > 9 s but 12 s in the five - repetition sit - to - stand test , a score of 3 was>12 s but 15 s in the five - repetition sit - to - stand test , a score of 4 was>15 s in the five - repetition sit - to - stand test , and a score of 5 was unable to complete the five - repetition sit - to - stand test . the strengths of our study were as follows : first , it was the first prospective cohort study about the incidence of falls and related clinical factors for older chinese veterans in military communities . in summary , the present study revealed the incidence of falls and fallers to be 342/1,000 py and 249/1,000 py , which were lower than those in hong kong and in western countries . the clinical risk factors for falls among older chinese veterans in military communities were poor senses , stroke , lumbar diseases , taking sedatives , fall history in the past 6 months , having multiple diseases , taking multiple drugs , and poor physical function . the preventive strategies targeting the above risk factors are very significant for reducing falls in older chinese veterans .

the restoration of upright balance after a perturbation relies on highly automated and , to a large extent , stereotyped postural responses , involving a complex pattern of activation of upper and lower leg , trunk , shoulder , and neck muscles ( allum et al . 2002 ; bloem et al . 2000 ; carpenter et al . 2004 ; mcilroy and maki 1995 ; moore et al . 1988 . these responses typically occur at onset latencies of 100 ms . in response to a perturbation , both feet - in - place and stepping strategies can be used to recover balance , with the incidence of stepping responses becoming larger as the perturbation magnitude increases ( hsiao and robinovitch , 1998 ; mcilroy and maki 1993 ) . these responses are triggered and modulated on the basis of sensory information , with lower extremity and trunk proprioception as well as vestibular inputs as possible sources ( allum and honegger 1998 ; bloem et al . 2000 ; do et al . 1988 ) . although the onsets of automated postural responses occur before voluntary ( cognitive ) control comes into play and the characteristics of the responses are distinctly different from those of voluntary movements ( nashner and cordo 1981 ) , previous studies have shown that higher brain levels , presumably involving the cerebral cortex , can modulate postural responses by changing the activity of the pathways that are involved in their generation ( for review see jacobs and horak 2007 ) . in 1976 , nashner was the first to report that experience causes functional adaptations in response amplitudes following an unexpected change of support surface motion . subsequent studies have shown that response amplitudes depend on the predictability of perturbation magnitudes ( beckley et al . 1989 ) , restrictions on the balance recovery strategy ( feet - in - place vs. stepping ; burleigh and horak 1996 ; burleigh et al . 1994 ; mcilroy and maki 1993 ) , and the task of holding an object ( bateni et al . 2004 1981 ) . in general , these factors do not cause corresponding changes in onset latencies and activation sequences . on the other hand , both response amplitudes and onset latencies may be affected by advanced warning of the perturbation ( mawston et al . 2007 ; mcchesney et al . 1996 ) and increased postural anxiety ( carpenter et al . 2004 ) . hence , the literature indicates that changes in initial contexts generally affect response amplitudes , preserve activation sequences , and have limited influence on onset latencies . overall , this pattern of results suggests that the sensory information as generated by the induced perturbation to upright balance inevitably launches a postural response directed toward recovering or maintaining an upright stance , that can be scaled by cognitive set ( by changing the gain ) , but not fully suppressed . however , in all of these previous studies the final goal was the same in every condition , namely balance recovery . it has been postulated ( nashner and mccollum 1985 ) that in order to reduce the degrees of freedom , the cns composes complex postural responses from a combination of stereotyped synergies . when a synergy is used to recover balance in response to a perturbation , selective cancellation of single muscle activation within the synergy might not be possible . this raises the question of whether postural responses are also immutable when they are not necessarily helpful in the execution of a task . would it be possible to turn off these ( highly automated ) postural responses when functionally undesired , or can they be integrated into the motor output in a meaningful way ? the answer could provide important knowledge of the functional organization of the central nervous system in such conflicts . in order to obtain insight into this issue , an experiment would be needed to study postural responses to identical mechanical perturbations , but with distinct task demands . instruction - related modulation of long - latency stretch reflexes in response to identical single - joint mechanical perturbations has been extensively studied by instructing participants either to resist or not to resist the perturbation ( e.g. hammond 1956 ; rothwell et al . these studies demonstrated that response amplitudes of the stretched muscles ( except for the flexor pollicis longus ) were heavily modulated as a result of the instruction , characterized by divergence between instructions ( almost ) immediately after response onset . whether such instruction - related modulation also applies to automated postural responses is not known . in the present study , the effects of instruction on automated postural responses in neck , trunk , shoulder , and leg muscles were investigated when people were either instructed to recover balance after being released from an inclined standing posture , or not to recover at all and fall onto a safety mattress in the most comfortable way . it was hypothesized that instruction would have a profound influence on the whole - body postural response . two possible scenarios were anticipated . in the first scenario , the instruction of not recovering balance would result in completely different muscle onset latencies , activation sequences , and amplitudes , indicative of a separate motor program ( i.e. a set of muscle commands that are structured before a movement sequence begins ) to prepare for a safe landing and , consequently , a cancellation of the automated postural response . in the second scenario , the instruction would differentially affect response amplitudes , with no or only limited changes in onset latencies or activation patterns . this pattern of results would imply that the automated postural response could not be suppressed , but could be tailored by differential ( feedback and/or feedforward ) gain settings in order to meet specific task constraints or demands . a total of 10 healthy young adults [ 3 women , 7 men , mean age 28.3 4.3 years ( range 2237 ) , height 1.74 0.13 m , weight 68 13 kg ] participated in this study . they all provided written informed consent to participate , and the study was approved by the simon fraser university office of research ethics . the participants stood barefoot on a wooden block ( length width height : 60 38 30 cm ) located flush with a gymnasium mattress ( length width height : 480 240 30 cm ) . a tether was attached at one end to an electromagnetic brake ( warner electric model pb500 , south beloit , il ) and at the other end to a chest harness worn by the participant ( fig . the participants placed their feet at a fixed position on the wooden block and the length of the tether was adjusted such that it supported the participant in a 15 backward or leftward - inclined position ( by means of visual comparison of the lean angle with a reference line ) . for backward trials , the tether was attached to the front of the harness , whereas for leftward trials , it was attached to the right side of the harness . postural perturbations were induced by sudden release of the tether ( 90% decay time in tether force = 15 ms ) . b raw data from a typical backward balance recovery trial ( dark gray area and black , dashed lines ) and a fall trial ( light grey area and gray , solid lines ) , showing left sternocleidomastoid emg ( scl ) , anterior deltoid emg ( dal ) , rectus femoris emg ( rfl ) , right tibialis anterior ( tar ) , lateral movement of the left elbow marker , and upward movement of the right foot marker . tether release is at time = 0 ms a schematic diagram of the experimental setup ( backward perturbation position illustrated ) . b raw data from a typical backward balance recovery trial ( dark gray area and black , dashed lines ) and a fall trial ( light grey area and gray , solid lines ) , showing left sternocleidomastoid emg ( scl ) , anterior deltoid emg ( dal ) , rectus femoris emg ( rfl ) , right tibialis anterior ( tar ) , lateral movement of the left elbow marker , and upward movement of the right foot marker . tether release is at time = 0 ms prior to each trial , the participant was instructed to either recover balance [ balance recovery ( br ) trials ] or to avoid balance recovery attempts and to focus on landing safely [ fall ( f ) trials ] after the perturbation . the participants were instructed to lean into the tether and maintain their hips and knees extended , arms flexed , and hands resting on their chest . before starting the trial , the experimenter confirmed by visual inspection that the head , trunk , and legs were aligned . the participants were verbally notified of this event . after the start of the trial , the tether was released at a random time interval , varying between 1 and 7 s. release from the 15 lean angle exceeded the maximum value previously observed where young adults can recover using feet - in - place strategies ( hsiao and robinovitch 2001 ) . hence , in order to recover , it was necessary for participants to take at least one step . after release , they were also free to move their arms . in the f trials , participants were not restricted in their use of safe landing responses ( such as breaking the fall with the outstretched hands ) , except that they were not allowed to rotate around the longitudinal axis . each participant first performed the series of backward perturbations , followed by the series of leftward perturbations . muscle activities in the left and right sternocleidomastoid ( scl and scr ) , anterior deltoid ( dal and dar ) , posterior deltoid ( dpl and dpr ) , rectus abdominis ( abl and abr ) , rectus femoris ( rfl and rfr ) , and tibialis anterior ( tal and tar ) were measured through surface electromyography ( emg ; bagnoli , delsys inc . , boston , ma ) . we considered collecting data from other muscles likely involved in postural responses ( e.g. hip abductors and calf muscles ) as well . however , in order to prevent harm to both the participants and equipment , we decided not to place electrodes on potential impact sites . the emg signals were amplified , band - pass filtered ( 20450 hz ) and sampled at 960 hz . in addition , the 3d positions of skin surface markers were recorded at 240 hz with an 8-camera motion analysis system ( motion analysis inc . , santa rosa , ca ) . markers were located at the top of the head , sacrum ( l5/s1 junction ) , and bilaterally at the acromion process , lateral epicondyle of the humerus , distal end of the radius , anterior - superior - iliac spine , greater trochanter , lateral epicondyle of the femur , lateral malleolus , and fifth metatarsal . furthermore , the tether force was recorded at 960 hz from a miniature load cell ( sensotec , model 31 ) . figure 1b shows an example of emg and kinematic data ( 0200 ms after tether release ) for backward br and f trials . emg signals were full - wave rectified and low - pass filtered at 25 hz ( zero - lag , second order butterworth filter ) . for each muscle , the mean baseline and standard deviation ( sd ) in emg activity over 1 s prior to tether release were calculated . the muscle onset latency was determined by a combination of computer algorithm and visual inspection ( to ensure data quality ) on a single trial basis . onset latency for a specific muscle was defined as the time between tether release and the instant the emg amplitude for that muscle was greater than 4sd s above its baseline value , for at least 30 ms . in addition , for each trial average emg amplitudes were determined for eight bins following tether release ( one bin from 0 to 60 ms , and seven consecutive 20-ms bins from 60 to 200 ms ) . we only analyzed the first 200 ms after tether release , because we were particularly interested in whether people would be able to exert cognitive control over the initial part of the postural response , which is presumably highly automated and not generated at the level of the cerebral cortex . as it is well known that reactions at 150200 ms can certainly be cognitively controlled , emg signals after 200 ms were no longer of interest with respect to our primary research question . we also calculated several kinematic variables to determine whether they would correspond to potential differences in emg parameters . the instant of step initiation was determined as the first sample after release in which the ankle marker started moving in upward direction . the instant of initial arm abduction was determined as the first lateral movement of the elbow marker . furthermore , we quantified the initial arm movements by calculating the difference between shoulder flexion and abduction angles at tether release and at 200 ms post - release . paired t tests were used to identify differences in the instants of tether release after the start of the trial and onsets of arm abduction movements between br and f trials . analyses of variance ( anova ) for repeated measures were used with post hoc paired t tests ( with alpha levels adjusted to 0.01 to correct for repeated testing ) to detect differences in baseline activity levels and response latencies between instructions . within - subjects factors were instruction ( br and f ) , and muscle ( 12 muscles ) . anovas for repeated measures were also used for statistical analysis of response amplitudes for each muscle . within - subjects factors were instruction ( br and f ) , and bins of emg activity ( 8 bins following release ) . when significant instruction bin interaction effects were present , post hoc reverse helmert contrasts were used to determine the first bin in which the amplitudes started to deviate between instructions . in addition , anovas for repeated measures were used with post hoc paired t tests ( with alpha levels adjusted to 0.01 to correct for repeated testing ) to detect the differences in maximum amplitudes and the time of this maximum amplitude between instructions , with instruction ( br and f ) and muscle ( 12 muscles ) as within - subjects factors . finally , paired t tests were used to determine whether changes in arm positions were differentially influenced by instruction . a total of 10 healthy young adults [ 3 women , 7 men , mean age 28.3 4.3 years ( range 2237 ) , height 1.74 0.13 m , weight 68 13 kg ] participated in this study . they all provided written informed consent to participate , and the study was approved by the simon fraser university office of research ethics . the participants stood barefoot on a wooden block ( length width height : 60 38 30 cm ) located flush with a gymnasium mattress ( length width height : 480 240 30 cm ) . a tether was attached at one end to an electromagnetic brake ( warner electric model pb500 , south beloit , il ) and at the other end to a chest harness worn by the participant ( fig . the participants placed their feet at a fixed position on the wooden block and the length of the tether was adjusted such that it supported the participant in a 15 backward or leftward - inclined position ( by means of visual comparison of the lean angle with a reference line ) . for backward trials , the tether was attached to the front of the harness , whereas for leftward trials , it was attached to the right side of the harness . postural perturbations were induced by sudden release of the tether ( 90% decay time in tether force = 15 ms ) . b raw data from a typical backward balance recovery trial ( dark gray area and black , dashed lines ) and a fall trial ( light grey area and gray , solid lines ) , showing left sternocleidomastoid emg ( scl ) , anterior deltoid emg ( dal ) , rectus femoris emg ( rfl ) , right tibialis anterior ( tar ) , lateral movement of the left elbow marker , and upward movement of the right foot marker . tether release is at time = 0 ms a schematic diagram of the experimental setup ( backward perturbation position illustrated ) . b raw data from a typical backward balance recovery trial ( dark gray area and black , dashed lines ) and a fall trial ( light grey area and gray , solid lines ) , showing left sternocleidomastoid emg ( scl ) , anterior deltoid emg ( dal ) , rectus femoris emg ( rfl ) , right tibialis anterior ( tar ) , lateral movement of the left elbow marker , and upward movement of the right foot marker . tether release is at time = 0 ms prior to each trial , the participant was instructed to either recover balance [ balance recovery ( br ) trials ] or to avoid balance recovery attempts and to focus on landing safely [ fall ( f ) trials ] after the perturbation . the participants were instructed to lean into the tether and maintain their hips and knees extended , arms flexed , and hands resting on their chest . before starting the trial , the experimenter confirmed by visual inspection that the head , trunk , and legs were aligned . after the participants indicated the participants were verbally notified of this event . after the start of the trial , the tether was released at a random time interval , varying between 1 and 7 s. release from the 15 lean angle exceeded the maximum value previously observed where young adults can recover using feet - in - place strategies ( hsiao and robinovitch 2001 ) . hence , in order to recover , it was necessary for participants to take at least one step . after release , they were also free to move their arms . in the f trials , participants were not restricted in their use of safe landing responses ( such as breaking the fall with the outstretched hands ) , except that they were not allowed to rotate around the longitudinal axis . each participant first performed the series of backward perturbations , followed by the series of leftward perturbations . muscle activities in the left and right sternocleidomastoid ( scl and scr ) , anterior deltoid ( dal and dar ) , posterior deltoid ( dpl and dpr ) , rectus abdominis ( abl and abr ) , rectus femoris ( rfl and rfr ) , and tibialis anterior ( tal and tar ) were measured through surface electromyography ( emg ; bagnoli , delsys inc . , we considered collecting data from other muscles likely involved in postural responses ( e.g. hip abductors and calf muscles ) as well . however , in order to prevent harm to both the participants and equipment , we decided not to place electrodes on potential impact sites . the emg signals were amplified , band - pass filtered ( 20450 hz ) and sampled at 960 hz . in addition , the 3d positions of skin surface markers were recorded at 240 hz with an 8-camera motion analysis system ( motion analysis inc . , santa rosa , ca ) . markers were located at the top of the head , sacrum ( l5/s1 junction ) , and bilaterally at the acromion process , lateral epicondyle of the humerus , distal end of the radius , anterior - superior - iliac spine , greater trochanter , lateral epicondyle of the femur , lateral malleolus , and fifth metatarsal . furthermore , the tether force was recorded at 960 hz from a miniature load cell ( sensotec , model 31 ) . figure 1b shows an example of emg and kinematic data ( 0200 ms after tether release ) for backward br and f trials . emg signals were full - wave rectified and low - pass filtered at 25 hz ( zero - lag , second order butterworth filter ) . for each muscle , the mean baseline and standard deviation ( sd ) in emg activity over 1 s prior to tether release were calculated . the muscle onset latency was determined by a combination of computer algorithm and visual inspection ( to ensure data quality ) on a single trial basis . onset latency for a specific muscle was defined as the time between tether release and the instant the emg amplitude for that muscle was greater than 4sd s above its baseline value , for at least 30 ms . in addition , for each trial average emg amplitudes were determined for eight bins following tether release ( one bin from 0 to 60 ms , and seven consecutive 20-ms bins from 60 to 200 ms ) . we only analyzed the first 200 ms after tether release , because we were particularly interested in whether people would be able to exert cognitive control over the initial part of the postural response , which is presumably highly automated and not generated at the level of the cerebral cortex . as it is well known that reactions at 150200 ms can certainly be cognitively controlled , emg signals after 200 ms were no longer of interest with respect to our primary research question . we also calculated several kinematic variables to determine whether they would correspond to potential differences in emg parameters . the instant of step initiation was determined as the first sample after release in which the ankle marker started moving in upward direction . the instant of initial arm abduction was determined as the first lateral movement of the elbow marker . furthermore , we quantified the initial arm movements by calculating the difference between shoulder flexion and abduction angles at tether release and at 200 ms post - release . paired t tests were used to identify differences in the instants of tether release after the start of the trial and onsets of arm abduction movements between br and f trials . analyses of variance ( anova ) for repeated measures were used with post hoc paired t tests ( with alpha levels adjusted to 0.01 to correct for repeated testing ) to detect differences in baseline activity levels and response latencies between instructions . within - subjects factors were instruction ( br and f ) , and muscle ( 12 muscles ) . anovas for repeated measures were also used for statistical analysis of response amplitudes for each muscle . within - subjects factors were instruction ( br and f ) , and bins of emg activity ( 8 bins following release ) . when significant instruction bin interaction effects were present , post hoc reverse helmert contrasts were used to determine the first bin in which the amplitudes started to deviate between instructions . in addition , anovas for repeated measures were used with post hoc paired t tests ( with alpha levels adjusted to 0.01 to correct for repeated testing ) to detect the differences in maximum amplitudes and the time of this maximum amplitude between instructions , with instruction ( br and f ) and muscle ( 12 muscles ) as within - subjects factors . finally , paired t tests were used to determine whether changes in arm positions were differentially influenced by instruction . the average lean angles prior to release were indeed close to the intended 15 , as revealed by analysis of kinematic data from bilateral malleolus and acromion markers . average lean angles were 15.4 0.6 ( se ) for backward and 16.9 0.6 for leftward trials . there were no significant differences between lean angles in br and f trials in both backward and leftward perturbations ( p = 0.087 and p = 0.427 , respectively ) . analysis also did not yield significant differences in the instants of tether release relative to the start of the trial ( backward : 4.1 0.24 s vs. 4.1 0.27 s for br and f trials , respectively , p = 0.955 ; leftward 4.3 0.19 s vs. 4.0 0.23 s , p = 0.080 ) . of the total 120 br trials collected in this study , furthermore , in only 3 out of 120 f trials a recovery attempt ( step initiation ) could be observed . in the backward br trials , 9 participants stepped back with the right leg first , whereas 1 participant stepped with the left leg first . for this participant , in the statistical analysis of backward perturbations the muscles on the right side the stepping foot was the dominant foot ( i.e. the foot they use to kick a ball ) , whereas two participants used their non - dominant foot to step . foot lift in the backward trials occurred at , on average , 180 5 ( se ) ms after release . in backward f trials , most participants landed with near - simultaneous impact to the buttocks and the hands / forearms , similar to the backward landing configuration reported by hsiao and robinovitch ( 1998 ) . in leftward br trials , all participants recovered balance by stepping with the left leg first . left foot lift occurred at 237 6 ms after tether release . in leftward f trials , participants usually landed on the left knee , left hip , and on both left and right hands / forearms . in response to the perturbation , arm abduction movements were commonly observed in both br and f trials , but were earlier and more pronounced in br than in f trials . in backward trials , bilateral arm abduction movements occurred at on average 154 5 ms after release in br trials and at 172 8 ms in f trials ( p = 0.018 ) . in leftward trials , clear abduction movements movement onsets in br trials occurred at an average 159 4 ms after release and in f trials at 179 8 ms ( p = 0.009 ) . onsets of emg activity in response to the perturbation could generally be detected in all the muscles within 200 ms . in backward br trials , neck , trunk and leg muscles showed similar onsets ( 7080 ms after tether release ) , followed by dpl and dpr at 8090 ms and dal and dar at 90100 ms , on average ( fig . 2 ) . in the backward f trials , the overall mean onset latencies of the 12 muscles measured were delayed by 4.1 ms ( se 2.4 ms ) compared to the br trials [ f(1,8 ) = 5.601 , p = 0.045 , fig . 2 ] . at the level of the individual muscles , onsets were not significantly different between the instructions ( all p values > 0.045 ) . as can be seen from fig . 2 , the overall activation sequence remained similar between instructions . 2average onset latencies ( se ) in response to backward and leftward perturbations for left and right sternocleidomastoid ( scl and scr ) , anterior deltoid ( dal and dar ) , posterior deltoid ( dpl and dpr ) , rectus abdominis ( abl and abr ) , rectus femoris ( rfl and rfr ) , and tibialis anterior ( tal and tar ) . data from balance recovery trials are shown as black diamonds , and data from fall trials are shown as gray squares . * p < 0.01 average onset latencies ( se ) in response to backward and leftward perturbations for left and right sternocleidomastoid ( scl and scr ) , anterior deltoid ( dal and dar ) , posterior deltoid ( dpl and dpr ) , rectus abdominis ( abl and abr ) , rectus femoris ( rfl and rfr ) , and tibialis anterior ( tal and tar ) . data from balance recovery trials are shown as black diamonds , and data from fall trials are shown as gray squares . * p < 0.01 with respect to muscle activation sequences in leftward br trials , scl and scr were activated first at 7580 ms after tether release , followed by abl , abr , tal , tar , dpl , and dpr at 8090 ms , dal and dar at 90100 ms , and rfl and rfr at 100110 ms . overall , mean onset latencies of the 12 muscles were delayed by 9.3 ms ( se 4.8 ms ) in the f trials compared to the br trials [ f(1,8 ) = 20.373 , p = 0.002 , fig . 2 ) . the activation sequence was again generally preserved in the leftward f trials , with the exception of scr . the onset of this individual muscle was substantially delayed in f trials compared to br trials ( mean difference se , 18.6 5.1 ms , p = 0.007 ) . a less pronounced but significant delay was also observed in dar ( mean difference se , 9.7 2.8 ms , p = 0.006 ) . with respect to emg amplitudes , there were no significant differences in baseline activity levels between br and f trials [ f(1,9 ) = 2.149 , p = 0.177 ] . hence , the analysis of response amplitudes was not compromised by instruction - related baseline differences . emg amplitudes after tether release were generally higher in br than in f trials and these instruction - related differences could often be detected shortly after onset ( mostly within 40 ms ) . for backward trials , significant instruction bin interactions were found for all muscles [ values for f(7,63 ) ranging from 2.964 to 19.863 , all p values < 0.010 ] , except abl , abr , and rfr [ values for f(7,63 ) ranging from 0.433 to 1.815 , p values > 0.100 ] . post hoc contrasts revealed that emg amplitudes started to increase more steeply in br than in f trials at 6080 ms after release for scr , at 80100 ms after release for scl and tal , followed by dal , rfl , and tar at 100120 ms , and dar at 120140 ms [ values for f(1,9 ) ranging from 5.946 to 18.575 , p values between 0.002 and 0.037 ] ( fig . 3emg amplitudes of balance recovery ( br ) trials minus the amplitudes in fall ( f ) trials in response to backward and leftward perturbations . average differences ( se ) are shown as a function of time after tether release for bilateral sternocleidomastoid ( sc ) , anterior deltoid ( da ) , posterior deltoid ( dp ) , rectus abdominis ( ab ) , rectus femoris ( rf ) , and tibialis anterior ( ta ) . muscles on the left side of the body are shown as black dashed lines , muscles on the right are shown as gray solid lines . the arrows indicate at which instant emg amplitudes started to deviate significantly between br and f trials emg amplitudes of balance recovery ( br ) trials minus the amplitudes in fall ( f ) trials in response to backward and leftward perturbations . average differences ( se ) are shown as a function of time after tether release for bilateral sternocleidomastoid ( sc ) , anterior deltoid ( da ) , posterior deltoid ( dp ) , rectus abdominis ( ab ) , rectus femoris ( rf ) , and tibialis anterior ( ta ) . muscles on the left side of the body are shown as black dashed lines , muscles on the right are shown as gray solid lines . the arrows indicate at which instant emg amplitudes started to deviate significantly between br and f trials for leftward trials , analysis yielded significant instruction bin interactions for all the muscles [ values for f(7,63 ) ranging from 2.424 to 18.294 , all p values < 0.030 ] , except rfl and tar [ values for f(7,63 ) of 1.317 and 1.198 , p values of 0.257 and 0.317 , respectively ] . higher emg amplitudes were observed in br than in f trials , starting at 80100 ms for scl , scr , dal , and abr , followed by dar and dpr at 100120 ms , abl and rfr at 120140 ms , dpl at 140160 ms , and tal at 180200 ms [ values for f(1,9 ) ranging from 5.157 to 21.745 , p values between 0.001 and 0.049 ] ( fig . 3 ) . in both backward and leftward perturbations , maximum emg values ( table 1 ) were also significantly larger in br than in f trials [ f(1,9 ) = 27.430 , p = 0.001 and f(1,9 ) = 33.627 , p the time of maximum emg was not significantly different between backward br and f trials [ f(1,9 ) = 4.198 , p = 0.071 ] . in leftward trials , analysis yielded a main effect of instruction on the time at which maximum emg values were reached [ ( f(1,9 ) = 8.417 , p = 0.018 ] . post hoc paired t tests revealed that rfr reached peak activity significantly earlier in f than br trials ( p = 0.003 ) . continued high rfr activity levels in br trials probably indicate preparation for the stepping movement of the left leg . activity levels in the absence of stepping responses ( f trials ) started to decrease at 150 ms . table 1mean se of maximum emg amplitude and the instant after release at which the maximum value was reached for balance recovery ( br ) and fall ( f ) trialsbackwardleftwardbrfbrfmaximum emg ( mv)time of max . ( ms)scl217 21 * 149 5124 21138 5136 17 * 141 349 10140 11scr196 25 * 155 2124 28138 4112 23 * 151 832 9139 10dal249 48 * 160 395 14144 7286 53 * 163 4 66 20144 5dar239 50 * 152 551 9148 6106 21 148 570 32139 4108 45144 456 26144 3dpr208 50141 5120 29137 6211 51 * 147 371 21140 11abl124 42128 8139 40138 869 22 140 8 23 7113 6abr101 25131 687 15136 857 17 120 529 9112 7rfl179 36 148 7119 20128 629 5144 627 7152 4rfr93 19121 475 15134 5202 63 * 173 3 * 24 7156 4tal271 49 * 162 6180 44140 8270 48 * 167 7 179 37140 6tar322 51 * 152 5167 35131 8147 30139 4122 26139 5 * p < 0.01 mean se of maximum emg amplitude and the instant after release at which the maximum value was reached for balance recovery ( br ) and fall ( f ) trials hence , many of the muscles recorded showed significantly higher emg amplitudes in br than in f trials , with the earliest and most consistent differences between instructions being present in sternocleidomastoid . however , instruction - related differences in rectus abdominis were only present in leftward perturbations . furthermore , in both perturbation directions , emg amplitude in rectus femoris of the stance limb was greater in br than f trials . finally , instruction had differential effects on shoulder muscle emg amplitudes for the two perturbation directions . analysis of shoulder kinematics revealed that the differential effects of instruction on emg amplitudes also resulted in corresponding changes in shoulder abduction and flexion angles within 200 ms after tether release ( fig . 4 ) . in backward trials , the deltoids ( shoulder abductor muscles ) showed higher activity in br than f trials . this corresponded to increases in shoulder abduction angles in br trials that were more than twice as large compared to f trials ( left : 10.7 vs. 4.7 , se of the difference 2.1 , p = 0.018 ; right : 8.0 vs. 3.5 , se of the difference 1.5 , p = 0.014 , fig . 4average changes in flexion and abduction angles ( se ) of the left ( l ) and right ( r ) arm between tether release and 200 ms post - release for balance recovery ( diamonds ) and fall trials ( squares ) . * p < 0.05 average changes in flexion and abduction angles ( se ) of the left ( l ) and right ( r ) arm between tether release and 200 ms post - release for balance recovery ( diamonds ) and fall trials ( squares ) . * p < 0.05 in leftward trials , instruction - related differences , with larger amplitudes in br than f trials , were most pronounced in dpr ( shoulder abductor and extensor ) and dal ( shoulder abductor and flexor ) . these differences were observed in conjunction with larger shoulder extension movements of the right arm in br trials ( 8.9 vs. 4.2 , se of the difference 1.4 , p = 0.008 ) , as well as larger abduction movements of both the left and right arm ( left : 9.5 vs. 4.3 , se of the difference 1.0 , p = 0.001 ; right : 1.7 vs. 0.7 , se of the difference 0.7 , p = 0.007 , fig . was slightly adducted in f trials , as shown by the average right arm abduction angle being left of the zero - abduction line in fig . the aim of the present study was to investigate the influence of instruction on automated postural responses . participants were instructed to either recover balance or simply fall onto a gymnasium mat following a sudden postural perturbation . at the behavioral level , participants were highly successful in following these instructions , consistently exhibiting stepping movements for balance recovery in br trials , and suppressing stepping in the f trials . yet emg recordings revealed similar postural responses with onset latencies between 70130 ms in both br and f trials . these onset latencies are in line with those previously reported for forward tether release balance perturbations ( do et al . 1988 ; thelen et al . 2000 ) . the presently observed onset latencies are also in the same order of magnitude as the medium - latency responses ( 80120 ms ) reported for support surface perturbations ( allum et al . 2004 ) , despite the different nature of the perturbations . only limited instruction - related changes in onset latencies were observed , with slightly delayed responses in f trials , in combination with generally preserved activation sequences . in contrast , very pronounced and early differences were observed between br and f trials in response amplitudes , which were generally much higher in br than in f trials , but with clear differentiation between muscles and perturbation directions . hence , our results support the hypothesis that instruction influences the early - stage response to a postural perturbation by adjusting multiple gain settings , resulting in targeted scaling across muscles of response amplitudes . first , it should be pointed out that the higher response amplitudes in br compared to f trials could indeed be interpreted as an effect of instruction . methodological aspects , such as the analysis procedure , can not explain these amplitude differences . instruction had no effect on pre - release emg activity , hence the analysis of response amplitudes was not compromised by instruction - related baseline differences . furthermore , instruction - related differences in muscle response amplitudes could not be explained by a general time shift of the response , as maximum amplitudes were larger in br than in f trials and in most muscles , occurred at a similar time after onset of the perturbation . it may be argued that modulation of response amplitudes could arise from differences in fear of falling between conditions . carpenter and co - workers ( 2004 ) showed that increased levels of postural anxiety resulted in larger response amplitudes after a support surface rotation , in combination with shorter onset latencies in deltoid muscles . in the present study , fear of falling might be expected to be most pronounced in the f trials . in these trials , however , response amplitudes were reduced and anterior deltoid latencies were delayed . hence , it seems unlikely that the differences in response amplitudes can be explained by unequal levels of fear of falling between the conditions . this may reflect that , in our experiments , the anxiety associated with the task of recovering balance following sudden release of the tether ( although different in nature ) may have matched that associated with the task of falling onto the mat , which may of course not be the case during a real - life fall onto a hard surface . the finding that the two different sets of instructions resulted in modulation of response amplitudes , rather than having major effects on onset latencies and activation sequences , is similar to observations from previous studies ( bateni et al . 2004 ; burleigh and horak 1996 ; burleigh et al . 1994 ; horak et al . 1989 ; mcilroy and maki 1993 ; nashner 1976 ) . in these previous studies , a postural response was always required in order to meet the general task demands of maintaining upright balance . cognitive set resulted in the modulation of response amplitudes of specific muscles within the automated postural response ( bateni et al . 2004 ; burleigh and horak 1996 ; burleigh et al . 1994 ; horak et al . 1989 ; mcilroy and maki 1993 ; nashner 1976 ) . the present study provides an important addition to the current body of knowledge by indicating that , even when the occurrence of any postural response may not be desired , it can not be fully suppressed by instruction , as responses also occurred when people were not supposed to recover , but to fall . the apparently reflex - like generation of postural responses , however , does not seem to interfere with the goal of falling , as participants clearly succeeded following the instruction . it shows that in balance recovery , we can rely on highly automated responses , but these responses can also be effectively downregulated by the central nervous system when they are not desired . the present finding that emg amplitudes were differentially modulated across muscles has been reported previously ( bateni et al . 2004 ; burleigh et al . 1994 ; mcilroy and maki 1993 ; nashner 1976 ) and it has been suggested that this represents a goal - directed interaction ( burleigh et al . the differential effects of instruction , as observed in the present study , are in line with this idea of goal - related changes in response gains . one result to support the goal - relatedness of emg modulation was the differential modulation of rectus femoris amplitudes . the higher rectus femoris activation of the stance limb in br ( compared to f ) trials can be interpreted as a preparatory action , in order to carry the body weight on one leg to allow stepping with the other . in contrast , in f trials , no such preparation for weightbearing was needed , which explains the much larger attenuation of the rectus femoris response in the stance than in the stepping leg . the asymmetrical maximum emg amplitudes of rfl and rfr in the backward f trials , however , seem to indicate that some preparatory activity for stepping may have been present in these trials as well . as previously suggested by maki et al . ( 1993 ) , participants may have been unable to suppress the initiation of the preparatory changes in limb loading , but they still may have been able to abort the stepping reaction prior to foot - lift . furthermore , the differential modulation of shoulder muscle responses in leftward trials was presumably goal - related . in leftward trials , the early deviations between instructions of both the shoulder muscle emg and the corresponding kinematic patterns seemed to be related to positioning the arms to prepare for impact in the f trials , as participants tended to utilize the active response of impacting the ground with the outstretched hands . ( 2004 ) , who observed modulation of early deltoid responses when a handrail was available for grasping when balance was perturbed . in their study , arm movements were always directed toward the handrails , irrespective of the direction of the perturbation . it is insightful to consider the potential mechanisms underlying the slight ( and probably not functionally meaningful ) delay in onset latencies observed in f trials . this delay can not be explained by differences in preparation time to the upcoming perturbation , as the instants of tether release were not significantly different between the conditions . this does not exclude the possibility , however , that the delayed responses in f trials may be related to differences in preparation alertness . the time - critical nature of the br trials requires a high degree of readiness in order to recover balance successfully after the perturbation , whereas this high time pressure is not present in f trials . this difference in time pressure may have induced higher levels of alertness in the br trials , which is known to facilitate any response ( posner and boies 1971 ) . such an effect of alertness on postural responses has been previously reported by mcchesney et al . ( 1996 ) , who found reduced onset latencies when a pre - perturbation warning signal was provided . the present observation that the slight delay in onset latencies was not accompanied by major changes in activation sequences provides additional support for such a generic alertness - related mechanism . a limitation of the present study was that a relatively small set of muscles was sampled , because emg electrodes were not positioned on potential impact sites . as such , data could not be collected from the prime movers in response to leftward perturbations ( i.e. hip abductors ) , so it can not be completely excluded that activation patterns in these perturbations may have been changed as a result of the instruction . furthermore , forward perturbations ( with ventral impact sites in f trials ) were not conducted . although it is conceivable that the presently observed instruction - based modulation of response amplitudes would also apply to forward perturbations , this remains to be determined experimentally . this may have influenced the degree to which the reaction could be suppressed , because it allowed participants to preplan their reactions , which is different than most real - life falls or imbalance episodes . it can be expected , however , that these are the most ideal circumstances for suppression of automated postural responses in the f trials , yet they still occurred in response to the perturbation . it appears that these self - protection mechanisms are so deeply wired in our system that , even under rather optimal conditions , they can not easily be de - activated . in conclusion , the present study indicates that automated postural responses occur when balance is perturbed , irrespective of whether people are instructed to recover or not to recover balance . this suggests that the triggering of postural responses is organized in a reflex - like manner , with supra - spinal control primarily contributing to adjust these responses in a functional and goal - oriented way .

the restoration of upright balance after a perturbation relies on highly automated and , to a large extent , stereotyped postural responses . although these responses occur before voluntary control comes into play , previous research has shown that they can be functionally modulated on the basis of cognitive set ( experience , advanced warning , instruction , etc . ) . it is still unknown , however , how the central nervous system deals with situations in which the postural response is not necessarily helpful in the execution of a task . in the present study , the effects of instruction on automated postural responses in neck , trunk , shoulder , and leg muscles were investigated when people were either instructed to recover balance after being released from an inclined standing posture [ balance recovery ( br ) trials ] , or not to recover at all and fall onto a safety mattress in the most comfortable way [ fall ( f ) trials ] , in both backward and leftward directions . participants were highly successful in following the instructions , consistently exhibiting stepping responses for balance recovery in br trials , and suppressing stepping in the f trials . yet emg recordings revealed similar postural responses with onset latencies between 70 and 130 ms in both br and f trials , with slightly delayed responses in f trials . in contrast , very pronounced and early differences were observed between br and f trials in response amplitudes , which were generally much higher in br than in f trials , but with clear differentiation between muscles and perturbation directions . these results indicate that a balance perturbation always elicits a postural response , irrespective of the task demands . however , when a specific balance recovery response is not desired after a perturbation , postural responses can be selectively downregulated and integrated into the motor output in a functional and goal - oriented way .

Introduction Methods Participants Procedure Data analysis Statistical analysis Results Discussion

the restoration of upright balance after a perturbation relies on highly automated and , to a large extent , stereotyped postural responses , involving a complex pattern of activation of upper and lower leg , trunk , shoulder , and neck muscles ( allum et al . in response to a perturbation , both feet - in - place and stepping strategies can be used to recover balance , with the incidence of stepping responses becoming larger as the perturbation magnitude increases ( hsiao and robinovitch , 1998 ; mcilroy and maki 1993 ) . these responses are triggered and modulated on the basis of sensory information , with lower extremity and trunk proprioception as well as vestibular inputs as possible sources ( allum and honegger 1998 ; bloem et al . although the onsets of automated postural responses occur before voluntary ( cognitive ) control comes into play and the characteristics of the responses are distinctly different from those of voluntary movements ( nashner and cordo 1981 ) , previous studies have shown that higher brain levels , presumably involving the cerebral cortex , can modulate postural responses by changing the activity of the pathways that are involved in their generation ( for review see jacobs and horak 2007 ) . overall , this pattern of results suggests that the sensory information as generated by the induced perturbation to upright balance inevitably launches a postural response directed toward recovering or maintaining an upright stance , that can be scaled by cognitive set ( by changing the gain ) , but not fully suppressed . when a synergy is used to recover balance in response to a perturbation , selective cancellation of single muscle activation within the synergy might not be possible . this raises the question of whether postural responses are also immutable when they are not necessarily helpful in the execution of a task . would it be possible to turn off these ( highly automated ) postural responses when functionally undesired , or can they be integrated into the motor output in a meaningful way ? in the present study , the effects of instruction on automated postural responses in neck , trunk , shoulder , and leg muscles were investigated when people were either instructed to recover balance after being released from an inclined standing posture , or not to recover at all and fall onto a safety mattress in the most comfortable way . in the first scenario , the instruction of not recovering balance would result in completely different muscle onset latencies , activation sequences , and amplitudes , indicative of a separate motor program ( i.e. in the second scenario , the instruction would differentially affect response amplitudes , with no or only limited changes in onset latencies or activation patterns . tether release is at time = 0 ms prior to each trial , the participant was instructed to either recover balance [ balance recovery ( br ) trials ] or to avoid balance recovery attempts and to focus on landing safely [ fall ( f ) trials ] after the perturbation . in the f trials , participants were not restricted in their use of safe landing responses ( such as breaking the fall with the outstretched hands ) , except that they were not allowed to rotate around the longitudinal axis . we only analyzed the first 200 ms after tether release , because we were particularly interested in whether people would be able to exert cognitive control over the initial part of the postural response , which is presumably highly automated and not generated at the level of the cerebral cortex . paired t tests were used to identify differences in the instants of tether release after the start of the trial and onsets of arm abduction movements between br and f trials . within - subjects factors were instruction ( br and f ) , and muscle ( 12 muscles ) . within - subjects factors were instruction ( br and f ) , and bins of emg activity ( 8 bins following release ) . in addition , anovas for repeated measures were used with post hoc paired t tests ( with alpha levels adjusted to 0.01 to correct for repeated testing ) to detect the differences in maximum amplitudes and the time of this maximum amplitude between instructions , with instruction ( br and f ) and muscle ( 12 muscles ) as within - subjects factors . tether release is at time = 0 ms prior to each trial , the participant was instructed to either recover balance [ balance recovery ( br ) trials ] or to avoid balance recovery attempts and to focus on landing safely [ fall ( f ) trials ] after the perturbation . in the f trials , participants were not restricted in their use of safe landing responses ( such as breaking the fall with the outstretched hands ) , except that they were not allowed to rotate around the longitudinal axis . we only analyzed the first 200 ms after tether release , because we were particularly interested in whether people would be able to exert cognitive control over the initial part of the postural response , which is presumably highly automated and not generated at the level of the cerebral cortex . paired t tests were used to identify differences in the instants of tether release after the start of the trial and onsets of arm abduction movements between br and f trials . within - subjects factors were instruction ( br and f ) , and muscle ( 12 muscles ) . within - subjects factors were instruction ( br and f ) , and bins of emg activity ( 8 bins following release ) . in addition , anovas for repeated measures were used with post hoc paired t tests ( with alpha levels adjusted to 0.01 to correct for repeated testing ) to detect the differences in maximum amplitudes and the time of this maximum amplitude between instructions , with instruction ( br and f ) and muscle ( 12 muscles ) as within - subjects factors . there were no significant differences between lean angles in br and f trials in both backward and leftward perturbations ( p = 0.087 and p = 0.427 , respectively ) . analysis also did not yield significant differences in the instants of tether release relative to the start of the trial ( backward : 4.1 0.24 s vs. 4.1 0.27 s for br and f trials , respectively , p = 0.955 ; leftward 4.3 0.19 s vs. 4.0 0.23 s , p = 0.080 ) . of the total 120 br trials collected in this study , furthermore , in only 3 out of 120 f trials a recovery attempt ( step initiation ) could be observed . in response to the perturbation , arm abduction movements were commonly observed in both br and f trials , but were earlier and more pronounced in br than in f trials . in backward trials , bilateral arm abduction movements occurred at on average 154 5 ms after release in br trials and at 172 8 ms in f trials ( p = 0.018 ) . in leftward trials , clear abduction movements movement onsets in br trials occurred at an average 159 4 ms after release and in f trials at 179 8 ms ( p = 0.009 ) . in backward br trials , neck , trunk and leg muscles showed similar onsets ( 7080 ms after tether release ) , followed by dpl and dpr at 8090 ms and dal and dar at 90100 ms , on average ( fig . in the backward f trials , the overall mean onset latencies of the 12 muscles measured were delayed by 4.1 ms ( se 2.4 ms ) compared to the br trials [ f(1,8 ) = 5.601 , p = 0.045 , fig . 2average onset latencies ( se ) in response to backward and leftward perturbations for left and right sternocleidomastoid ( scl and scr ) , anterior deltoid ( dal and dar ) , posterior deltoid ( dpl and dpr ) , rectus abdominis ( abl and abr ) , rectus femoris ( rfl and rfr ) , and tibialis anterior ( tal and tar ) . * p < 0.01 average onset latencies ( se ) in response to backward and leftward perturbations for left and right sternocleidomastoid ( scl and scr ) , anterior deltoid ( dal and dar ) , posterior deltoid ( dpl and dpr ) , rectus abdominis ( abl and abr ) , rectus femoris ( rfl and rfr ) , and tibialis anterior ( tal and tar ) . overall , mean onset latencies of the 12 muscles were delayed by 9.3 ms ( se 4.8 ms ) in the f trials compared to the br trials [ f(1,8 ) = 20.373 , p = 0.002 , fig . emg amplitudes after tether release were generally higher in br than in f trials and these instruction - related differences could often be detected shortly after onset ( mostly within 40 ms ) . post hoc contrasts revealed that emg amplitudes started to increase more steeply in br than in f trials at 6080 ms after release for scr , at 80100 ms after release for scl and tal , followed by dal , rfl , and tar at 100120 ms , and dar at 120140 ms [ values for f(1,9 ) ranging from 5.946 to 18.575 , p values between 0.002 and 0.037 ] ( fig . 3emg amplitudes of balance recovery ( br ) trials minus the amplitudes in fall ( f ) trials in response to backward and leftward perturbations . the arrows indicate at which instant emg amplitudes started to deviate significantly between br and f trials emg amplitudes of balance recovery ( br ) trials minus the amplitudes in fall ( f ) trials in response to backward and leftward perturbations . the arrows indicate at which instant emg amplitudes started to deviate significantly between br and f trials for leftward trials , analysis yielded significant instruction bin interactions for all the muscles [ values for f(7,63 ) ranging from 2.424 to 18.294 , all p values < 0.030 ] , except rfl and tar [ values for f(7,63 ) of 1.317 and 1.198 , p values of 0.257 and 0.317 , respectively ] . higher emg amplitudes were observed in br than in f trials , starting at 80100 ms for scl , scr , dal , and abr , followed by dar and dpr at 100120 ms , abl and rfr at 120140 ms , dpl at 140160 ms , and tal at 180200 ms [ values for f(1,9 ) ranging from 5.157 to 21.745 , p values between 0.001 and 0.049 ] ( fig . in both backward and leftward perturbations , maximum emg values ( table 1 ) were also significantly larger in br than in f trials [ f(1,9 ) = 27.430 , p = 0.001 and f(1,9 ) = 33.627 , p the time of maximum emg was not significantly different between backward br and f trials [ f(1,9 ) = 4.198 , p = 0.071 ] . table 1mean se of maximum emg amplitude and the instant after release at which the maximum value was reached for balance recovery ( br ) and fall ( f ) trialsbackwardleftwardbrfbrfmaximum emg ( mv)time of max . ( ms)scl217 21 * 149 5124 21138 5136 17 * 141 349 10140 11scr196 25 * 155 2124 28138 4112 23 * 151 832 9139 10dal249 48 * 160 395 14144 7286 53 * 163 4 66 20144 5dar239 50 * 152 551 9148 6106 21 148 570 32139 4108 45144 456 26144 3dpr208 50141 5120 29137 6211 51 * 147 371 21140 11abl124 42128 8139 40138 869 22 140 8 23 7113 6abr101 25131 687 15136 857 17 120 529 9112 7rfl179 36 148 7119 20128 629 5144 627 7152 4rfr93 19121 475 15134 5202 63 * 173 3 * 24 7156 4tal271 49 * 162 6180 44140 8270 48 * 167 7 179 37140 6tar322 51 * 152 5167 35131 8147 30139 4122 26139 5 * p < 0.01 mean se of maximum emg amplitude and the instant after release at which the maximum value was reached for balance recovery ( br ) and fall ( f ) trials hence , many of the muscles recorded showed significantly higher emg amplitudes in br than in f trials , with the earliest and most consistent differences between instructions being present in sternocleidomastoid . furthermore , in both perturbation directions , emg amplitude in rectus femoris of the stance limb was greater in br than f trials . 4average changes in flexion and abduction angles ( se ) of the left ( l ) and right ( r ) arm between tether release and 200 ms post - release for balance recovery ( diamonds ) and fall trials ( squares ) . * p < 0.05 average changes in flexion and abduction angles ( se ) of the left ( l ) and right ( r ) arm between tether release and 200 ms post - release for balance recovery ( diamonds ) and fall trials ( squares ) . * p < 0.05 in leftward trials , instruction - related differences , with larger amplitudes in br than f trials , were most pronounced in dpr ( shoulder abductor and extensor ) and dal ( shoulder abductor and flexor ) . these differences were observed in conjunction with larger shoulder extension movements of the right arm in br trials ( 8.9 vs. 4.2 , se of the difference 1.4 , p = 0.008 ) , as well as larger abduction movements of both the left and right arm ( left : 9.5 vs. 4.3 , se of the difference 1.0 , p = 0.001 ; right : 1.7 vs. 0.7 , se of the difference 0.7 , p = 0.007 , fig . the aim of the present study was to investigate the influence of instruction on automated postural responses . participants were instructed to either recover balance or simply fall onto a gymnasium mat following a sudden postural perturbation . at the behavioral level , participants were highly successful in following these instructions , consistently exhibiting stepping movements for balance recovery in br trials , and suppressing stepping in the f trials . yet emg recordings revealed similar postural responses with onset latencies between 70130 ms in both br and f trials . only limited instruction - related changes in onset latencies were observed , with slightly delayed responses in f trials , in combination with generally preserved activation sequences . in contrast , very pronounced and early differences were observed between br and f trials in response amplitudes , which were generally much higher in br than in f trials , but with clear differentiation between muscles and perturbation directions . furthermore , instruction - related differences in muscle response amplitudes could not be explained by a general time shift of the response , as maximum amplitudes were larger in br than in f trials and in most muscles , occurred at a similar time after onset of the perturbation . in the present study , fear of falling might be expected to be most pronounced in the f trials . this may reflect that , in our experiments , the anxiety associated with the task of recovering balance following sudden release of the tether ( although different in nature ) may have matched that associated with the task of falling onto the mat , which may of course not be the case during a real - life fall onto a hard surface . the present study provides an important addition to the current body of knowledge by indicating that , even when the occurrence of any postural response may not be desired , it can not be fully suppressed by instruction , as responses also occurred when people were not supposed to recover , but to fall . it shows that in balance recovery , we can rely on highly automated responses , but these responses can also be effectively downregulated by the central nervous system when they are not desired . the differential effects of instruction , as observed in the present study , are in line with this idea of goal - related changes in response gains . the higher rectus femoris activation of the stance limb in br ( compared to f ) trials can be interpreted as a preparatory action , in order to carry the body weight on one leg to allow stepping with the other . in contrast , in f trials , no such preparation for weightbearing was needed , which explains the much larger attenuation of the rectus femoris response in the stance than in the stepping leg . the asymmetrical maximum emg amplitudes of rfl and rfr in the backward f trials , however , seem to indicate that some preparatory activity for stepping may have been present in these trials as well . in leftward trials , the early deviations between instructions of both the shoulder muscle emg and the corresponding kinematic patterns seemed to be related to positioning the arms to prepare for impact in the f trials , as participants tended to utilize the active response of impacting the ground with the outstretched hands . this does not exclude the possibility , however , that the delayed responses in f trials may be related to differences in preparation alertness . the time - critical nature of the br trials requires a high degree of readiness in order to recover balance successfully after the perturbation , whereas this high time pressure is not present in f trials . it can be expected , however , that these are the most ideal circumstances for suppression of automated postural responses in the f trials , yet they still occurred in response to the perturbation . in conclusion , the present study indicates that automated postural responses occur when balance is perturbed , irrespective of whether people are instructed to recover or not to recover balance . this suggests that the triggering of postural responses is organized in a reflex - like manner , with supra - spinal control primarily contributing to adjust these responses in a functional and goal - oriented way .

it is a great desire of humans to find golden ways to solve major problems , especially to treat severe diseases effectively in order to prolong life , whenever possible without creating additional risks or side effects . in the past , omega-3 polyunsaturated fatty acids ( -3 ) , especially eicosapentaenoic acid ( epa ) and docosahexaenoic acid ( dha ) , appeared to be compounds having the potential to fulfill this dream , if supplemented to daily nutrition in sufficient amounts . consequently , a huge amount of data has been accumulated on this topic and many reviews and meta - analyses have been published ( bucher et al . , 2002 ; leaf et al . , 2003 ; whelton et al . , 2004 ; yzebe and lievre , 2004 ; dhein et al . , 2005 ; hooper et al . , 2006 ; reiffel and mcdonald , 2006 ; wang et al . , 2006 ; lombardi and terranova , 2007 ; cheng and santoni , 2008 ; jenkins et al . , 2008a ; len et al . , 2008 ; siddiqui et al . , 2008 ; marik and varon , 2009 ; zhao et al . , 2009 ; based on this background the purpose of the present paper is not to again review all the available data on -3 effects or to discuss omega-3 unsaturated fatty acids as essential compounds in human and animal biology . this paper focuses on the effects of supplementation with -3 on cardiac rhythm and discusses the potential clinical consequences of recent clinical studies that do not support the existence of this golden -3 way . furthermore , the complexity of the biological interactions of -3 as well as the variation of potential clinical settings are outlined in order to explain that supplementation with -3 does not necessarily result in an overall beneficial clinical effect in every condition . an inverse relationship between consumption of fish oil and cardiovascular risk was shown in early observational , case control , and cohort studies , with respect to the occurrence of cardiovascular disease ( whelton et al . , 2004 ) , sudden cardiac death ( scd ) and non - scd from coronary heart disease ( daviglus et al . , 1997 ) , and with regard to scd in apparently healthy persons ( siscovick et al . , 1995 ; albert et al . , 1998 , 2002 ; -3 levels in erythrocyte membranes were directly associated with a reduced rate of primary cardiac arrest ( siscovick et al . , 1995 ) . similarly , elevated -3 blood levels were associated with a reduced risk of sudden death among men without evidence of prior cardiovascular disease ( albert et al . , 2002 ) . these data were supported by prospective and randomized nutritional intervention studies of secondary prevention after acute myocardial infarction ( ami ) . in the diet and reinfarction trial ( dart ) a diet rich in fish and cereals was associated with a significant 29% reduction of all - cause mortality within 2 years after ami ( burr et al . , 1989 ) . in the lyon diet heart study the mediterranean diet group [ diet enriched by alpha - linolenic acid ( ala , -3 ) and olive oil , combined with an increased intake of cereals , fresh fruit , vegetables and fish , but limited intake of saturated fatty acids and linoleic acid ( -6 ) ] had a significantly lower rate of the combined endpoint cardiac death and non fatal myocardial infarction , if compared to the control group taking a prudent western - type diet ( p = 0.0001 ; follow - up 27 months ; de lorgeril et al . a predefined supplementation with -3 was used in the large placebo - controlled , open labeled gissi prevenzione trial ( epa + dha 1 g / day , vitamin e 300 mg / day , a combination of both , or placebo ; gissi - prevenzione investigators , 1999 ) , focusing on secondary prevention after ami . in this study the intervention arms using -3 showed a significant reduction of scd though this was not the primary endpoint of the gissi prevenzione trial . in parallel to these encouraging clinical data , animal studies ( mostly using the rat or canine model ) supported an anti - arrhythmic effect of -3 especially with respect to ischemia - induced ventricular tachycardia ( vt ) or ventricular fibrillation ( vf ) ( matthan et al . the clearest effect in the prevention of vf by -3 could be shown in infusion studies using a special experimental canine model . in this model acute myocardial ischemia was induced at a site distant from a previous myocardial infarction during submaximal exercise thereby activating the autonomic nervous system ( billman , 2006 ) and inducing vf . however , these clear effects of -3 under well - defined experimental conditions can not simply be translated into the clinical situation , and several aspects have to be considered ( billman , 2006 ) : in this canine model not only superfusion with -3 but also the application of -receptor antagonists , calcium - channel blockers , and endurance exercise training all interventions that are routinely used in actual clinical practice were effective in vf prevention . animals resistant to vf were characterized by reduced -receptor responsiveness and an intact parasympathetic regulation , indicating that these are first line mechanisms to prevent ischemia - induced tachyarrhythmias . finally , incorporation of -3 into the phospholipid bilayer can be expected to be significantly less in infusion studies as compared to feeding studies . feeding studies more closely may imitate the clinical situation , and under these conditions -3 can be expected to exert their effect primarily after being incorporated into the cellular membrane . numerous animal feeding studies have been published between 1987 and 1999 , and the results showed a considerable heterogeneity . still , a meta - analysis of these studies suggests fish oil does prevent ischemia and ischemia - reperfusion induced vt / vf ( matthan et al . , 2005 ) . heterogeneity of experimental results also can be seen in more recent studies . in isolated hearts of pigs fed with fish oil for 8 weeks , spontaneous ischemia - induced sustained vt / vf was facilitated in the -3 group ( coronel et al . , 2007 ) . other studies report increased resistance to ischemia - reperfusion injury after dietary -3 application , which also could be a basis to protect against reperfusion arrhythmias ( abdukeyum et al . , 2008 ; zeghichi - hamri et al . , 2010 ) . the results of the animal studies and their apparent inconsistencies may be remembered when judging the data of recent prospective , randomized , double - blind clinical studies that interrupted the long list of positive results of older studies investigating the effect of -3 on cardiovascular risk . in the following three randomized prospective studies evaluating the effect of high doses of -3 in patients with icd - devices failed to give homogeneous results . in one study recurrent vt events not due to myocardial ischemia were more common in patients treated with fish oil ( 1.3 g -3 per day during a period of two years ; p < 0.007 ; raitt et al . , 2005 ) . however , in another study predominantly including patients with coronary artery disease , -3 supplementation was associated with a significant risk reduction for the primary endpoint ( time to first icd - event or death from any cause ) by 31% ( p = 0.033 ) . remarkably in this study no significant effect of -3 could be shown in the subgroups of patients without coronary artery disease or with a left ventricular ejection fraction above 30% ( leaf et al . , 2005a ) . finally the sofa - study did not show a significant effect of -3 supplementation on the primary endpoint ( appropriate icd - interventions for recurrent vt / vf or death from any cause ; hazard ratio 0.86 , 95% ci 0.641.16 ) . the majority of the patients included in the sofa - study had coronary artery disease , more than 60% with previous myocardial infarction ; almost 40% of the study participants had various forms of cardiomyopathy or valvular heart disease ( brouwer et al . , 2006 ) . in a meta - analysis of these three studies all - cause mortality did not significantly differ between the fish oil and the control groups ( relative risk 0.70 ; 95% ci 0.421.15 ; jenkins et al . finally , in a substudy of the gissi - hf trial ( 566 heart failure patients with implanted icd - devices , 57% with previous myocardial infarction , mean follow - up 928 days ) a statistically non - significant trend toward a lower risk of icd - discharge in patients treated with -3 was shown [ adjusted hazard ratio ( hr ) 0.80 ; 95% ci 0.591.09 ; p = 0.152 ] . however , mortality was similar in both groups [ total mortality : -3 ( 26.6% ) , placebo ( 24.3% ) ; adjusted hr 1.25 ; 95% ci 0.891.75 ; p = 0.19 ; mortality for arrhythmias : -3 ( 3.6% ) , placebo ( 2.1% ) ; adjusted hr 1.84 ; 95% ci 0.675.05 ; finzi et al . , the apparent heterogeneity in the response of icd - patients to fish oil supplementation could be a consequence of different study populations and different arrhythmic origins ( ischemic versus non - ischemic ) . heterogeneity also could be the result of different concomitant medications of the study populations including -blockers , digoxin , amiodarone , and sotalol . a potential influence of medication on the effect of fish oil supplementation may be indicated by the results of the dart 2 study ( burr et al . , 2003 ) . in the controlled prospective dart 2 trial conducted with general practitioners of south wales male patients with stable angina ( n = 3,114 , under 70 years of age ) were randomly allocated to four study groups with specific nutritional advises including the advise to eat oily fish or take fish oil capsules in two of the study groups . the risk of scd was significantly increased in the group taking oily fish or fish oil capsules ( hr 1.54 ; 95% ci 1.062.23 ; burr et al . , 2003 ) . the adverse effects of fish or fish oil capsules only occurred in men not taking beta - blockers or dihydropyridine calcium - channel blockers , and were increased in patients taking digoxin ( burr et al . , 2005 ) . unfortunately , a conclusive interpretation of the dart 2 data is seriously limited as patient s recruitment and monitoring was interrupted for 1 year , long - term compliance was uncertain , and sudden death could not be ascertained in all cases ( burr et al . , 2003 ) . in the large scale multicenter japan epa lipid intervention study ( jelis ; yokoyama et al . , 2007 ) consumption of 1.8 g epa per day over a mean period of 4.6 years in hyperlipidemic patients ( no ami in the last 6 months , no serious heart disease ) treated with statins resulted in a reduction of major cardiovascular events ( combined endpoint including scd , fatal and non - fatal myocardial infarction , and other non - fatal events including unstable angina , angioplasty , stenting , or coronary bypass grafting ) from 3.5 to 2.8% ( p = 0.011 ; hr 0.81 ; 95% ci 0.690.95 ) . however , there was neither a reduction of scd ( 0.2% in both study arms ; hr 1.06 ; 95% ci 0.5520.07 ) nor of coronary death ( 0.3% in both study arms ; hr 0.94 ; 95% ci 0.571.56 ) or all - cause death ( control 2.8% , epa - group 3.1% ; hr 1.09 ; 95% ci 0.921.28 ) . compared to the gissi prevenzione trial death rates and especially the rates of scd were very low in both groups and therefore may be difficult to be reduced further by any intervention ( rates for scd : jelis 0.2% in both groups ; gissi 2.2%/2.9% -3 versus control ; gissi - prevenzione investigators , 1999 ) . furthermore , these low event rates may at least in part be the result of a high fish consumption of the japanese population at baseline . as most risk reduction already occurs at about 250 mg epa / dha intake per day ( mozaffarian and rimm , 2006 ) , a further increase of -3 intake may not have a substantial additional effect on cardiac death reduction ( mozaffarian , 2007 ) . in the alpha - omega - study , a multicenter , double - blind , placebo - controlled trial , 4,837 patients in the chronic stable phase after myocardial infarction ( average 3.7 years after ami ) and 6080 years of age ( 21.8% women ) were randomly assigned to one of four trial arms . margarine was used in all trial arms , supplemented with either epa / dha ( daily intake aimed to be 400 mg ) , alpha - linolenic acid ( ala , daily intake aimed to be 2 g ) , epa / dha + ala , or placebo , respectively ( kromhout et al . , 2010 ) . after a follow - up of 40 months , 13.9% of the patients had a major cardiovascular event ( death , non - fatal cardiovascular events , or cardiac intervention ) . in addition , in all secondary endpoints , including ventricular - arrhythmia and total death , there was no significant difference between the study groups . importantly , a high percentage of the patients received state of the art medication , including statins . in a post hoc analysis after unblinding of the data in the subgroup of patients with diabetes ventricular - arrhythmia - related events tended to be reduced in the epa / dha group ( hr 0.51 ; 95% ci 0.241.11 ) and significantly were reduced in the ala group ( hr 0.39 ; 95% ci 0.170.88 ) . in a secondary analysis of the alpha - omega trial taking high risk patients with previous myocardial infarction and diabetes the epa / dha + ala group experienced significantly less ventricular - arrhythmia - related events ( hr 0.16 ; 95% ci 0.040.69 ; kromhout et al . , 2011 ) . these differential results support the necessity to exactly define the clinical conditions under which supplementation of -3 may be beneficial . in the omega trial the effect of supplementation with 1 g / day of esterified epa / dha on the rate of scd and other clinical events within 1 year after ami was tested in 3,851 patients ( 25.6% female , mean age 64.0 years ; rauch et al . a 1-year follow - up was chosen , as the risk of cardiac death after ami including a presumed arrhythmic death is highest in the first 3 months after the event ( solomon et al . , 2005 ; pouleur et al . , 2010 furthermore , in the gissi trial , the significance in lowering scd by -3 had already been reached within 120 days ( marchioli et al . , 2002 ) . following guidelines for the management of ami and secondary prevention 77% of the patients in the omega trial received acute percutaneous coronary intervention , and/or thrombolysis ( 8.3% ) . at hospital discharge the following medications were prescribed for almost all patients : beta - blockers ( 94% ) , ace - inhibitors ( 83% ) , arbs ( 8% ) , statins ( 94% ) , acetylsalicylic acid ( 95% ) , and clopidogrel ( 88% ) . under these conditions , the rates of scd were 1.5% in both study groups ( or 0.95 ; 95% ci 0.561.60 ) and total mortality was 4.6% in the -3 group and 3.7% in the control group ( or 1.25 ; 95% ci 0.901.72 ) . in none of the predefined secondary endpoints , including total death , major adverse cardiovascular and cerebrovascular events , and revascularization procedures in survivors , was found a significant difference between the study groups , and not even a trend in favor of the -3 group could be observed . in addition , icd - terminated vt or vf in survivors was 0.1% ( n = 2/1,654 ) in the control group but 0.5% ( n = 9/1,705 ) in the -3 group [ p = 0.06 ; or ( 95% ci ) : 4.47 ( 0.9720.74 ) ] . furthermore , there was no significant difference between the study groups with regard to scd or total death in any of the predefined subgroups of patients with higher risk ( diabetes , age > 70 years , no acute revascularization , ejection fraction < 35% ) . despite these apparently homogeneous results their interpretation is limited as the case estimate in the omega - study was based on an overestimation of the rate of scd in the control group , thereby leading to an underpowering of the study . two other randomized controlled trials published recently also failed to show a clear beneficial effect of -3 supplementation . in 563 elderly norwegian men at high cardiovascular risk a non - significant tendency to a reduced all - cause mortality could be observed ( hr 0.53 ; 95% ci 0.271.04 ) , but the rate of cardiovascular events remained unchanged ( hr 0.89 ; 95% ci 0.551.45 , follow - up 3 years ; einvik et al . , 2010 ) . in 2,501 patients with a history of myocardial infarction , unstable angina or ischemic stroke supplementation with epa / dha was not associated with a significant decrease of major vascular events during a follow - up of 4.7 years ( hr 1.08 ; 95% ci 0.791.47 ; galan et al . , 2010 ) . which conclusions may be drawn from the clinical studies and the animal studies discussed above ? the effect of -3 supplementation may depend on the background diet and the pre - existent intake of fish oil ( mozaffarian and rimm , 2006 ; reiffel and mcdonald , 2006 ; mozaffarian , 2007 ) . with regard to earlier studies , treatment of patients with coronary artery disease , especially treatment of patients with myocardial infarction has improved markedly . in the gissi trial ( inclusion period october 1993 to september 1995 ) only 4.4% of the patients had acute coronary revascularization at baseline , and only 4.7% were on cholesterol - lowering drugs at hospital discharge , increasing to only 46% after 42 months of follow - up ( gissi - prevenzione investigators , 1999 ) . furthermore , only 43.9% of the patients included in the gissi trial were on beta - blocker treatment at the start of the study , and this percentage decreased during follow - up . it therefore may be speculated that up - to - date guideline adjusted treatment of ami ( including acute revascularization , medical treatment , and support of life style changes ) may interfere with molecular and cellular -3 interactions thereby weakening or competing with a potential beneficial -3 effect . although the available data not homogeneously support this hypothesis ( marchioli et al . , 2007 ) , this aspect should strongly be considered in future research . the anti - arrhythmic effect of -3 may depend on the pathophysiological conditions that facilitate arrhythmias . the clinical and experimental data outlined above suggest that -3 supplementation may especially protect against ischemia - induced arrhythmias . therefore , prevention of ischemia by modern treatments ( i.e. , revascularization , beta - blockers , statins , ace - inhibitors , inhibition of thrombocyte aggregation , physical exercise ) could attenuate a potentially beneficial effect of -3 . beta - blockers are well known to prevent sudden death , and even statins could have some anti - arrhythmic effects ( anh and marine , 2004 ; lorenz et al . , 2005 ) . potential anti - arrhythmic effects of -3 by augmentation of vagal activity ( mozaffarian et al . , 2005 ; okeefe et al . , 2006 ) may be blunted by beta - blocker treatment and increased physical training during cardiac rehabilitation ( nolan et al . the anti - arrhythmic effect proven under experimental conditions in animal models and suggested in the earlier clinical studies appears to depend on the clinical conditions being studied . these clinical conditions are determined by the type and stage of the underlying myocardial disease and represent a sum of various pathophysiological conditions ( including ischemia , reperfusion , ischemic preconditioning , scar tissue , inflammation , congenital defects , etc . ) and the effects of modern medication including beta - blockers , ace - inhibitors , statins , and other interventions potentially interfering with the arrhythmic risk , such as exercise training . these considerations may also apply to the role of -3 in the prevention of atrial fibrillation . positive results ( mozaffarian et al . , 2004 , primary prevention in patients > 65 years of age ; cal et al . , 2005 , patients undergoing coronary artery surgery ; macchia et al . , 2008 , postmyocardial infarction patients ) were not confirmed in more recent studies and meta - analyses ( kowey et al . , 2010 ; saravanan et al . , 2010 ; bianconi et al . , 2011 ; farquharson et al . , 2011 ; liu et al . , 2011 ) . still , it was demonstrated recently , that the use of fish oil ( dha 1.5 g and epa 0.3 g daily ) resulted in a prolongation and reduced dispersion of pulmonary venous and left atrial effective refractory periods in patients with paroxysmal atrial fibrillation ( kumar et al . , 2011 ) . angiotensin aldosterone system inhibitor , taking -3 ( 2 g / day ) improved the probability of maintaining sinus rhythm after direct current cardioversion ( nodari et al . , 2011 ) . similar to the prevention of ventricular tachyarrhythmias , prevention of atrial fibrillation therefore may depend on distinct clinical and pathophysiological conditions and concomitant medication . the ongoing opera - trial , including a total of 1,516 patients scheduled for cardiac surgery and in sinus rhythm , will give more insight into the potential role of -3 supplementation to prevent post - operative atrial fibrillation ( mozaffarian et al . for understanding the seemingly heterogeneous efficacy of -3 supplementation in preventing tachyarrhythmias , it is important to reflect on their molecular and cellular interactions as has been delineated extensively in recent reviews ( leaf et al . , 2003 , 2005b ; dhein et al . , 2005 ; , 2007 ; lombardi and terranova , 2007 ; siddiqui et al . , 2008 ) . in the following , only some aspects of potential relevance for interpretation of the clinical data are discussed : there are three major ways in which -3 may interfere with cellular and membrane function , thereby potentially moderating cardiac rhythm : direct interactions of -3 with membrane bound proteins like the fast sodium channel , the voltage - gated l - type ca channel , specific potassium channels , and the na / ca - exchanger ( hallaq et al . , 1990 , 1992 ; honore et al . , 1994 ; xiao et al . , 1995 , 1997 ; kang and leaf , 1996 ; leifert et al . , 1999 ; leaf et al . , 2003 ; den ruijter et al . , 2007 ; wang et al . , 2010 ) such interactions may occur predominantly with circulating -3 when it is delivered by acute administration and infusion . incorporation into the phospholipid bilayer , thereby potentially changing membrane fluidity , and/or forming -3 rich micro - domains , and/or interacting with internal binding sites . this may result in a change of the function of membrane bound proteins like ion channels , receptors and signal transduction systems ( mcmurchie et al . , 1988 ; croset and kinsella , 1989 ; kinoshita et al . , 1994 ; grynberg et al . , 1996 ; , 1999 , 2000b ; mclennan , 2001 ; den ruijter et al . , 2007 ) . incorporation into the cellular membranes predominantly is achieved by dietary long - term administration of -3 . interaction with intracellular pathways including gene expression and metabolism of phosphoinositides ( jud et al . , circulating -3 compounds are likely to have different electrophysiological effects , compared to -3 incorporated into the membranes ( den ruijter et al . , 2007 for review ) . for example , peak cardiac sodium current was reduced by 51% after acute administration of epa and dha in neonatal rat cardiomyocytes ( xiao et al . , 1995 ) , but remained unaffected by -3 incorporated in pig and rat cardiomyocytes ( leifert et al . differential effects of circulating versus incorporated -3 have also been demonstrated with respect to various potassium channels and the regulation of calcium homeostasis ( den ruijter et al . incorporated -3 , however , also may prevent further action potential ( ap ) shortening induced by circulating -3 . patients with high levels of incorporated -3 therefore may not have a further benefit from short term -3 supplementation ( den ruijter et al . , 2010 ) . this could be of a direct clinical relevance , as acute -3 supplementation may be used for prevention of atrial fibrillation induced by cardiac surgery , which is being investigated in the opera - trial ( mozaffarian et al . , 2011b ) . apart from these considerations the molecular interactions of -3 and their effects on cardiac rhythm may be influenced by a large variety of additional conditions : the various kinds of -3 formulations being used ( re - esterified triacylglycerides , ethyl - esters or phospholipids ; neubronner et al . the activity state of membrane bound proteins and ligand occupation of specific receptors involved in signal transduction ( rauch et al . , 1989 ; xiao et al . , 1998 ; den ruijter et al . , 2007 ) , or the increased responsiveness of inhibitory g - proteins after ischemic preconditioning ( niroomand et al . , the activity of cellular phospholipases and the presence of lysophosphatides that change phospholipid environment and function of membrane bound proteins ( chien et al . , 1981 ; corr et al . , 1984 ; rauch et al . , 1994 ) , and may even vary between different myocardial regions depending on the degree of ischemia and/or inflammation . the heterogeneity of electrical stability of myocardial cells in the diseased heart muscle due to regional differences with regard to various degrees of ischemia and tissue damage , ischemic preconditioning , etc . it should also be remembered , that in patients with coronary artery disease , myocardium is not presenting as a homogeneous and healthy tissue experiencing acute ischemia in a well - defined area , but rather as a mixture of healthy myocardium , hypertrophied tissue , scar tissue , and ischemic myocardium and includes areas of tissue with ischemic preconditioning , inflammation , various degrees of membrane phospholipid degradation and with more or less acute or chronic stretch , etc . the characteristics of aps vary significantly between human and various animal myocardial cells and with gender ( karagueuzian et al . , 1982 ; shattock and bers , 1989 ; cheng , 2006 ; tanaka et al . , 2008 ) . vt or vf are predominantly caused by triggered activity or by re - entry mechanisms . fish oil shortens cardiac ap and accentuates the ap notch , which may lead to depression or even loss of the ap dome ( verkerk et al . , 2006 , 2007 ) . under clinical conditions where the ap is prolonged triggered activity may be the predominant pro - arrhythmic mechanism , which could be inhibited in isolated cardiomyocytes from rabbits and from patients with end stage heart failure by superfusion with -3 ( den ruijter et al . , . in keeping with these experimental results -3 were effective in reducing the arrhythmic risk in patients with idiopathic dilated cardiomyopathy ( nodari et al . , 2009 ) . conversely , ap shortening also may be pro - arrhythmic by reducing the refractory period and thereby promoting re - entry . supplementation with -3 may increase a preexisting heterogeneity in ap duration and repolarization ( verkerk et al . , 2007 ) , as can be seen , for example , in acute ischemia ( yan et al . , 2004 ) . in this way the occurrence of unidirectional block and re - entry may be facilitated ( janse and wit , 1989 ) . in the clinical situation therefore supplementation with -3 may prevent or facilitate ventricular tachyarrhythmias depending on the predominant underlying arrhythmic mechanism ( den ruijter et al . , 2007 ) . based on these considerations it becomes apparent that -3 do not have a specific way to act , but rather possess multiple sites of potential actions , that may be influenced by a number of external conditions at the cellular and molecular level . multiple sites of interaction between -3 and myocardial tissue in combination with various possible ways of interference with these biochemical interactions are unlikely to result in an unequivocally predictable and homogeneous beneficial effect on clinical outcomes . -3 clearly interfere with the physiology of myocardial cell membranes through a variety of specific and unspecific pathways , and thereby exhibit anti - arrhythmic effects under certain well - defined experimental and clinical conditions this complexity makes it difficult to predict the effects of -3 supplementation on cardiac rhythm within the wide variety of conditions that represent clinical practice . for the future it will be necessary to define exactly the clinical conditions in which supplementation with of -3 is beneficial , and without potentially harmful effects bernhard rauch has no conflicts of interest ; jochen senges received honoraria for educational presentations from trommsdorff gmbh & co. kg arzneimittel , alsdorf , germany and pronova biopharma , lysaker , norway

supplementation of omega-3 fatty acids ( -3 ) has been associated with a decreased cardiovascular risk , thereby concentrating attention on a potentially preventive effect regarding tachyarrhythmias and sudden cardiac death . however , recent randomized controlled trials challenge the efficacy of the additional application of -3 and its anti - arrhythmic effect under certain clinical conditions . the present paper reflects the results of earlier and recent clinical studies with respect to the individual background conditions that may determine the clinical outcome of -3 supplementation and thereby explain apparently conflicting clinical results . it is concluded that the efficacy of -3 supplementation to prevent cardiac arrhythmias strongly depends on the underlying clinical and pharmacological conditions , a hypothesis that also is supported by data from experimental animal studies and by molecular interactions of -3 at the cellular level .

Introduction Earlier Clinical Studies Animal Studies Recent Clinical Studies Some Aspects of the Molecular and Cellular Interactions of -3 to Explain Heterogeneous Clinical Results Concluding Remarks Conflict of Interest Statement

it is a great desire of humans to find golden ways to solve major problems , especially to treat severe diseases effectively in order to prolong life , whenever possible without creating additional risks or side effects . in the past , omega-3 polyunsaturated fatty acids ( -3 ) , especially eicosapentaenoic acid ( epa ) and docosahexaenoic acid ( dha ) , appeared to be compounds having the potential to fulfill this dream , if supplemented to daily nutrition in sufficient amounts . consequently , a huge amount of data has been accumulated on this topic and many reviews and meta - analyses have been published ( bucher et al . , 2009 ; based on this background the purpose of the present paper is not to again review all the available data on -3 effects or to discuss omega-3 unsaturated fatty acids as essential compounds in human and animal biology . this paper focuses on the effects of supplementation with -3 on cardiac rhythm and discusses the potential clinical consequences of recent clinical studies that do not support the existence of this golden -3 way . furthermore , the complexity of the biological interactions of -3 as well as the variation of potential clinical settings are outlined in order to explain that supplementation with -3 does not necessarily result in an overall beneficial clinical effect in every condition . an inverse relationship between consumption of fish oil and cardiovascular risk was shown in early observational , case control , and cohort studies , with respect to the occurrence of cardiovascular disease ( whelton et al . , 2004 ) , sudden cardiac death ( scd ) and non - scd from coronary heart disease ( daviglus et al . , 1998 , 2002 ; -3 levels in erythrocyte membranes were directly associated with a reduced rate of primary cardiac arrest ( siscovick et al . similarly , elevated -3 blood levels were associated with a reduced risk of sudden death among men without evidence of prior cardiovascular disease ( albert et al . these data were supported by prospective and randomized nutritional intervention studies of secondary prevention after acute myocardial infarction ( ami ) . in the diet and reinfarction trial ( dart ) a diet rich in fish and cereals was associated with a significant 29% reduction of all - cause mortality within 2 years after ami ( burr et al . in the lyon diet heart study the mediterranean diet group [ diet enriched by alpha - linolenic acid ( ala , -3 ) and olive oil , combined with an increased intake of cereals , fresh fruit , vegetables and fish , but limited intake of saturated fatty acids and linoleic acid ( -6 ) ] had a significantly lower rate of the combined endpoint cardiac death and non fatal myocardial infarction , if compared to the control group taking a prudent western - type diet ( p = 0.0001 ; follow - up 27 months ; de lorgeril et al . a predefined supplementation with -3 was used in the large placebo - controlled , open labeled gissi prevenzione trial ( epa + dha 1 g / day , vitamin e 300 mg / day , a combination of both , or placebo ; gissi - prevenzione investigators , 1999 ) , focusing on secondary prevention after ami . in this study the intervention arms using -3 showed a significant reduction of scd though this was not the primary endpoint of the gissi prevenzione trial . in parallel to these encouraging clinical data , animal studies ( mostly using the rat or canine model ) supported an anti - arrhythmic effect of -3 especially with respect to ischemia - induced ventricular tachycardia ( vt ) or ventricular fibrillation ( vf ) ( matthan et al . however , these clear effects of -3 under well - defined experimental conditions can not simply be translated into the clinical situation , and several aspects have to be considered ( billman , 2006 ) : in this canine model not only superfusion with -3 but also the application of -receptor antagonists , calcium - channel blockers , and endurance exercise training all interventions that are routinely used in actual clinical practice were effective in vf prevention . animals resistant to vf were characterized by reduced -receptor responsiveness and an intact parasympathetic regulation , indicating that these are first line mechanisms to prevent ischemia - induced tachyarrhythmias . finally , incorporation of -3 into the phospholipid bilayer can be expected to be significantly less in infusion studies as compared to feeding studies . feeding studies more closely may imitate the clinical situation , and under these conditions -3 can be expected to exert their effect primarily after being incorporated into the cellular membrane . numerous animal feeding studies have been published between 1987 and 1999 , and the results showed a considerable heterogeneity . still , a meta - analysis of these studies suggests fish oil does prevent ischemia and ischemia - reperfusion induced vt / vf ( matthan et al . the results of the animal studies and their apparent inconsistencies may be remembered when judging the data of recent prospective , randomized , double - blind clinical studies that interrupted the long list of positive results of older studies investigating the effect of -3 on cardiovascular risk . however , in another study predominantly including patients with coronary artery disease , -3 supplementation was associated with a significant risk reduction for the primary endpoint ( time to first icd - event or death from any cause ) by 31% ( p = 0.033 ) . remarkably in this study no significant effect of -3 could be shown in the subgroups of patients without coronary artery disease or with a left ventricular ejection fraction above 30% ( leaf et al . finally the sofa - study did not show a significant effect of -3 supplementation on the primary endpoint ( appropriate icd - interventions for recurrent vt / vf or death from any cause ; hazard ratio 0.86 , 95% ci 0.641.16 ) . the majority of the patients included in the sofa - study had coronary artery disease , more than 60% with previous myocardial infarction ; almost 40% of the study participants had various forms of cardiomyopathy or valvular heart disease ( brouwer et al . finally , in a substudy of the gissi - hf trial ( 566 heart failure patients with implanted icd - devices , 57% with previous myocardial infarction , mean follow - up 928 days ) a statistically non - significant trend toward a lower risk of icd - discharge in patients treated with -3 was shown [ adjusted hazard ratio ( hr ) 0.80 ; 95% ci 0.591.09 ; p = 0.152 ] . heterogeneity also could be the result of different concomitant medications of the study populations including -blockers , digoxin , amiodarone , and sotalol . a potential influence of medication on the effect of fish oil supplementation may be indicated by the results of the dart 2 study ( burr et al . in the controlled prospective dart 2 trial conducted with general practitioners of south wales male patients with stable angina ( n = 3,114 , under 70 years of age ) were randomly allocated to four study groups with specific nutritional advises including the advise to eat oily fish or take fish oil capsules in two of the study groups . unfortunately , a conclusive interpretation of the dart 2 data is seriously limited as patient s recruitment and monitoring was interrupted for 1 year , long - term compliance was uncertain , and sudden death could not be ascertained in all cases ( burr et al . however , there was neither a reduction of scd ( 0.2% in both study arms ; hr 1.06 ; 95% ci 0.5520.07 ) nor of coronary death ( 0.3% in both study arms ; hr 0.94 ; 95% ci 0.571.56 ) or all - cause death ( control 2.8% , epa - group 3.1% ; hr 1.09 ; 95% ci 0.921.28 ) . furthermore , these low event rates may at least in part be the result of a high fish consumption of the japanese population at baseline . as most risk reduction already occurs at about 250 mg epa / dha intake per day ( mozaffarian and rimm , 2006 ) , a further increase of -3 intake may not have a substantial additional effect on cardiac death reduction ( mozaffarian , 2007 ) . in the alpha - omega - study , a multicenter , double - blind , placebo - controlled trial , 4,837 patients in the chronic stable phase after myocardial infarction ( average 3.7 years after ami ) and 6080 years of age ( 21.8% women ) were randomly assigned to one of four trial arms . after a follow - up of 40 months , 13.9% of the patients had a major cardiovascular event ( death , non - fatal cardiovascular events , or cardiac intervention ) . importantly , a high percentage of the patients received state of the art medication , including statins . in a post hoc analysis after unblinding of the data in the subgroup of patients with diabetes ventricular - arrhythmia - related events tended to be reduced in the epa / dha group ( hr 0.51 ; 95% ci 0.241.11 ) and significantly were reduced in the ala group ( hr 0.39 ; 95% ci 0.170.88 ) . in a secondary analysis of the alpha - omega trial taking high risk patients with previous myocardial infarction and diabetes the epa / dha + ala group experienced significantly less ventricular - arrhythmia - related events ( hr 0.16 ; 95% ci 0.040.69 ; kromhout et al . these differential results support the necessity to exactly define the clinical conditions under which supplementation of -3 may be beneficial . in the omega trial the effect of supplementation with 1 g / day of esterified epa / dha on the rate of scd and other clinical events within 1 year after ami was tested in 3,851 patients ( 25.6% female , mean age 64.0 years ; rauch et al . a 1-year follow - up was chosen , as the risk of cardiac death after ami including a presumed arrhythmic death is highest in the first 3 months after the event ( solomon et al . under these conditions , the rates of scd were 1.5% in both study groups ( or 0.95 ; 95% ci 0.561.60 ) and total mortality was 4.6% in the -3 group and 3.7% in the control group ( or 1.25 ; 95% ci 0.901.72 ) . in none of the predefined secondary endpoints , including total death , major adverse cardiovascular and cerebrovascular events , and revascularization procedures in survivors , was found a significant difference between the study groups , and not even a trend in favor of the -3 group could be observed . furthermore , there was no significant difference between the study groups with regard to scd or total death in any of the predefined subgroups of patients with higher risk ( diabetes , age > 70 years , no acute revascularization , ejection fraction < 35% ) . despite these apparently homogeneous results their interpretation is limited as the case estimate in the omega - study was based on an overestimation of the rate of scd in the control group , thereby leading to an underpowering of the study . two other randomized controlled trials published recently also failed to show a clear beneficial effect of -3 supplementation . in 563 elderly norwegian men at high cardiovascular risk a non - significant tendency to a reduced all - cause mortality could be observed ( hr 0.53 ; 95% ci 0.271.04 ) , but the rate of cardiovascular events remained unchanged ( hr 0.89 ; 95% ci 0.551.45 , follow - up 3 years ; einvik et al . in 2,501 patients with a history of myocardial infarction , unstable angina or ischemic stroke supplementation with epa / dha was not associated with a significant decrease of major vascular events during a follow - up of 4.7 years ( hr 1.08 ; 95% ci 0.791.47 ; galan et al . which conclusions may be drawn from the clinical studies and the animal studies discussed above ? the effect of -3 supplementation may depend on the background diet and the pre - existent intake of fish oil ( mozaffarian and rimm , 2006 ; reiffel and mcdonald , 2006 ; mozaffarian , 2007 ) . in the gissi trial ( inclusion period october 1993 to september 1995 ) only 4.4% of the patients had acute coronary revascularization at baseline , and only 4.7% were on cholesterol - lowering drugs at hospital discharge , increasing to only 46% after 42 months of follow - up ( gissi - prevenzione investigators , 1999 ) . furthermore , only 43.9% of the patients included in the gissi trial were on beta - blocker treatment at the start of the study , and this percentage decreased during follow - up . it therefore may be speculated that up - to - date guideline adjusted treatment of ami ( including acute revascularization , medical treatment , and support of life style changes ) may interfere with molecular and cellular -3 interactions thereby weakening or competing with a potential beneficial -3 effect . the anti - arrhythmic effect of -3 may depend on the pathophysiological conditions that facilitate arrhythmias . the clinical and experimental data outlined above suggest that -3 supplementation may especially protect against ischemia - induced arrhythmias . , revascularization , beta - blockers , statins , ace - inhibitors , inhibition of thrombocyte aggregation , physical exercise ) could attenuate a potentially beneficial effect of -3 . beta - blockers are well known to prevent sudden death , and even statins could have some anti - arrhythmic effects ( anh and marine , 2004 ; lorenz et al . potential anti - arrhythmic effects of -3 by augmentation of vagal activity ( mozaffarian et al . the anti - arrhythmic effect proven under experimental conditions in animal models and suggested in the earlier clinical studies appears to depend on the clinical conditions being studied . these clinical conditions are determined by the type and stage of the underlying myocardial disease and represent a sum of various pathophysiological conditions ( including ischemia , reperfusion , ischemic preconditioning , scar tissue , inflammation , congenital defects , etc . ) and the effects of modern medication including beta - blockers , ace - inhibitors , statins , and other interventions potentially interfering with the arrhythmic risk , such as exercise training . these considerations may also apply to the role of -3 in the prevention of atrial fibrillation . , 2008 , postmyocardial infarction patients ) were not confirmed in more recent studies and meta - analyses ( kowey et al . similar to the prevention of ventricular tachyarrhythmias , prevention of atrial fibrillation therefore may depend on distinct clinical and pathophysiological conditions and concomitant medication . the ongoing opera - trial , including a total of 1,516 patients scheduled for cardiac surgery and in sinus rhythm , will give more insight into the potential role of -3 supplementation to prevent post - operative atrial fibrillation ( mozaffarian et al . for understanding the seemingly heterogeneous efficacy of -3 supplementation in preventing tachyarrhythmias , it is important to reflect on their molecular and cellular interactions as has been delineated extensively in recent reviews ( leaf et al . in the following , only some aspects of potential relevance for interpretation of the clinical data are discussed : there are three major ways in which -3 may interfere with cellular and membrane function , thereby potentially moderating cardiac rhythm : direct interactions of -3 with membrane bound proteins like the fast sodium channel , the voltage - gated l - type ca channel , specific potassium channels , and the na / ca - exchanger ( hallaq et al . , 2010 ) such interactions may occur predominantly with circulating -3 when it is delivered by acute administration and infusion . incorporation into the phospholipid bilayer , thereby potentially changing membrane fluidity , and/or forming -3 rich micro - domains , and/or interacting with internal binding sites . this may result in a change of the function of membrane bound proteins like ion channels , receptors and signal transduction systems ( mcmurchie et al . incorporation into the cellular membranes predominantly is achieved by dietary long - term administration of -3 . differential effects of circulating versus incorporated -3 have also been demonstrated with respect to various potassium channels and the regulation of calcium homeostasis ( den ruijter et al . incorporated -3 , however , also may prevent further action potential ( ap ) shortening induced by circulating -3 . patients with high levels of incorporated -3 therefore may not have a further benefit from short term -3 supplementation ( den ruijter et al . this could be of a direct clinical relevance , as acute -3 supplementation may be used for prevention of atrial fibrillation induced by cardiac surgery , which is being investigated in the opera - trial ( mozaffarian et al . apart from these considerations the molecular interactions of -3 and their effects on cardiac rhythm may be influenced by a large variety of additional conditions : the various kinds of -3 formulations being used ( re - esterified triacylglycerides , ethyl - esters or phospholipids ; neubronner et al . fish oil shortens cardiac ap and accentuates the ap notch , which may lead to depression or even loss of the ap dome ( verkerk et al . under clinical conditions where the ap is prolonged triggered activity may be the predominant pro - arrhythmic mechanism , which could be inhibited in isolated cardiomyocytes from rabbits and from patients with end stage heart failure by superfusion with -3 ( den ruijter et al . conversely , ap shortening also may be pro - arrhythmic by reducing the refractory period and thereby promoting re - entry . in the clinical situation therefore supplementation with -3 may prevent or facilitate ventricular tachyarrhythmias depending on the predominant underlying arrhythmic mechanism ( den ruijter et al . based on these considerations it becomes apparent that -3 do not have a specific way to act , but rather possess multiple sites of potential actions , that may be influenced by a number of external conditions at the cellular and molecular level . multiple sites of interaction between -3 and myocardial tissue in combination with various possible ways of interference with these biochemical interactions are unlikely to result in an unequivocally predictable and homogeneous beneficial effect on clinical outcomes . -3 clearly interfere with the physiology of myocardial cell membranes through a variety of specific and unspecific pathways , and thereby exhibit anti - arrhythmic effects under certain well - defined experimental and clinical conditions this complexity makes it difficult to predict the effects of -3 supplementation on cardiac rhythm within the wide variety of conditions that represent clinical practice . for the future it will be necessary to define exactly the clinical conditions in which supplementation with of -3 is beneficial , and without potentially harmful effects bernhard rauch has no conflicts of interest ; jochen senges received honoraria for educational presentations from trommsdorff gmbh & co. kg arzneimittel , alsdorf , germany and pronova biopharma , lysaker , norway

naf is superior to tc - labeled phosphate agents in terms of bone image quality ( 1 , 2 ) . furthermore , the fast kinetics of naf enables image acquisition as early as 30 minutes post injection , improving patient compliance ( 3 , 4 ) . in fact , naf has been introduced as a bone imaging agent for positron emission tomography ( pet ) since the 1960s ( 5 ) . however , bone pet using naf could not be routinely used in clinical practice during those years due to the undeveloped pet imaging technology . the advent of gamma imaging technology and the development of tc - labeled phosphate agents in the 1970s have almost replaced bone pet with the bone scan ( 6 ) . the recent innovative advancement of the pet technique allowed naf to resurface as a promising bone imaging agent ( 3 , 7 ) . pet integrated with ct , yielding pet / ct , further improved the lesion detection rate with the aid of anatomical information from the ct . thanks to the effective attenuation correction algorithm , pet generated a more accurate quantitative image data set than gamma camera imaging ( 7 - 9 ) . furthermore , the supply crisis of mo , the parent radionuclide of tc , in 2010 had many physicians pay more attention to the usefulness of bone pet using naf ( 1 , 4 ) . bone pet is able to effectively evaluate not only malignant diseases ( 8 - 10 ) , but also benign bone diseases ( 11 - 13 ) . previously , we reported the utility of bone pet for the evaluation of bone abnormalities for the first time in korea ( 14 ) . however , in the previous study , bone pet was not directly compared to the bone scan for the detection rate of bone metastasis ( bm ) . thus , in the current study , we aimed to compare the diagnostic accuracies between bone pet and bone scan in cancer patients who underwent both imaging studies . sixteen cancer patients ( m : f = 10:6 , mean age = 60 12 ) were enrolled in this study . eleven of these patients were included in our previous report ( 14 ) , which did not involve the head - to - head comparison between the bone scan and the bone pet . underlying diseases of the patients included breast cancer ( n = 5 ) , prostate cancer ( n = 5 ) , thyroid cancer ( n = 3 ) , gastric cancer ( n = 1 ) , colon cancer ( n = 1 ) , and larynx cancer ( n = 1 ) ( table 1 ) . the bone scan and the bone pet were performed within 1 month apart from each other . the reasons for the bone evaluation were regular follow - up ( n = 10 ) , presence of suspicious bm ( n = 5 ) , and post - therapeutic evaluation of the known bm ( n = 1 ) . this study was approved by the institutional review board of seoul national university bundang hospital . a bone scan was performed using tc - hydroxymethylenediphosphonic acid ( hdp , mallinckrodt , st . whole body bone scan images were acquired using a dual - head gamma camera ( forte , adac - philips , holt , mo , usa ) equipped with low energy high resolution collimator 3 hours post tc - hdp injection . naf was generated from a reaction of o ( p , n)f using an in - house cyclotron ( kotron13 , kirams , seoul , korea ) . ten minutes later , furosemide ( 10 mg , lasix ) was injected in order to flush the activity of the renal pelvis or urinary excretory system . bone pet images were obtained from the skull base to the upper thigh or from the skull vertex to the feet , as requested by the physicians who had set the orders starting 30 minutes post naf injection . a dedicated pet scanner ( allegro , philips medical systems , andover , ma , usa ) was used for 9 patients prior to the year 2009 ( 14 ) and a pet / ct scanner ( dvct , ge healthcare , milwaukee , wi , usa ) was used for 7 patients afterwards . pet images from the dedicated pet scanner were attenuation - corrected using cs transmission scans and then reconstructed using an iterative algorithm ( row - action maximum - likelihood , philips medical systems , andover , ma , usa ) . pet / ct images were attenuation - corrected using the ct scan and then reconstructed using a 3-dimensional ordered - subset iteration algorithm ( vue point , ge healthcare , milwaukee , wi , usa ) . the resolutions of the reconstructed trans - axial images were 4.8 mm and 5.1 mm for pet and pet / ct , respectively . a positive finding for bm was defined as the presence of an abnormally high bony uptake , which is not associated with typical degenerative , traumatic or periarticular lesions ( 8) . diagnostic accuracies were also analyzed using the receiver operating characteristic ( roc ) curve analysis by utilizing a 4-point grading system ; definite , probable , less likely , and no evidence of bm . consensus was reached by two nuclear medicine physicians in order to call a lesion bm . a gold standard for bm was either the presence of typical findings compatible with bm in at least 2 imaging studies among mri , f - fdg pet / ct or i whole body scan , or the presence of a clinical progression causing a change of treatment plan during at least a one - year follow - up . those who had at least one proven bm lesion were considered bm positive patients regardless of the presence of any false positive bm findings . mcnemar 's chi - square test or fisher 's exact test or roc analysis was used for the comparison of the diagnostic accuracy . sixteen cancer patients ( m : f = 10:6 , mean age = 60 12 ) were enrolled in this study . eleven of these patients were included in our previous report ( 14 ) , which did not involve the head - to - head comparison between the bone scan and the bone pet . underlying diseases of the patients included breast cancer ( n = 5 ) , prostate cancer ( n = 5 ) , thyroid cancer ( n = 3 ) , gastric cancer ( n = 1 ) , colon cancer ( n = 1 ) , and larynx cancer ( n = 1 ) ( table 1 ) . the bone scan and the bone pet were performed within 1 month apart from each other . the reasons for the bone evaluation were regular follow - up ( n = 10 ) , presence of suspicious bm ( n = 5 ) , and post - therapeutic evaluation of the known bm ( n = 1 ) . this study was approved by the institutional review board of seoul national university bundang hospital . a bone scan was performed using tc - hydroxymethylenediphosphonic acid ( hdp , mallinckrodt , st . whole body bone scan images were acquired using a dual - head gamma camera ( forte , adac - philips , holt , mo , usa ) equipped with low energy high resolution collimator 3 hours post tc - hdp injection . naf was generated from a reaction of o ( p , n)f using an in - house cyclotron ( kotron13 , kirams , seoul , korea ) . ten minutes later , furosemide ( 10 mg , lasix ) was injected in order to flush the activity of the renal pelvis or urinary excretory system . bone pet images were obtained from the skull base to the upper thigh or from the skull vertex to the feet , as requested by the physicians who had set the orders starting 30 minutes post naf injection . a dedicated pet scanner ( allegro , philips medical systems , andover , ma , usa ) was used for 9 patients prior to the year 2009 ( 14 ) and a pet / ct scanner ( dvct , ge healthcare , milwaukee , wi , usa ) was used for 7 patients afterwards . pet images from the dedicated pet scanner were attenuation - corrected using cs transmission scans and then reconstructed using an iterative algorithm ( row - action maximum - likelihood , philips medical systems , andover , ma , usa ) . pet / ct images were attenuation - corrected using the ct scan and then reconstructed using a 3-dimensional ordered - subset iteration algorithm ( vue point , ge healthcare , milwaukee , wi , usa ) . the resolutions of the reconstructed trans - axial images were 4.8 mm and 5.1 mm for pet and pet / ct , respectively . a positive finding for bm was defined as the presence of an abnormally high bony uptake , which is not associated with typical degenerative , traumatic or periarticular lesions ( 8) . diagnostic accuracies were also analyzed using the receiver operating characteristic ( roc ) curve analysis by utilizing a 4-point grading system ; definite , probable , less likely , and no evidence of bm . consensus was reached by two nuclear medicine physicians in order to call a lesion bm . a gold standard for bm was either the presence of typical findings compatible with bm in at least 2 imaging studies among mri , f - fdg pet / ct or i whole body scan , or the presence of a clinical progression causing a change of treatment plan during at least a one - year follow - up . those who had at least one proven bm lesion were considered bm positive patients regardless of the presence of any false positive bm findings . mcnemar 's chi - square test or fisher 's exact test or roc analysis was used for the comparison of the diagnostic accuracy . of the 16 patients in the current study , 8 ( 50.0% ) proved to be bm positive and 8 ( 50.0% ) as bm negative . of the 8 bm ( + ) patients , 2 had disseminated metastatic lesions , as shown in figure 1 . all of the 8 bm ( + ) patients were categorized as having bm in the bone pet , yielding 100% sensitivity , whereas 7 of the 8 bm ( + ) patients were positive in the bone scan , yielding an 87.5% sensitivity ; the sensitivities were not statistically different between the bone pet and the bone scan ( p > 0.05 ) . of the 8 bm ( - ) patients , 7 were correctly categorized as having no bm in the bone pet , yielding an 87.5% ( 7/8 ) specificity , whereas only 2 were correctly diagnosed in the bone scan , yielding a 25.0% ( 2/8 ) specificity . as a result , a bone pet had a significantly higher specificity than a bone scan in the patient - based analysis ( p < 0.05 ) . a case showing higher specificity of a bone pet than a bone scan is demonstrated in figure 2 . overall , the accuracy of bone pet ( 93.8% = 15/16 ) was significantly greater than that of the bone scan ( 56.3% = 9/16 , p < 0.05 ) in the patient - based analysis ( table 2 ) . lesion - based analysis was conducted for the bone lesions identified by imaging studies , namely bone pet , bone scan , or ct . this is because benign bone lesions without abnormal findings in the imaging studies can not be the lesions of interest in clinical practice . table 1 ) were excluded in the lesion - based analysis because it was not sensible to identify a few more lesions in the clinical context of disseminated bm . as a result , 43 bone lesions were found in 14 patients . of the 43 lesions , 31 were proven to be malignant and 12 were benign . all of the 31 bm lesions were positive in the bone pet ( sensitivity 100% = 31/31 ) , whereas only 12 bm lesions were positive in the bone scan ( sensitivity 38.7% = 12/31 ) . as a result , bone pet was found to be significantly more sensitive than the bone scan ( p < 0.01 ) ( fig . specificity was also significantly greater for bone pet ( 75% = 9/12 ) than the bone scan ( 8.3% = 1/12 ) ( p < 0.05 ) . overall , the accuracy of bone pet ( 93.0% = 40/43 ) was significantly greater than that of the bone scan ( 30.2% = 13/43 , p < 0.005 ) in the lesion - based analysis ( table 3 ) . three false positive bone pet lesions were noted in 3 patients ( nos . 5 , 6 , and 9 ) . the first two were sacrum and rib lesions from patients with spinal bone metastases proven by a spine mri . with regard to the other proven bone metastases , the two lesions were strongly suggested to be clinical metastatic lesions . however , bone pet positive sacral lesion was negative in f - fdg pet / ct , while other bone pet positive lesions were all positive in the same patient . in addition , bone pet positive rib lesion was clinically stable without any evidence of a disease progression for 4 years and 2 months afterward . thus , the 2 lesions were considered to be benign . for the last bone lesion in patient no.9 ( fig . 4 ) , the rib lesion appeared to be positive in both the bone scan and the bone pet ; however , it was negative on the f - fdg pet / ct and the i whole body scan . serum thyroglobulin ( < 0.2 ng / ml ) and anti - thyroglobulin antibody ( < 25 u / ml ) were undetectable . the lesion was considered to be a benign lesion because other imaging studies were all negative ; moreover , there was no evidence of a disease progression thereafter for more than one year . in the roc curve analyses , the bone pet was significantly more accurate than the bone scan for the detection of bm . in the patient - based analysis , the area under the curve ( auc ) of the bone pet ( auc = 0.992 , standard error [ se ] = 0.0110 , 95% confidence interval [ ci ] = 0.781 - 1.000 ) was significantly higher than that of the bone scan ( auc = 0.750 , se = 0.118 , 95% ci = 0.476 - 0.927 ) ( p = 0.0306 ) . in addition , in the lesion - based analysis , the auc of the bone pet ( auc = 0.976 , se = 0.0155 , 95% ci = 0.876 - 0.999 ) was significantly greater than that of the bone scan ( auc = 0.691 , se = 0.0752 , 95% ci = 0.532 - 0.823 ) ( p = 0.0001 ) ( fig . 5 ) . no difference was observed between the two studies in both patient and lesion - based analyses ( table 4 ) . of the 16 patients in the current study , 8 ( 50.0% ) proved to be bm positive and 8 ( 50.0% ) as bm negative . of the 8 bm ( + ) patients , 2 had disseminated metastatic lesions , as shown in figure 1 . all of the 8 bm ( + ) patients were categorized as having bm in the bone pet , yielding 100% sensitivity , whereas 7 of the 8 bm ( + ) patients were positive in the bone scan , yielding an 87.5% sensitivity ; the sensitivities were not statistically different between the bone pet and the bone scan ( p > 0.05 ) . of the 8 bm ( - ) patients , 7 were correctly categorized as having no bm in the bone pet , yielding an 87.5% ( 7/8 ) specificity , whereas only 2 were correctly diagnosed in the bone scan , yielding a 25.0% ( 2/8 ) specificity . as a result , a bone pet had a significantly higher specificity than a bone scan in the patient - based analysis ( p < 0.05 ) . a case showing higher specificity of a bone pet than a bone scan is demonstrated in figure 2 . overall , the accuracy of bone pet ( 93.8% = 15/16 ) was significantly greater than that of the bone scan ( 56.3% = 9/16 , p < 0.05 ) in the patient - based analysis ( table 2 ) . lesion - based analysis was conducted for the bone lesions identified by imaging studies , namely bone pet , bone scan , or ct . this is because benign bone lesions without abnormal findings in the imaging studies can not be the lesions of interest in clinical practice . table 1 ) were excluded in the lesion - based analysis because it was not sensible to identify a few more lesions in the clinical context of disseminated bm . as a result , 43 bone lesions were found in 14 patients . of the 43 lesions , 31 were proven to be malignant and 12 were benign . all of the 31 bm lesions were positive in the bone pet ( sensitivity 100% = 31/31 ) , whereas only 12 bm lesions were positive in the bone scan ( sensitivity 38.7% = 12/31 ) . as a result , bone pet was found to be significantly more sensitive than the bone scan ( p < 0.01 ) ( fig . specificity was also significantly greater for bone pet ( 75% = 9/12 ) than the bone scan ( 8.3% = 1/12 ) ( p overall , the accuracy of bone pet ( 93.0% = 40/43 ) was significantly greater than that of the bone scan ( 30.2% = 13/43 , p < 0.005 ) in the lesion - based analysis ( table 3 ) . three false positive bone pet lesions were noted in 3 patients ( nos . 5 , 6 , and 9 ) . the first two were sacrum and rib lesions from patients with spinal bone metastases proven by a spine mri . with regard to the other proven bone metastases , the two lesions were strongly suggested to be clinical metastatic lesions . however , bone pet positive sacral lesion was negative in f - fdg pet / ct , while other bone pet positive lesions were all positive in the same patient . in addition , bone pet positive rib lesion was clinically stable without any evidence of a disease progression for 4 years and 2 months afterward . thus , the 2 lesions were considered to be benign . for the last bone lesion in patient no.9 ( fig . 4 ) , the rib lesion appeared to be positive in both the bone scan and the bone pet ; however , it was negative on the f - fdg pet / ct and the i whole body scan . serum thyroglobulin ( < 0.2 ng / ml ) and anti - thyroglobulin antibody ( < 25 u / ml ) were undetectable . the lesion was considered to be a benign lesion because other imaging studies were all negative ; moreover , there was no evidence of a disease progression thereafter for more than one year . in the roc curve analyses , the bone pet was significantly more accurate than the bone scan for the detection of bm . in the patient - based analysis , the area under the curve ( auc ) of the bone pet ( auc = 0.992 , standard error [ se ] = 0.0110 , 95% confidence interval [ ci ] = 0.781 - 1.000 ) was significantly higher than that of the bone scan ( auc = 0.750 , se = 0.118 , 95% ci = 0.476 - 0.927 ) ( p = 0.0306 ) . in addition , in the lesion - based analysis , the auc of the bone pet ( auc = 0.976 , se = 0.0155 , 95% ci = 0.876 - 0.999 ) was significantly greater than that of the bone scan ( auc = 0.691 , se = 0.0752 , 95% ci = 0.532 - 0.823 ) ( p = 0.0001 ) ( fig . no difference was observed between the two studies in both patient and lesion - based analyses ( table 4 ) . bone pet has been proven to be a useful bone imaging modality worldwide ( 1 , 4 ) . the combination of excellent physico - chemical characteristics of naf and robust imaging techniques of modern pet scanner enabled the bone pet to be the most promising tool for bone imaging . not only malignant bone diseases ( 8 - 10 ) , but benign metabolic bone diseases ( 11 - 13 ) have also been successfully investigated using the bone pet . furthermore , pet integrated with ct , yielding pet / ct , increased the diagnostic accuracy of bone pet in a variety of bone diseases ( 16 ) . in korea , there has been only one clinical report on naf bone pet ( 14 ) . the study investigated only the feasibility of naf bone pet in both cancer and benign bone disease patients . however , a critical question regarding the diagnostic accuracy of bone pet vs. bone scan for the detection of bm has not been addressed in the previous report . thus , in the current study , we performed a direct comparison between the diagnostic accuracies of naf bone pet vs. tc - hdp bone scan in cancer patients who underwent both imaging studies . the major finding of the current study is that the bone pet was found to be more accurate than the bone scan in the evaluation of bm and further , bone pet was shown to improve the sensitivity ( in the lesion - based analysis ) and specificity for bm . bone pet could effectively exclude non - metastatic bone lesions , which was reflected by its high specificity , compared to the bone scan in both patient- and lesion - based analyses . the improved specificity of the bone pet compared to the bone scan seems to be mostly due to the tomographic nature of a pet and/or supplemental information from a ct ( 1 , 4 , 15 ) . planar bone scan images often fail to give confidence to readers regarding the exact location of the bone lesions due to the lack of depth information . on the contrary , of course , if we had adopted a single photon emission computed tomography ( spect ) instead of the simple planar bone scan , the diagnostic accuracy of tc - hdp bone imaging would have been better ( 16 ) , although a whole body coverage by spect might have been unrealistic due to the limited axial field of view of the current gamma camera . another reason for the high specificity of bone pet compared to a bone scan also could be explained by the way the patients were recruited to the current study . bone scan has already been proven to be useful as an initial screening test for bm evaluation ( 6 , 15 ) , and patients without any abnormality on their bone scans are not regarded as candidates for further tests , such as bone pet . therefore , due to the test referral bias , true negative bone scan cases are typically excluded in this kind of retrospective study , resulting in a low specificity of the bone scan . however , bone pet and bone scan had comparable sensitivities in the patient - based analysis ( table 2 ) . with regard to the sensitivity of bm , bone pet seems to have a physico - chemical advantage over the bone scan ( 8 , 17 ) . the high first - pass extraction , rapid blood clearance , and low protein binding of naf provide bone pet imaging with a high image - quality and a high lesion - contrast ( 3 , 7 ) . in our cases , bone pet clearly showed a more prominent uptake pattern compared to the bone scan in most of the bm lesions ( figs . 1 - 3 ) , providing the readers with more confidence in the reading nevertheless , given the comparable sensitivity in the patient level ( table 2 ) and the relatively low cost , the bone scan may continue to be the primary screening study for the evaluation of bm for the time being . in fact , one crucial obstacle in the clinical application of bone pet is the cost - effectiveness of bone pet over the bone scan ( 10 ) . first , pet imaging is quantitative , whereas gamma camera imaging is not ( 1 , 4 , 7 ) . bone blood flow and bone turnover could be quantitatively measured using dynamic naf pet studies in malignant bone disease , paget disease , or renal osteodystropy ( 3 , 11 , 18 , 19 ) . second , patient compliance for a bone pet should be better than that for a bone scan because it only takes about 0.5 - 1 hour for a bone pet acquisition in order to get started after the injection , which is shorter than the time for a bone scan , which takes at least 3 - 4 hours . third , none - medical issues , such as the supply problem of mo and the parent radionuclide of tc may advance the clinical use of naf bone pet faster than expected . in this regard , physicians need to increase their awareness and pay closer attention to the development of naf bone pet in the future . in conjunction with all the mentioned points above , the unique strength of the current study could be the comparison of the diagnostic accuracies between bone pet / ct and bone pet . although the analyzed number of patients ( n = 16 ) or lesions ( n = 43 ) were quite low compared to other studies , the competency of bone pet was not inferior to that of bone pet / ct ( table 4 ) . although bone pet / ct may be more accurate for the identification of a particular bm lesion than the bone pet ( fig . 2 ) , overall , the two studies were equivalent in terms of detection of bm in the level of patients and individual lesions ( table 4 ) . if the results could be advocated in other larger scale studies , it might provide some chances of clinical utility to the out - dated dedicated pet scanners in a few hospitals in korea , including one in our hospital . the characteristics of bm ( i.e. , osteoblastic , osteolytic , or mixed ) or the known predilection site of bm may also have played some roles for determining the diagnostic accuracy of the bone pet . the heterogeneity of patient diseases also may have affected the low diagnostic accuracy of the bone scan ; particularly with single malignant disease patients , such as breast cancer , a bone scan may have a better diagnostic accuracy than other studies ( 20 , 21 ) . furthermore , the fact that not all of the suspicious lesions were pathologically proven is another critical limitation of the study . 100% sensitivity of bone pet in the current study may overestimate the genuine diagnostic accuracy of the bone pet . some bone metastatic lesions have been reported to be contained within the bone marrow without involving the cortical bone ( 22 ) . if we had performed more f - fdg pet / ct studies , such bm lesions may have been missed by the bone pet , but observed by f - fdg pet / ct , generating a sensitivity of bone pet to be lower than 100% . f - fdg pet has been reported to be more accurate than tc - mdp bone scan ( 21 ) ; however , f - fdg pet seems to have a lower accuracy for bm than naf bone pet ( 14 ) . as a matter of fact , not only f - fdg pet / ct , but also mri sometimes offers equivocal findings regarding the nature of bone lesions ( 20 ) . without a doubt , a bone biopsy would determine the exact nature of the bone lesion ; yet , it is not always a practical approach for patient management . furthermore , the cost - effectiveness of a bone pet was not evaluated in this study . in this regard , further large scale studies are required to determine the role of the bone pet for the evaluation of bm in the current medical reimbursement condition of korea . naf bone pet is more accurate than tc - hdp bone scan for the evaluation of bm . bone pet by itself has a potential to be a gold standard test for bm . however , without sufficient confirmatory data in korea , we claim , solely from our findings , that the accuracy of bone pet is superior to that of the bone scan . the characteristics of bm ( i.e. , osteoblastic , osteolytic , or mixed ) or the known predilection site of bm may also have played some roles for determining the diagnostic accuracy of the bone pet . the heterogeneity of patient diseases also may have affected the low diagnostic accuracy of the bone scan ; particularly with single malignant disease patients , such as breast cancer , a bone scan may have a better diagnostic accuracy than other studies ( 20 , 21 ) . furthermore , the fact that not all of the suspicious lesions were pathologically proven is another critical limitation of the study . 100% sensitivity of bone pet in the current study may overestimate the genuine diagnostic accuracy of the bone pet . some bone metastatic lesions have been reported to be contained within the bone marrow without involving the cortical bone ( 22 ) . if we had performed more f - fdg pet / ct studies , such bm lesions may have been missed by the bone pet , but observed by f - fdg pet / ct , generating a sensitivity of bone pet to be lower than 100% . f - fdg pet has been reported to be more accurate than tc - mdp bone scan ( 21 ) ; however , f - fdg pet seems to have a lower accuracy for bm than naf bone pet ( 14 ) . as a matter of fact , not only f - fdg pet / ct , but also mri sometimes offers equivocal findings regarding the nature of bone lesions ( 20 ) . without a doubt , a bone biopsy would determine the exact nature of the bone lesion ; yet , it is not always a practical approach for patient management . furthermore , the cost - effectiveness of a bone pet was not evaluated in this study . in this regard , further large scale studies are required to determine the role of the bone pet for the evaluation of bm in the current medical reimbursement condition of korea . naf bone pet is more accurate than tc - hdp bone scan for the evaluation of bm . bone pet by itself has a potential to be a gold standard test for bm . however , without sufficient confirmatory data in korea , we claim , solely from our findings , that the accuracy of bone pet is superior to that of the bone scan .

objectivena18f bone positron emission tomography ( bone pet ) is a new imaging modality which is useful for the evaluation of bone diseases . here , we compared the diagnostic accuracies between bone pet and bone scan for the detection of bone metastasis ( bm).materials and methodssixteen cancer patients ( m : f = 10:6 , mean age = 60 12 years ) who underwent both bone pet and bone scan were analyzed . bone pet was conducted 30 minutes after the injection of 370 mbq na18f , and a bone scan was performed 3 hours after the injection of 1295 mbq 99mtc - hydroxymethylene diphosphonate.resultsin the patient - based analysis ( 8 patients with bm and 8 without bm ) , the sensitivities of bone pet ( 100% = 8/8 ) and bone scan ( 87.5% = 7/8 ) were not significantly different ( p > 0.05 ) , whereas the specificity of bone pet ( 87.5% = 7/8 ) was significantly greater than that of the bone scan ( 25% = 2/8 ) ( p < 0.05 ) . in the lesion - based analysis ( 43 lesions in 14 patients ; 31 malignant and 12 benign ) , the sensitivity of bone pet ( 100% = 31/31 ) was significantly greater than that of bone scan ( 38.7% = 12/31 ) ( p < 0.01 ) , and the specificity of bone pet ( 75.0% = 9/12 ) was also significantly higher than that of bone scan ( 8.3% = 1/12 ) ( p < 0.05 ) . the receiver operating characteristic curve analysis showed that bone pet was significantly more accurate than the bone scan in the patient ( p = 0.0306 ) and lesion ( p = 0.0001 ) based analyses.conclusionna18f bone pet is more accurate than bone scan for bm evaluation .

INTRODUCTION MATERIALS AND METHODS Subjects Bone Scan Na Interpretation of Bone PET and Bone Scan Statistical Analysis RESULTS Patient-Based Analysis Lesion-Based Analysis False Positive Findings of Bone PET ROC Curve Analysis Bone PET vs. Bone PET/CT DISCUSSION Limitation Conclusion

in fact , naf has been introduced as a bone imaging agent for positron emission tomography ( pet ) since the 1960s ( 5 ) . the advent of gamma imaging technology and the development of tc - labeled phosphate agents in the 1970s have almost replaced bone pet with the bone scan ( 6 ) . bone pet is able to effectively evaluate not only malignant diseases ( 8 - 10 ) , but also benign bone diseases ( 11 - 13 ) . previously , we reported the utility of bone pet for the evaluation of bone abnormalities for the first time in korea ( 14 ) . however , in the previous study , bone pet was not directly compared to the bone scan for the detection rate of bone metastasis ( bm ) . thus , in the current study , we aimed to compare the diagnostic accuracies between bone pet and bone scan in cancer patients who underwent both imaging studies . sixteen cancer patients ( m : f = 10:6 , mean age = 60 12 ) were enrolled in this study . eleven of these patients were included in our previous report ( 14 ) , which did not involve the head - to - head comparison between the bone scan and the bone pet . underlying diseases of the patients included breast cancer ( n = 5 ) , prostate cancer ( n = 5 ) , thyroid cancer ( n = 3 ) , gastric cancer ( n = 1 ) , colon cancer ( n = 1 ) , and larynx cancer ( n = 1 ) ( table 1 ) . the bone scan and the bone pet were performed within 1 month apart from each other . the reasons for the bone evaluation were regular follow - up ( n = 10 ) , presence of suspicious bm ( n = 5 ) , and post - therapeutic evaluation of the known bm ( n = 1 ) . a bone scan was performed using tc - hydroxymethylenediphosphonic acid ( hdp , mallinckrodt , st . diagnostic accuracies were also analyzed using the receiver operating characteristic ( roc ) curve analysis by utilizing a 4-point grading system ; definite , probable , less likely , and no evidence of bm . mcnemar 's chi - square test or fisher 's exact test or roc analysis was used for the comparison of the diagnostic accuracy . sixteen cancer patients ( m : f = 10:6 , mean age = 60 12 ) were enrolled in this study . eleven of these patients were included in our previous report ( 14 ) , which did not involve the head - to - head comparison between the bone scan and the bone pet . underlying diseases of the patients included breast cancer ( n = 5 ) , prostate cancer ( n = 5 ) , thyroid cancer ( n = 3 ) , gastric cancer ( n = 1 ) , colon cancer ( n = 1 ) , and larynx cancer ( n = 1 ) ( table 1 ) . the bone scan and the bone pet were performed within 1 month apart from each other . the reasons for the bone evaluation were regular follow - up ( n = 10 ) , presence of suspicious bm ( n = 5 ) , and post - therapeutic evaluation of the known bm ( n = 1 ) . a bone scan was performed using tc - hydroxymethylenediphosphonic acid ( hdp , mallinckrodt , st . a dedicated pet scanner ( allegro , philips medical systems , andover , ma , usa ) was used for 9 patients prior to the year 2009 ( 14 ) and a pet / ct scanner ( dvct , ge healthcare , milwaukee , wi , usa ) was used for 7 patients afterwards . diagnostic accuracies were also analyzed using the receiver operating characteristic ( roc ) curve analysis by utilizing a 4-point grading system ; definite , probable , less likely , and no evidence of bm . mcnemar 's chi - square test or fisher 's exact test or roc analysis was used for the comparison of the diagnostic accuracy . of the 16 patients in the current study , 8 ( 50.0% ) proved to be bm positive and 8 ( 50.0% ) as bm negative . all of the 8 bm ( + ) patients were categorized as having bm in the bone pet , yielding 100% sensitivity , whereas 7 of the 8 bm ( + ) patients were positive in the bone scan , yielding an 87.5% sensitivity ; the sensitivities were not statistically different between the bone pet and the bone scan ( p > 0.05 ) . of the 8 bm ( - ) patients , 7 were correctly categorized as having no bm in the bone pet , yielding an 87.5% ( 7/8 ) specificity , whereas only 2 were correctly diagnosed in the bone scan , yielding a 25.0% ( 2/8 ) specificity . as a result , a bone pet had a significantly higher specificity than a bone scan in the patient - based analysis ( p < 0.05 ) . a case showing higher specificity of a bone pet than a bone scan is demonstrated in figure 2 . overall , the accuracy of bone pet ( 93.8% = 15/16 ) was significantly greater than that of the bone scan ( 56.3% = 9/16 , p < 0.05 ) in the patient - based analysis ( table 2 ) . lesion - based analysis was conducted for the bone lesions identified by imaging studies , namely bone pet , bone scan , or ct . table 1 ) were excluded in the lesion - based analysis because it was not sensible to identify a few more lesions in the clinical context of disseminated bm . of the 43 lesions , 31 were proven to be malignant and 12 were benign . all of the 31 bm lesions were positive in the bone pet ( sensitivity 100% = 31/31 ) , whereas only 12 bm lesions were positive in the bone scan ( sensitivity 38.7% = 12/31 ) . as a result , bone pet was found to be significantly more sensitive than the bone scan ( p < 0.01 ) ( fig . specificity was also significantly greater for bone pet ( 75% = 9/12 ) than the bone scan ( 8.3% = 1/12 ) ( p < 0.05 ) . overall , the accuracy of bone pet ( 93.0% = 40/43 ) was significantly greater than that of the bone scan ( 30.2% = 13/43 , p < 0.005 ) in the lesion - based analysis ( table 3 ) . 4 ) , the rib lesion appeared to be positive in both the bone scan and the bone pet ; however , it was negative on the f - fdg pet / ct and the i whole body scan . serum thyroglobulin ( < 0.2 ng / ml ) and anti - thyroglobulin antibody ( < 25 u / ml ) were undetectable . in the roc curve analyses , the bone pet was significantly more accurate than the bone scan for the detection of bm . in the patient - based analysis , the area under the curve ( auc ) of the bone pet ( auc = 0.992 , standard error [ se ] = 0.0110 , 95% confidence interval [ ci ] = 0.781 - 1.000 ) was significantly higher than that of the bone scan ( auc = 0.750 , se = 0.118 , 95% ci = 0.476 - 0.927 ) ( p = 0.0306 ) . in addition , in the lesion - based analysis , the auc of the bone pet ( auc = 0.976 , se = 0.0155 , 95% ci = 0.876 - 0.999 ) was significantly greater than that of the bone scan ( auc = 0.691 , se = 0.0752 , 95% ci = 0.532 - 0.823 ) ( p = 0.0001 ) ( fig . no difference was observed between the two studies in both patient and lesion - based analyses ( table 4 ) . of the 16 patients in the current study , 8 ( 50.0% ) proved to be bm positive and 8 ( 50.0% ) as bm negative . all of the 8 bm ( + ) patients were categorized as having bm in the bone pet , yielding 100% sensitivity , whereas 7 of the 8 bm ( + ) patients were positive in the bone scan , yielding an 87.5% sensitivity ; the sensitivities were not statistically different between the bone pet and the bone scan ( p > 0.05 ) . of the 8 bm ( - ) patients , 7 were correctly categorized as having no bm in the bone pet , yielding an 87.5% ( 7/8 ) specificity , whereas only 2 were correctly diagnosed in the bone scan , yielding a 25.0% ( 2/8 ) specificity . as a result , a bone pet had a significantly higher specificity than a bone scan in the patient - based analysis ( p < 0.05 ) . a case showing higher specificity of a bone pet than a bone scan is demonstrated in figure 2 . overall , the accuracy of bone pet ( 93.8% = 15/16 ) was significantly greater than that of the bone scan ( 56.3% = 9/16 , p < 0.05 ) in the patient - based analysis ( table 2 ) . lesion - based analysis was conducted for the bone lesions identified by imaging studies , namely bone pet , bone scan , or ct . table 1 ) were excluded in the lesion - based analysis because it was not sensible to identify a few more lesions in the clinical context of disseminated bm . of the 43 lesions , 31 were proven to be malignant and 12 were benign . all of the 31 bm lesions were positive in the bone pet ( sensitivity 100% = 31/31 ) , whereas only 12 bm lesions were positive in the bone scan ( sensitivity 38.7% = 12/31 ) . as a result , bone pet was found to be significantly more sensitive than the bone scan ( p < 0.01 ) ( fig . specificity was also significantly greater for bone pet ( 75% = 9/12 ) than the bone scan ( 8.3% = 1/12 ) ( p overall , the accuracy of bone pet ( 93.0% = 40/43 ) was significantly greater than that of the bone scan ( 30.2% = 13/43 , p < 0.005 ) in the lesion - based analysis ( table 3 ) . 4 ) , the rib lesion appeared to be positive in both the bone scan and the bone pet ; however , it was negative on the f - fdg pet / ct and the i whole body scan . serum thyroglobulin ( < 0.2 ng / ml ) and anti - thyroglobulin antibody ( < 25 u / ml ) were undetectable . in the roc curve analyses , the bone pet was significantly more accurate than the bone scan for the detection of bm . in the patient - based analysis , the area under the curve ( auc ) of the bone pet ( auc = 0.992 , standard error [ se ] = 0.0110 , 95% confidence interval [ ci ] = 0.781 - 1.000 ) was significantly higher than that of the bone scan ( auc = 0.750 , se = 0.118 , 95% ci = 0.476 - 0.927 ) ( p = 0.0306 ) . in addition , in the lesion - based analysis , the auc of the bone pet ( auc = 0.976 , se = 0.0155 , 95% ci = 0.876 - 0.999 ) was significantly greater than that of the bone scan ( auc = 0.691 , se = 0.0752 , 95% ci = 0.532 - 0.823 ) ( p = 0.0001 ) ( fig . no difference was observed between the two studies in both patient and lesion - based analyses ( table 4 ) . not only malignant bone diseases ( 8 - 10 ) , but benign metabolic bone diseases ( 11 - 13 ) have also been successfully investigated using the bone pet . furthermore , pet integrated with ct , yielding pet / ct , increased the diagnostic accuracy of bone pet in a variety of bone diseases ( 16 ) . however , a critical question regarding the diagnostic accuracy of bone pet vs. bone scan for the detection of bm has not been addressed in the previous report . thus , in the current study , we performed a direct comparison between the diagnostic accuracies of naf bone pet vs. tc - hdp bone scan in cancer patients who underwent both imaging studies . the major finding of the current study is that the bone pet was found to be more accurate than the bone scan in the evaluation of bm and further , bone pet was shown to improve the sensitivity ( in the lesion - based analysis ) and specificity for bm . bone pet could effectively exclude non - metastatic bone lesions , which was reflected by its high specificity , compared to the bone scan in both patient- and lesion - based analyses . the improved specificity of the bone pet compared to the bone scan seems to be mostly due to the tomographic nature of a pet and/or supplemental information from a ct ( 1 , 4 , 15 ) . planar bone scan images often fail to give confidence to readers regarding the exact location of the bone lesions due to the lack of depth information . on the contrary , of course , if we had adopted a single photon emission computed tomography ( spect ) instead of the simple planar bone scan , the diagnostic accuracy of tc - hdp bone imaging would have been better ( 16 ) , although a whole body coverage by spect might have been unrealistic due to the limited axial field of view of the current gamma camera . another reason for the high specificity of bone pet compared to a bone scan also could be explained by the way the patients were recruited to the current study . bone scan has already been proven to be useful as an initial screening test for bm evaluation ( 6 , 15 ) , and patients without any abnormality on their bone scans are not regarded as candidates for further tests , such as bone pet . therefore , due to the test referral bias , true negative bone scan cases are typically excluded in this kind of retrospective study , resulting in a low specificity of the bone scan . however , bone pet and bone scan had comparable sensitivities in the patient - based analysis ( table 2 ) . with regard to the sensitivity of bm , bone pet seems to have a physico - chemical advantage over the bone scan ( 8 , 17 ) . the high first - pass extraction , rapid blood clearance , and low protein binding of naf provide bone pet imaging with a high image - quality and a high lesion - contrast ( 3 , 7 ) . in our cases , bone pet clearly showed a more prominent uptake pattern compared to the bone scan in most of the bm lesions ( figs . 1 - 3 ) , providing the readers with more confidence in the reading nevertheless , given the comparable sensitivity in the patient level ( table 2 ) and the relatively low cost , the bone scan may continue to be the primary screening study for the evaluation of bm for the time being . in fact , one crucial obstacle in the clinical application of bone pet is the cost - effectiveness of bone pet over the bone scan ( 10 ) . second , patient compliance for a bone pet should be better than that for a bone scan because it only takes about 0.5 - 1 hour for a bone pet acquisition in order to get started after the injection , which is shorter than the time for a bone scan , which takes at least 3 - 4 hours . in conjunction with all the mentioned points above , the unique strength of the current study could be the comparison of the diagnostic accuracies between bone pet / ct and bone pet . although the analyzed number of patients ( n = 16 ) or lesions ( n = 43 ) were quite low compared to other studies , the competency of bone pet was not inferior to that of bone pet / ct ( table 4 ) . although bone pet / ct may be more accurate for the identification of a particular bm lesion than the bone pet ( fig . 2 ) , overall , the two studies were equivalent in terms of detection of bm in the level of patients and individual lesions ( table 4 ) . , osteoblastic , osteolytic , or mixed ) or the known predilection site of bm may also have played some roles for determining the diagnostic accuracy of the bone pet . the heterogeneity of patient diseases also may have affected the low diagnostic accuracy of the bone scan ; particularly with single malignant disease patients , such as breast cancer , a bone scan may have a better diagnostic accuracy than other studies ( 20 , 21 ) . 100% sensitivity of bone pet in the current study may overestimate the genuine diagnostic accuracy of the bone pet . if we had performed more f - fdg pet / ct studies , such bm lesions may have been missed by the bone pet , but observed by f - fdg pet / ct , generating a sensitivity of bone pet to be lower than 100% . f - fdg pet has been reported to be more accurate than tc - mdp bone scan ( 21 ) ; however , f - fdg pet seems to have a lower accuracy for bm than naf bone pet ( 14 ) . without a doubt , a bone biopsy would determine the exact nature of the bone lesion ; yet , it is not always a practical approach for patient management . furthermore , the cost - effectiveness of a bone pet was not evaluated in this study . in this regard , further large scale studies are required to determine the role of the bone pet for the evaluation of bm in the current medical reimbursement condition of korea . naf bone pet is more accurate than tc - hdp bone scan for the evaluation of bm . however , without sufficient confirmatory data in korea , we claim , solely from our findings , that the accuracy of bone pet is superior to that of the bone scan . , osteoblastic , osteolytic , or mixed ) or the known predilection site of bm may also have played some roles for determining the diagnostic accuracy of the bone pet . the heterogeneity of patient diseases also may have affected the low diagnostic accuracy of the bone scan ; particularly with single malignant disease patients , such as breast cancer , a bone scan may have a better diagnostic accuracy than other studies ( 20 , 21 ) . 100% sensitivity of bone pet in the current study may overestimate the genuine diagnostic accuracy of the bone pet . if we had performed more f - fdg pet / ct studies , such bm lesions may have been missed by the bone pet , but observed by f - fdg pet / ct , generating a sensitivity of bone pet to be lower than 100% . f - fdg pet has been reported to be more accurate than tc - mdp bone scan ( 21 ) ; however , f - fdg pet seems to have a lower accuracy for bm than naf bone pet ( 14 ) . without a doubt , a bone biopsy would determine the exact nature of the bone lesion ; yet , it is not always a practical approach for patient management . furthermore , the cost - effectiveness of a bone pet was not evaluated in this study . in this regard , further large scale studies are required to determine the role of the bone pet for the evaluation of bm in the current medical reimbursement condition of korea . naf bone pet is more accurate than tc - hdp bone scan for the evaluation of bm . however , without sufficient confirmatory data in korea , we claim , solely from our findings , that the accuracy of bone pet is superior to that of the bone scan .

malaria is one of the most deadly infectious diseases in human history with 3.2 billion people in 97 countries at risk . an estimated 444,000 deaths from malaria were reported by the who in 2015 and 90% of these occurred in sub - saharan africa , mostly among children under the age of five . human malaria , which is transmitted by the female anopheles mosquito , can be caused by five species of plasmodia ; however , plasmodium falciparum and plasmodium vivax are the most signficant.p . falciparum is dominant in africa and accounts for most of the deaths , while p. vivax has a larger global distribution . to simplify treatment options malaria is a treatable disease and malarial control programs depend on drug therapy for treatment and chemoprevention , and on insecticides ( including insecticide impregnated bed nets ) to prevent transmission . a large collection of drugs has been used for the treatment of malaria , but many of the most important compounds have been lost to drug resistance ( e.g. , chloroquine and pyrimethamine ) . artemisinin combination therapies ( act ) replaced older treatments , becoming highly effective , crucial tools in global efforts that have led to the decline in malaria deaths over the past decade . however , resistance to the artemisinin components ( associated with kelch13 propeller protein mutations ) has been found in southeast asia putting at risk malaria treatment programs . to combat drug resistance a significant effort is underway to identify new compounds that can be used for the treatment of malaria , with several new entities currently in clinical development . the triazolopyrimidine dsm265 ( 1 ) ( figure 1 ) developed by our group is the first antimalarial agent that targets dihydroorotate dehydrogenase ( dhodh ) to reach clinical development , validating this target for the treatment of malaria . dhodh is a mitochondrial enzyme that is required for the fourth step of de novo pyrimidine biosynthesis , catalyzing the flavin - dependent oxidation of dihydroorotate to orotic acid with mitochondrially derived coenzyme q ( coq ) serving as a second substrate . pyrimidines are essential for both rna and dna biosynthesis , and because plasmodia do not encode pyrimidine salvage enzymes , which are found in humans and other organisms , the de novo pyrimidine pathway and dhodh are essential to the parasite . we identified the triazolopyrimidine dhodh inhibitor series by a target - based high throughput screen , and the initial lead dsm1 ( 2 ) ( figure 1 ) was shown to selectively inhibit p. falciparum dhodh and to kill parasites in vitro , but it was ineffective in vivo due to poor metabolic properties . the series was subsequently optimized to improve its in vivo properties resulting in the identification of dsm74 ( 3 ) , which while metabolically stable lacked potency . x - ray structures of 2 and 3 bound to pfdhodh were then used to guide the medicinal chemistry program in the search for more potent analogues , resulting in the identification of 1 . structures of selected triazolopyrimidine pfdhodh inhibitors . shown structures include , 2-(1,1-difluoroethyl)-5-methyl - n-[4-(pentafluoro--sulfanyl)phenyl]-triazolo[1,5-a]pyrimidin-7-amine ( 1 ) , ( 5-methyltriazolo[1,5-a]pyrimidin-7-yl)naphthalen-2-ylamine ( 2 ) , and ( 5-methyltriazolo[1,5-a]pyrimidin-7-yl)(4-trifluoromethylphenyl)amine ( 3 ) . while 1 has progressed successfully to phase ii clinical trials , we sought to identify potential backup compounds if unforeseen issues arise during its clinical development . importantly , 1 has potent activity on pfdhodh and p. falciparum parasites in vivo , and its pharmacokinetic properties support its use as a single dose treatment or once weekly prophylactic . however , while 1 has excellent selectivity against human dhodh and thus is not expected to show on target activity in humans , it does have some activity against rodent dhodhs that has complicated animal toxicity testing . we therefore sought to identify compounds that have broad selectivity against all mammalian dhodhs , while if possible identifying compounds with higher potency against pfdhodh that could potentially reduce the dose required for treatment . we describe herein a series of triazolopyrimidines where the sf5-aniline moiety of 1 has been replaced by either substituted 1,2,3,4-tetrahydro-2-naphthyl or 2-indanyl amines . compounds from both series are inactive against a range of mammalian dhodhs , while several of the identified tetrahydronapthalenes are more potent than 1 against p. falciparum parasites in vitro and indeed are among the most potent compounds that have been identified in the series . however , these compounds are less metabolically stable than 1 in mouse liver microsomes , suggesting the likelihood of higher clearance in mice and explaining the requirement for higher doses to inhibit parasites in the in vivo mouse model of malaria . activity relationships ( sars ) of replacing the p - sf5-aniline of 1 with substituted 1,2,3,4-tetrahydro-2-naphthyl or 2-indanyl amines with the goal of eliminating activity on rodent dhodhs , while potentially improving potency against p. falciparum dhodh and thereby lowering the dose required for in vivo efficacy . the clinical candidate 1 is a potent inhibitor of pfdhodh with only minimal activity detected against human dhodh ( selectivity window > 5000-fold ; table 1 ) . however , 1 showed inhibitory activity against rodent and to a lesser extent dog dhodhs , although potency toward these enzymes remained considerably less than for pfdhodh . because these species are required for toxicity testing in preclinical studies we sought to eliminate rodent and dog dhodh inhibitory activity for any potential candidate that might be developed as a backup to 1 . we previously reported that substitution of the p - sf5-aniline with p - cf3 aniline in the context of either mono or difluoro groups at the meta position led to greater inhibition of the mammalian enzymes . x - ray structural data suggested that increasing hydrophobicity drove the interaction of these compounds with the mammalian enzymes . we hypothesized based on these structures that increasing the size of the aromatic amine might prevent binding to mammalian dhodhs , while increasing potency toward the parasite enzyme . we previously tested several tetrahydro-2-naphthyl and 2-indanyl amines as substitutes for the cf3-aniline of 3 but found these compounds lacked potency , and furthermore , the one analogue containing 1,2,3,4-tetrahydro-2-naphthyl also lacked metabolic stability in mouse microsomes . however , these amines were never tested within the context of haloalkyl groups ( cf2ch3 or cf3 ) at the c2 position of the triazolopyrimidine ring , which was a key substitution required to boost the potency of the series . we therefore synthesized a series of tetrahydro-2-naphthyl or 2-indanyl analoues with halo alkyl groups at the c2 position . substituents ( halo , haloalkyl , me , cf3 , ome , so2me , and so2nme2 ) were incorporated onto the tetrahydro-2-naphthyl or 2-indanyl aromatic rings to improve metabolic stability over the unsubstituted analogues . dhodh ic50 and p. falciparum ec50 values were determined from triplicate data points at each concentration in the dose titration . compound 1 has been previously profiled in these assays , and parasite data were previously reported . the synthetic strategy to generate the appropriately substituted triazolopyrimidines has previously been described . for the studies described herein , 7-chloro-2-(1,1-difluoroethyl)-5-methyltriazolo[1,5-a ] pyrimidine ( 4a ) and 7-chloro-2-(trifluoromethyl)-5-methyltriazolo[1,5-a ] pyrimidine ( 4b ) were prepared using these methods ( scheme 1 ) and were reacted with the requisite amines to afford the final products containing either the 1,2,3,4-tetrahydro-2-naphthyl ( 525 ) or 2-indanyl ( 2637 ) amines in place of the p - sf5-aniline of 1 . most amines utilized for the study were commercially available with the exception that amines for compounds 2937 were synthesized as described in scheme 2 and supplementary scheme s1 . the 1,2,3,4-tetrahydro-2-naphthyls contained a chiral center , and for select compounds , individual enantiomers were purified using a chiral column as described in the experimental section . reagents and conditions : ( i ) ac2o , naoac , acoh , rt , 12 h ; ( ii ) clso3h , chcl3 , rt , 1 h ; ( iii ) ( a ) sncl22h2o , acoh , hcl , rt , 2 h ; ( b ) naome , mei , meoh ; ( iv ) mcpba , chcl3 ; ( v ) 3 n hcl . the amine precursor 5-(methylsulfonyl)-2,3-dihydro-1h - inden-2-aminehcl 43 was prepared in five steps ( scheme 2 ) . first , 2-aminoindane 38 was acetylated with acetic anhydride - sodium acetate to yield the acetamido derivative 39 . further , regioselective chlorosulfonylation of 39 led to the sulfonyl chloride intermediate 40 . reduction of the sulfonyl chloride to the thiol was achieved by tin chloride , which was converted to a methylthio derivative 41 using sodium methoxide and methyl iodide . next , oxidation of methylthio intermediate 41 with mcpba gave the acetyl protected methanesulfonyl derivative 42 . upon deprotection of this acetyl group using 3 n hcl , the required amine precursor 43 was obtained . compounds were first analyzed for potency against p. falciparum , p. vivax , and human dhodh , and for activity on p. falciparum parasites in vitro ( table 1 ) . compounds in the series showed ic50s against pfdhodh in the range of 0.0058 m and ec50s against p. falciparum 3d7 parasites in whole cell assays between 0.000396.5 m ( table 1 ) . generally , the tetrahydro-2-naphthyl compounds ( 525 ) were more potent in both assays than the 2-indanyls ( 2637 ) . the most potent analogues based on the whole cell p. falciparum assay were 9 and 13 , both of which contain a halogen ( chloro or bromo , respectively ) in the 7 position . compound 13 is the most potent analogue identified in the triazolopyrimidine series that has been reported . overall the trend was for 7-substituted tetrahydro-2-naphthyls to be more potent than those with substitutions at the 6 position . compounds with br and cl substituents ( 7 , 9 , 11 , 13 ) were more potent than those with fluoro 6 , ome ( 15 , 16 ) or with disubstitutions ( 19 , 20 , 21 ) , with the least active tetrahydro-2-naphthyl 21 containing a 6-cf3 , 7-f . potency of 21 in the whole cell parasite assay was worse than expected based on the inhibitory activity against pfdhodh , suggesting that the compound may be poorly transported into the parasite . analogues with cf3 at c2 ( 22 , 23 , 24 ) on the triazolopyrimidine ring were less active than those with cf2ch3 ( 5 , 6 , 9 ) at this position . within the indanyl series the most potent compounds were 32 and 36 containing 4,7-dimethyl or 4-cf3 substituents , respectively , while the least active compounds were 30 and 31 containing 5-so2me or 5-so2nme2 substituents , respectively . for the tetrahydro-2-naphthyls where individual enantiomers were characterized , differences in potency between the active enantiomer ( 7 , 9 , 11 , 13 , 16 , 19 , 24 ) and the inactive enantiomer ( 8 , 10 , 12 , 14 , 17 , 18 , 25 ) ranged from 20- to 360-fold toward pfdhodh and from 30- to 2000-fold against p. falciparum 3d7 parasites . the small molecule x - ray structure of the active enantiomer 9 was solved demonstrating that it was in the s configuration ( figure s1 and table s1 ) . we did not isolate the individual enantiomers for compounds from the indane series ( 27 , 28 , 30 , 31 , 34 , and 36 ) , and it is likely that the purified active enantiomers would also show higher activity for these compounds as well . thus , the reported potency data for the racemic mixture likely overestimates the true ic50 by 2-fold . overall we observed a good correlation between potency on pfdhodh and potency on 3d7 p. there was a tendency for the most potent compounds on the parasite to show lower potency against pfdhodh , but this difference is likely caused by the complication of stoichiometric binding for compounds with ic50s at or below the concentration of enzyme used in the assay ( pfdhodh = 5 nm ) . in order to confirm that , for the most potent compounds , parasite killing is due to dhodh inhibition , we tested select compounds ( 9 and 13 ) for their ability to inhibit growth of a genetically engineered parasite strain that expresses yeast dhodh ( d10 ydhodh ) . this strain is resistant to dhodh inhibitors if their mechanism of action is on target . the ec50s ( d10 ydhodh ec50 = 8.6 and 7.4 m for 9 and 13 , respectively ) for both compounds were increased by > 600-fold against this parasite strain relative to the wild - type 3d7 parasites , supporting dhodh inhibition as their mechanism of action ( table 1 and figure s3 ) . the addition of proguanil did not reverse the resistance of this strain , which is also consistent with dhodh as the mechanism of parasite killing . in contrast , it has been previously reported that proguanil reverses the atovaquone resistance phenotype of the d10 ydhodh parasite strain , providing a mechanism to distinguish between dhodh and bc1 targeted parasite growth inhibition . the weak but detectable activity observed on the d10 ydhodh parasites for both compounds suggests that at high concentrations there is a secondary target . for a couple of select compounds ( 9 and 35 ) whole cell activity was also tested on several additional cell lines including chloroquine and pyrimethamine resistant parasites ( table s2 ) . species selectivity was first evaluated by testing against human and p. vivax dhodh ( table 1 ) and then for a selection of the more potent compounds , inhibitor activity was also measured for mouse , rat , and dog dhodh ( table 2 ) . none of the compounds showed any inhibition of human dhodh up to the highest tested concentration ( 100 m ) , which is similar to results for 1 . however , unlike 1 , none of the compounds showed any activity against either the rodent or dog dhodhs ( table 2 ) , demonstrating that we had achieved our objective of eliminating mammalian dhodh activity . measured ic50s for p. vivax dhodh ranged from 1.510-fold higher than for pfdhodh . the 7-position substituted tetrahydro-2-naphthyls , 9 and 13 showed minimal differences ( 1.53-fold ) between the two enzymes suggesting that they would show good activity against both p. falciparum and p. vivax parasites , whereas the 6 position compounds ( 7 and 11 ) had 58-fold lower activity on p. vivax than p. falciparum dhodh . each replicate ic50 was determined from triplicate data points at each concentration in the dose titration . in order to understand the structural basis for the superior potency of 13 we solved its x - ray structure bound to pfdhodh . the structure was solved to 2.32 resolution at an rwork and rfree of 0.18 and 0.21 , respectively ( figure 2 and table s3 ) . strong electron density was observed for the entirety of 13 ( figure s4a ) . compound 13 was oriented in the pocket similarly to other triazolopyrimidines ( e.g. , 13(12,17,18 ) ) with the triazolopyrimidine ring bound adjacent to the flavin cofactor in position to form h - bond interactions with arg-265 and his-185 ( figures 2 and s4b ) . the tetrahydro-2-naphthyl moiety bound in a hydrophobic pocket forming edge - to - face stacking interactions with phe-227 and phe-188 and was in a very similar orientation to what we previously observed for the naphthyl moiety of 2 ( figure 2 ) . some modest conformational differences in ring geometry were observed reflecting the difference between the fully planar and aromatic naphthyl and the puckered configuration of the nonaromatic ring of the tetrahydro-2-naphthyl . a halogen bond was observed between the 7-bromo group on the tetrahydro-2-naphthyl of 13 and cys-233 sh , which likely provides significant binding energy to the enzyme inhibitor interaction . x - ray structure of pfdhodh bound to 13 ( pfdhodh-13 ) . limited residues from the 4 shell around 13 are shown , and the structure has been aligned to the pfdhodh structure bound to 2 ( pdb 3i65 ) to allow comparison of the binding modes . only the inhibitor 2 from 3i65 is displayed . pfdhodh amino acid , fmn , and orotate carbons are shown in purple , the carbons of 13 are shown in tan , and the carbons of 2 are shown in green . nitrogens are blue , oxygens are red , sulfur is light yellow , fluorines are light blue , and bromine is deep red . selected compounds were evaluated for their physicochemical properties to determine if they had good drug - like properties . analysis included in silico calculations , chromatographic estimation of log d ( ph 7.4 ) , aqueous solubility , and plasma protein binding ( tables 3 and 4 ) . tested compounds had physicochemical properties that are suggestive of good oral absorption ( mw < 430 , h bond donors 2 , h bond acceptors 6 , polar surface area < 70 ) . log d ranged from 3.14.1 , and the more potent analogues tended to have higher log d values . aqueous solubility ( ph 6.5 ) was poor ( 1.66.3 g / ml ) to moderate ( 12.525 g / ml ) , and in general , compounds in this series had lower kinetic solubility than 1 ( table 3 ) . more extensive solubility studies in simulated gastric and intestinal fluids was conducted on a selection of the most potent analogues from the 2-indanyl ( 35 ) and the tetrahydro-2-naphthyl ( 7 , 9 , 11 , 13 ) series ( table 4 ) . solubility in fasted - state simulated intestinal fluid was similar to or slightly improved compared to 1 . the tetrahydro-2-naphthyl compounds substituted at the 6 position ( 11 and 7 ) showed considerably better solubility in fed - state simulated intestinal fluid than either 1 or the 7 position ( 9 and 13 ) substituted compounds . protein binding by ultracentrifugation in human ( h ) , rat ( r ) , and mouse ( m ) plasma . in vitro intrinsic clearance in human ( h ) , rat ( r ) , and mouse ( m ) liver microsomes . predicted in vivo intrinsic clearance obtained using physiological scaling factors . human , rat , and mouse plasma protein binding ( ppb ) was assessed for the more potent compounds that were progressed to in vivo pharmacokinetic testing ( 7 , 9 , 11 , 13 , 35 ) . the tetrahydro-2-naphthyl compounds substituted at the 6 position ( 7 and 11 ) showed higher protein binding compared to the compounds substituted at the 7 position ( 9 and 13 ) . binding was similar across the species tested for each of the compounds . by comparison , 1 exhibited high protein binding in human and mouse plasma but lower binding in rat plasma . the indanyl analogue 35 had considerably lower protein binding compared to either 1 or the tetrahydro-2-naphthyl compounds . to obtain a preliminary indication of the likelihood that compounds would show good in vivo pharmacokinetic properties , selected compounds were analyzed for metabolic stability in vitro using human , rat , and mouse liver microsomes . we previously found that the in vitro microsomal stability of compounds from the triazolopyrimidine series provided a good rank order estimation of which compounds would have the highest in vivo exposure . we report in table 3 the in vitro intrinsic clearance ( clint , l / min / mg microsomal protein ) and the predicted in vivo intrinsic clearance ( ml / min / kg ) obtained using physiologically based scaling factors as previously described . given the early stage of optimization for the series , protein binding was not determined for all compounds , and therefore , no attempt was made to predict the in vivo blood clearance ( which requires corrections for both plasma and microsomal binding ) . in vitro clint values of < 20 l / min / mg protein were taken as being suggestive of good metabolic stability , assuming that clearance only occurs via hepatic metabolism . overall , the substituted tetrahydro-2-naphthyls showed good metabolic stability in human liver microsomes , but they were less stable in mouse microsomes ( table 3 ) . chloro- , bromo- , and cf3-substituted compounds ( 7 , 9 , 11 , 12 , 13 , 20 , 21 ) were marginally more metabolically stable than the fluoro- ( 6 , 19 ) or ome - substituted ( 16 ) compounds . with the exception of 35 , the indanyl analogues were generally metabolically unstable showing high intrinsic clearance in mouse liver microsomes and moderate to high values in human and rat microsomes . the least stable compounds were the unsubstituted 2-indanyl 26 and the 4,7 dimethyl substituted derivative 32 . replacement of both methyl groups with fluorine 33 did not improve stability , although perhaps surprisingly the single 4-f derivative 34 was significantly more stable . substantial improvement in metabolic stability was obtained by moving the fluorine to the 5 and 6 positions of the ring 35 , leading to low intrinsic clearance in human and rat microsomes and moderate clearance in mouse microsomes . these data suggested that 35 would show good plasma exposure in both mice and rats . in order to evaluate any potential cardiac risks , select compounds were tested for inhibition of the human ether - a - go - go - related gene ( herg ) k channel in a standard patch clamp assay . herg channel inhibition has been associated with qt prolongation and arrhythmias , and patch clamp assays have become a routine method to provide an initial analysis that a compound may potentially be associated with qt syndromes in humans . compounds in both the tetrahydro-2-naphthyl and 2-indanyl series inhibited the herg channel with ic50s in the range of 0.51.8 m ( table 3 ) . in order to test for the potential for drug drug interactions , select compounds were tested for their ability to inhibit cytochrome p450 isoforms in human liver microsomes ( table 5 ) . the most significant inhibition was observed for 9 and 13 as both inhibited these two isoforms in the 24 m range . five of the most potent compounds were selected for assessment of their in vivo pharmacokinetic properties based on their good in vitro metabolic stability . mice were dosed orally while rats were dosed orally , and intravenously to allow determination of the clearance , volume of distribution , and oral bioavailability . compounds included one from the indanyl series ( 35 ) and four from the tetrahydro-2-naphthyl series ( 7 , 9 , 11 , 13 ) . all five compounds showed good exposure over 24 h in both mice and rats after oral administration ( tables 6 and 7 ; figures 3 and 4 ) . compounds were well tolerated at the administered doses , and no adverse reactions were observed . in mice , cmax and auc24h values were highest for 35 and lower for the tetrahydro-2-naphthyl compounds . compared to 1 and taking into account the differences in dose , total cmax and auc24h values were similar or lower for the tetrahydro-2-naphthyl compounds , whereas unbound cmax and auc24h were similar ( for 7 ) or somewhat higher ( 34-fold for 9 , 11 , and 13 ) . the indanyl ( 35 ) had the highest plasma exposure in mice in terms of both total and unbound cmax and auc24h and significantly higher exposure compared to 1 . similar to 1 , the terminal elimination half - life ( t1/2 ) in mice was 2 h for all five compounds ( table 6 and figure 3 ) . plasma concentration versus time profiles following a single oral dose of 20 mg / kg to male swiss outbred mice . data represent the mean concentrations for two mice at each time point . plasma concentration versus time profiles following a single intravenous ( iv ) or oral ( po ) dose to male sprague dawley rats . each data point represents the mean of data from two rats , and error bars represent the range . values in parentheses have been corrected for plasma protein binding and represent unbound values . protein binding in mouse plasma is an approximation based on rat and human plasma protein binding data . calculated unbound parameters ( in italics ) are therefore approximations only . values in parentheses have been corrected for plasma protein binding and represent unbound values . in rats , the oral bioavailability was high for all compounds tested , ranging from 63100% compared to 57% for 1 ( table 7 and figure 4 ) . the plasma clearance and volume of distribution were significantly lower for the tetrahydro-2-naphthyl compounds substituted on the 6 position ( 7 and 11 ) than for those substituted on the 7 position ( 9 and 13 ) ; however , when corrected for differences in plasma protein binding , values across the four tetrahydro - naphthyl compounds were comparable ( within 2-fold ) . the 6 position compounds also had lower blood to plasma partitioning consistent with their higher plasma protein binding compared to the 7 position analogues . unbound clearance values for the tetrahydro-2-naphthyl compounds were somewhat lower for the two chloro - substituted compounds ( 438 and 589 ml / min / kg for 7 and 9 , respectively ) compared to the bromo compounds ( 933 and 800 ml / min / kg for 11 and 13 , respectively ) , and all were higher than for 1 ( 263 ml / min / kg ) . as a result , unbound auc values for the tetrahydro-2-naphthyls were approximately 24-fold lower than 1 after oral dosing at approximately 20 mg / kg . the t1/2 for the tetrahydro-2-naphthyl compounds ranged from 6 to 7 h in comparison to 13 h for 1 . the indanyl compound ( 35 ) had low unbound clearance and volume of distribution , and overall unbound oral exposure was superior to 1 and to the tetrahydro-2-naphthyls ( table 7 ) . based on the observation of good oral exposure after dosing in mice , we tested three compounds in the scid mouse p. falciparum efficacy model , which has become the standard model for evaluating the efficacy of new antimalarial compounds . the tetrahydro-2-naphthyl compounds substituted at the 7 position ( 9 and 13 ) were more potent against p. falciparum in the in vitro parasite assay than those substituted on the 6 position ( 7 and 11 ) and showed fairly equivalent exposure in mice after oral dosing . we therefore decided to test both a 6- and 7-substituted tetrahydro-2-naphthyl ( 7 and 9 ) in the p. because 35 showed significantly higher plasma exposure in mice than the remaining compounds , we also selected this compound for an efficacy study despite the 10-fold lower potency in the p. falciparum parasite assay . all three compounds were dosed orally once a day for 4 days and tested for efficacy and blood concentrations at two doses ( 10 and 30 mg / kg ) . each was well tolerated , and there were no adverse reactions . the effective dose that led to 90% parasite clearance ( ed90 ) was calculated 24 h after the last dose ( table 8 and figure 5a ) . pharmacokinetic sampling was performed on day 1 to allow the total blood auc at the ed90 to be assessed ( tables 8 and s4 ; figure 5b ) . because of the complicated nature of blood in the scid mouse model ( i.e. , mice contain a mixture of human and mouse plasma and erythrocytes and a hematocrit of 7080% ) , it was not possible to accurately convert blood concentrations to plasma concentrations or to correct concentrations for differences in plasma protein binding . mice were infected with p. falciparum pf3d70087/n9 parasites by intravenous injection on day zero , and dosing was once daily for 4 days starting on day 3 postinfection . 1 qd data were taken from ref ( 11 ) and converted to different units for consistency . 1 bid and chloroquine data were taken from ref ( 12 ) and converted to different units for consistency . all three compounds showed efficacy in this model ; however , 9 significantly outperformed the other two compounds based on ed90 ( tables 8 and s4 ; figure 5 ) . the ed90 was higher for all three compounds than for 1 ranging from 19 mg / kg for 9 to > 30 mg / kg for 7 and 35 . the pharmacokinetic data indicated that , as expected , 35 exhibited higher total blood concentrations than the other two compounds . both 7 and 9 were approximately dose linear for both cmax and auc , whereas 35 showed significant nonlinearity and evidence of a second peak at 8 h at the lower dose ( table s4 ) . despite requiring a higher dose , 9 showed higher apparent efficacy compared to 1 based on the total blood auc at the ed90 , which was 5-fold lower than that required for 1 in the once daily dosing regimen ( table 8) . however , when accounting for differences in the plasma unbound fractions for 1 and 9 ( approximately 0.002 for 1 and 0.03 for 9 , table 3 ) , 1 likely has lower unbound concentrations at the ed90 and hence higher intrinsic efficacy . in contrast , 35 was significantly less effective than 1 suggesting that the lower intrinsic potency of 35 could not be overcome by higher exposure levels . the pharmacokinetic / pharmacodynamic relationship for 7 could not be evaluated in detail as the ed90 was not reached in this study . malaria is one of the most serious global infectious diseases , and while a number of effective drugs have been used to treat malaria , drug resistance has led to the loss of most agents that have been in widespread use . dhodh has emerged as a key new target in efforts to identify antimalarial drugs that work on clinical isolates resistant to standard therapies , and as noted , the triazolopyrimidine 1 is currently in clinical development . in order to identify potential backup compounds to 1 in case issues arise in its clinical development , we sought to identify additional compounds within the triazolopyrimidine series with the potential to advance into preclinical development . one identified potential liability of 1 has been the finding that , while it does not inhibit human dhodh , it does show moderate inhibition of the mouse and rat enzymes , which complicates toxicity testing in animals . we therefore sought to identify new derivatives that showed broad selectivity against all of the key mammalian enzymes . herein we describe a series of tetrahydro-2-naphthyl and 2-indanyl triazolopyrimidine analogues , which display a range of potencies against plasmodium dhodh but importantly lack activity against human dhodh or any of the key mammalian enzymes from species that are important for toxicological profiling ( mouse , rat , and dog ) . pharmacologic profiling and in vivo efficacy assays suggest that several of the identified analogues have potential , with 9 showing the most promising profile . in evaluating the tetrahydro-2-naphthyl and 2-indanyl triazolopyrimidine analogues we found that the tetrahydro-2-naphthyls ( e.g. , 7 , 9 , 11 , 13 ) had significantly greater potency on pfdhodh and plasmodium parasites in whole cell assays than the 2-indanyl analogues ( e.g. , 35 ) . the most potent of the tetrahydro-2-naphthyl derivatives also showed good activity ( within 1.5-fold ) against p. vivax dhodh suggesting that they would be useful for the treatment of both p. falciparum and p. vivax . in contrast , the best of the 2-indanyl analogues ( 35 ) was 7-fold less active on p. vivax dhodh suggesting it might not have good activity against the p. vivax parasite . the x - ray structure of the 7-bromo analogue 13 showed that , in addition to the previously observed h - bonds and stacking interactions observed for other analogues in the series , a good halogen bond was formed between the bromo group of 13 and cys-233 that is likely to provide considerable binding energy to the interaction . indeed halogen bonding to protein residues that include lewis bases ( e.g. , sulfur ) are commonly observed in protein structures deposited in the pdb . the overall binding mode of 13 is very similar to that of 2 , which was expected based on their close structural similarity . like the naphthyl of 2 , the tetrahydro-2-naphthyl of 13 better fills the available binding pocket than the sf5-aniline of 1 , which supports our initial hypothesis that compounds with improved potency could be identified by substitution of the sf5-aniline with bulkier aromatic groups . comparison of the tetrahydro-2-naphthyl compounds containing 7 position ( 9 and 13 ) versus 6 position ( 7 and 11 ) substitutions identified a number of interesting differences . analogues substituted in the 7 position were more potent than 6 position analogues , and indeed 13 with subnanomolar activity against the parasite in whole cell assays is the most potent analogue that has been identified in the series . however , these assays did not take into account potential differences in protein binding . the 6 position analogues exhibited consistently higher plasma protein binding than the 7 position compounds and a similar trend is also likely for binding to albumax present in the in vitro test medium . assessment of the pharmacologic properties showed that while the 7 position tetrahydro-2-naphthyls had greater apparent antimalarial potency , analogues substituted at the 6 position had somewhat improved physicochemical properties . the 6 position analogues 7 and 11 were more soluble ( in 0.1 n hcl and fessif media ) than either of the 7 position analogues ( 9 and 13 ) . while the total plasma exposure ( auc and cmax ) in rats was higher for the 6 position analogues , correction of the data for protein binding indicated that the unbound exposure and unbound clearance were similar ( within 2-fold ) for the 6 and 7 position analogues with the chloro - substituted compounds ( 7 and 9 ) showing about 2-fold lower unbound clearance than the bromo - substituted compounds ( 11 and 13 ) . so while total exposure appears greater for the 6 position analogues , unbound concentrations are comparable across the tetrahydro - naphthyl compounds tested . protein binding data in mouse plasma was only available for 9 , 11 , and 35 , and binding for each of these compounds was similar in each of the three species tested . mouse plasma protein binding was therefore estimated for 7 and 13 using the average of the human and rat values , suggesting that unbound concentrations for each of the compounds are likely to be within a similar range ( table 6 ) . the best of the 2-indanyl analogues ( 35 ) had considerably higher unbound plasma exposure in rats compared to the tetrahydro-2-naphthyls and to 1 and had similarly low unbound clearance . the in vitro data generated in liver microsomes provided a mechanism to rank compounds based on their relative metabolic stability . the compounds selected for in vivo testing were expected to have good exposure , and this was seen following dosing to both rats and mice indicating that , qualitatively , the in vitro data were informative . converting the in vitro intrinsic clearance determined in rat microsomes to a plasma clearance using physiologically based scaling factors and taking into account binding to rat plasma and microsomal proteins ( as per ref ( 21 ) ) and blood to plasma partitioning ratio led to an under prediction of the actual in vivo clearance by a factor of 3- to 16-fold for the five compounds tested . these trends are similar to what had been observed previously for 1(16 ) and suggest that in vivo clearance mechanisms for the series also involve additional non - cyp pathways not fully represented by the microsomal test system . in the case of 1 , hepatocyte assays also led to an under prediction of in vivo clearance highlighting the difficulties in obtaining accurate clearance predictions based on in vitro data alone . further studies with the current compounds using hepatocytes may have provided additional insight into their clearance mechanisms . in vivo efficacy of the three tested compounds , 9 ( 7 position ) had the most potent in vivo activity . the total blood auc required to reach ed90 was lower than for 1 ; however , it took a higher dose ( 19 mg / kg versus 8.1 mg / kg for qd dosing ) to achieve this activity , possibly due to a higher unbound clearance of 9 compared to 1 as seen in rats . the binding trends in human and mouse plasma would suggest that unbound concentrations of 9 in the scid mouse are likely to be considerably higher compared to those for 1 at the same total blood concentration . therefore , the true in vivo potency of 1 based on unbound concentrations is likely to be greater than that for 9 . in spite of the high blood exposure , the 2-indanyl ( 35 ) lacked sufficient intrinsic potency to provide good activity in the in vivo model . in order to improve patient compliance , single dose treatments are being sought by the international organizations that are promoting new drug discovery for malaria . while all three tested compounds ( 7 , 9 , and 35 ) showed in vivo antimalarial activity , it is unlikely that any of these compounds have the necessary properties to support a single dose treatment regimen at a practical dose level in humans . preliminary safety analysis included characterizing herg channel activity to assess potential cardiac effects and cyp inhibition studies to determine the potential for drug drug interactions . all five of the profiled compounds showed some cyp inhibition , with cyp isoform 2d6 being inhibited to the greatest extent . cyp2d6 inhibition was modest with the most potent inhibition observed in the 36 m range , but does suggest some potential for drug drug interactions with compounds that are substrates for this enzyme . tested compounds in these series also showed some herg inhibition ( 0.51.5 m range ) . this inhibitory activity is similar to what was observed for 1 , where subsequent studies using the rabbit wedge model and dog cardiovascular studies showed no evidence for qt prolongation , arrhythmias , or cardiac effects . additionally , for 1 the unbound plasma concentration at likely therapeutic doses is 70320-fold lower than the ic50 for herg channel inhibition , suggesting it would be very unlikely that 1 would inhibit the channel in vivo . insufficient efficacy data is available for compounds 7 and 35 to determine the safety margin relative to unbound concentrations at an efficacious dose ; however , for 9 the cmax at the ed90 in the scid study ( unbound concentration of approximately 0.05 m ) is 30-fold lower than the ic50 on the herg channel , again suggesting a low likelihood the channel would be inhibited in vivo . however , given the herg channel activity , careful evaluation of cardiac safety would be an important component of preclinical development if any of these compounds were to be advanced . first , only the chloro - substituted tetrahydro-2-naphthyls were tested in the scid efficacy model , leaving open the possibility that one of the bromo analogues might have shown better in vivo efficacy . additional optimization of the series might focus on reducing lipophilicity as this could lead to reduced intrinsic clearance and better in vivo exposure and also could lead to reduced herg activity . reduced lipophilicity would also likely result in improved aqueous solubility thereby simplifying formulation approaches for oral administration . decreasing lipophilicity will likely require the sf5-anline of 1 to be replaced with more hydrophilic aromatic amines , provided potency can be maintained . within the tetrahydro-2-naphthyls , it might yet be possible to identify more metabolically stable analogues by identifying the specific sites of metabolism and attempting to block them . we have described the identification of several tetrahydro-2-naphthyl and 2-indanyl triazolopyrimidine analogues that are potent inhibitors of plasmodium dhodh and that have improved species selectivity over the mammalian enzymes in comparison to the clinical candidate 1 . the compounds have potent activity against p. falciparum in parasite assays and showed antimalarial activity in the p. falciparum scid mouse model of disease . these compounds showed good plasma exposure after oral dosing in mice and rats and were well tolerated in the in vivo studies that were performed . several of the identified compounds , most notably 9 , have the potential to be further developed for treatment of malaria . however , the findings that 9 is less intrinsically potent than 1 in vivo ( based on expected unbound concentrations ) and that the unbound clearance in rat is 2-fold higher than for 1 suggest that this compound is unlikely to meet the strict criteria required for a single dose cure at a reasonable dose . plasmodium and mammalian dhodhs were expressed in e. coli as n - terminal truncations ( lacking the mitochondrial transmembrane domains ) fused to his6-purification tags as previously described . protein ( pfdhodh384413 ) for crystallization studies was further truncated to remove a proteolytically sensitive loop ( amino acids 384421 ) that prevents crystallization as previously described . proteins were expressed in e. coli bl21 phage - resistant cells ( novagen ) , and they were purified by ni - agarose column chromatography ( ge healthcare life sciences , histrap hp ) and gel - filtration column chromatography ( ge healthcare life sciences , hiload 16/600 superdex 200 pg ) as previously described . protein was concentrated to 1030 mg / ml and stored at 80 c . steady - state kinetic analysis was performed using a dye - based spectrophotometric method in assay buffer ( 100 mm hepes , ph 8.0 , 150 mm nacl , 10% glycerol , 0.05% triton x-100 ) as previously described . enzyme and substrate concentrations were dhodh ( e = 5 nm ) , substrates ( 0.2 mm l - dihydroorotate , 0.02 mm coqd , and 0.12 mm 2,5-dichloroindophenol ( dcip ) ) . inhibitor stock solutions ( 100 mm ) were made in dmso and protected from light in dark amber vials . serial dilutions were than performed to generate a 3-fold dilution series of 100 stocks also in dmso that were used in the final assay ( final inhibitor concentration range was 0.01100 m ) . data were fitted to the log[i ] vs response ( three parameters ) equation to determine the concentration that gave 50% enzyme inhibition ( ic50 ) except for compounds where the ic50 > 10 m , which were instead fitted to the standard ic50 equation ( y = 1/(1 + x / ic50 ) ) in graphpad prism . falciparum parasites were grown in rpmi-1640 containing 0.5% albumax - ii as previously described . inhibitor stock solutions were prepared as described above for dhodh kinetic analysis except that a 500 dilution series prepared in dmso was used to generate a 10 dilution series in rpmi so that final dmso concentration in the media was 0.2% . growth assays for p. falciparum 3d7 cells and drug - resistant parasites were performed using the sybr - green 72 h growth assay . data were fitted to the log[i ] vs response variable slope ( four parameter ) equation in graph pad prism to determine the effective concentration ( ec50 ) that led to 50% reduction in parasitemia . selected compounds were incubated at a concentration of 1 m with mouse , rat , or pooled human liver microsomes ( bd gentest , woburn , ma or xenotech llc , lenexa , kansas city ) at a microsomal protein concentration of 0.4 mg / ml . substrate depletion was assessed by lc ms . measured in vitro intrinsic clearance values ( l / min / mg microsomal protein ) were converted to predicted in vivo intrinsic clearances ( ml compound stock solutions were prepared in dmso , which was then spiked into ph 6.5 phosphate buffer giving a final dmso concentration of 1% . media included 0.1 n hcl to represent a simulated gastric condition , fasted ( fassif , ph 6.5 ) and fed ( fessif , ph 5.0 or 5.8 ) state simulated intestinal fluids prepared as described previously . solid material was incubated in media for a period of 56 h at 37 c with periodic mixing and sample supernatant concentrations determined by hplc with uv detection following two separate centrifugations to remove undissolved compound . select compounds were analyzed for plasma protein binding ( ppb ) in human , rat , or mouse plasma using an ultracentrifugation method at 37 c as previously described followed by lc protein binding was calculated by comparing the unbound concentration in the ultracentrifuged samples to the total plasma concentration in control samples incubated at 37 c for the same time period . for compounds progressing to rat pharmacokinetic studies , blood to plasma partitioning ratios in rat blood pharmacokinetic studies for select compounds were performed in mice and rats in accordance with the australian code of practice for the care and use of animals for scientific purposes , and the study protocols were approved by the monash institute of pharmaceutical sciences animal ethics committee . oral doses were administered as a suspension in a vehicle containing 0.5% w / v carboxymethylcellulose , 0.5% v / v benzyl alcohol ( as a preservative ) , and 0.4% v / v tween 80 , and intravenous doses were administered in cosolvent vehicle containing 40% propylene glycol , 10% ethanol , 0.4% tween 80 in water . iv doses to rats were administered as a 10 min constant rate infusion of 1 ml into an indwelling jugular vein cannula , and oral doses were administered by gavage as 10 ml / kg ( both rats and mice ) . blood was collected from rats via a cannula inserted into the carotid artery on the day prior to dosing , and from mice via either submandibular bleed or terminal cardiac puncture ( maximum of two samples per mouse ) into tubes containing heparin as an anticoagulant , and plasma was separated by centrifugation . ms using a waters xevo tq mass spectrometer coupled to a waters acquity uplc . chromatography was conducted using a supelco ascentis express rp amide column ( 50 2.1 mm , 2.7 m ) with a mobile phase consisting of 0.05% formic acid in water and 0.05% formic acid in methanol mixed via a linear gradient with a cycle time of 4 min . the flow rate was 0.4 ml / min , and the injection volume was 3 l . detection was via positive electrospray ionization mass spectrometry with multiple - reaction monitoring using a cone voltage of 3040 v and collision energy of 2535 v. calibration curves were prepared using blank rat or mouse plasma , and validation studies confirmed the accuracy , precision , and limit of quantitation to be within acceptable ranges . inhibition of cytochrome p450 ( cyp ) enzymes was assessed in human liver microsomes using a substrate specific approach where the formation of metabolites specific to a particular cyp isoform was monitored . microsomes were suspended in phosphate buffer and incubated at 37 c in the presence of probe substrates , and reactions were initiated by the addition of an nadph - regenerating system . ms . the ic50 value for each compound or control inhibitor was the concentration at which there was a 50% reduction in the amount of metabolite formed relative to the maximum metabolite formation in the absence of inhibitor . select compounds were tested for activity against the human ether - a - go - go related gene ( herg ) k channel in a ionworks patch clamp electrophysiology assay under contract to essen bioscience ( welwyn garden city hertfordshire , uk ) . nod - scid il-2rnull ( nsg ) mice ( jackson laboratory , usa ) ( 2336 g ) were engrafted with human erythrocytes and then infected with p. falciparum pf3d70087/n9 parasites ( 20 10 ) by intravenous injection as described . compounds ( 7 , 9 , and 35 ) were administered by oral gavage once daily ( qd ) for four consecutive days starting on day 3 postinfection in vehicle ( 0.5% w / v sodium carboxymethylcellulose , 0.5% v / v benzyl alcohol , 0.4% v / v tween 80 in water ) . compound levels in the formulations or in the blood of infected mice were measured by lc human biological samples were sourced ethically , and their research use was in accordance with the terms of the informed consents . compound 9 was crystallized from ethanol . a colorless prism , measuring 0.20 0.13 0.10 mm was mounted on a loop with oil . data was collected at 173 c on a bruker apex ii single crystal x - ray diffractometer , mo - radiation . crystal - to - detector distance was 40 mm , and exposure time was 15 s per frame for all sets . the scan width was 0.5. data collection was 99.9% complete to 25 in . a total of 113483 reflections were collected covering the indices , h = 11 to 11 , k = 38 to 38 , l = 18 to 18 . of that , 16915 reflections were symmetry independent and the rint = 0.0703 indicated that the data was of average quality ( 0.07 ) . 4 ) . the data were integrated and scaled using saint aand sadabs within the apex2 software package by bruker . solution by direct methods ( shelxs , sir97 ) produced a complete heavy atom phasing model consistent with the proposed structure . the structure was completed by difference fourier synthesis with shelxl97 . hydrogen atoms were placed in geometrically idealized positions and constrained to ride on their parent atoms with ch distances in the range 0.951.00 . isotropic thermal parameters ueq were fixed such that they were 1.2ueq of their parent atom ueq for ch s and 1.5ueq of their parent atom ueq in the case of methyl groups . all non - hydrogen atoms were refined anisotropically by full - matrix least - squares . preliminary crystallization conditions were found using the random crystallization screen cryos suite ( nextal ) . then the condition was refined by variation of ph , precipitant , and protein concentrations to find optimal conditions . crystals of pfdhodh384413 - 13 complex grew at condition with 0.16 m ammonium sulfate , 0.1 m sodium acetate , ph 4.2 , 9.5% peg4000 ( w / v ) , 24% glycerol ( v / v ) , and 10 mm dtt . the crystallization drop was mixed with an equal volume of reservoir solution and pfdhodh384413 ( 27 mg / ml ) pre - equilibrated with 1 mm 13 ( 0.1 m stock solution in dmso ) and 2 mm dihydroorotate ( dho , 0.1 m stock solution in dmso ) . diffraction data were collected at 100 k on beamline 19id at advanced photon source ( aps ) using an adsc q315 detector . the crystal of pfdhodh384413 - 13 diffracted to 2.32 and has a space group of p64 with the cell dimension of a = b = 86.1 , c = 139.1 ( table s3 ) . crystallographic phases for pfdhodh inhibitors were solved by molecular replacement with phaser using the previously reported structure of pfdhodh384413 bound to dsm1 ( pdb i d 3i65(17 ) ) as a search model ( ligands were removed from the search model ) . structures were rebuilt with coot and refined in phenix to rwork and rfree of 0.18 and 0.21 , respectively ( table s3 ) . electron density for residues 159160 , 344354 , and 567569 were missing and thus not built into the final structure . atomic representations were created using pymol molecular graphics system ( version 1.7 , schrdinger ) . all reagents and starting materials were obtained from commercial suppliers and used without further purification unless otherwise stated . reaction progress was monitored by thin layer chromatography ( tlc ) on preloaded silica gel 60 f254 plates . flash chromatography was carried out using prepacked teledyne isco redisep rf silica - gel columns as the stationary phase and analytical grade solvents as the eluent unless otherwise stated . h nmr spectra were recorded on bruker avance 300 and 500 mhz spectrometer at ambient temperature . chemical shifts are reported in parts per million ( ) and coupling constants in hz . h nmr spectra were referenced to the residual solvent peaks as internal standards ( 7.26 ppm for cdcl3 , 2.50 ppm for dmso - d6 , and 3.34 ppm for meod ) . spin multiplicities are described as s ( singlet ) , brs ( broad singlet ) , d ( doublet ) , t ( triplet ) , q ( quartet ) , dd ( doublet of doublets ) , dt ( doublet of triplets ) , and m ( multiplet ) . total ion current traces were obtained for electrospray positive and negative ionization ( es+/es ) on a bruker esquire liquid chromatograph - ion trap mass spectrometer . analytical chromatographic conditions used for the lc ms analysis are as follows : column , zorbax extend c18 from agilent technologies , 2.1 100 mm . solvents were a , aqueous solvent = water + 5% acetonitrile + 1% acetic acid ; b , organic solvent = acetonitrile + 1% acetic acid ; methods , 14 min run time ( 010 min 20100% b , flow rate 0.275 ml / min ; 1012 min 100% b , flow rate 0.350 ml / min ; 1212.50 min 10020% b , flow rate 0.350 ml / min ; 12.5014.0 min 20% b , flow rate 0.350 ml / min ) . the following additional parameters were used : injection volume ( 10 l ) , column temperature ( 30 c ) , uv wavelength range ( 254330 nm ) . analytical hplc analyses were performed on a supelcosil lc18 column ( 5 m , 4.6 mm 25 cm ) with a linear elution gradient ranging from 0 to 100% acn over 27 min , using a supelcosil lc18 column ( 5 m , 4.6 mm 25 cm ) at a flow rate of 1 ml / min . a purity of > 95% has been established for all reported compounds ( table 1 ) . the two enantiomers were separated for selected compounds on a semipreparative chiral hplc on chiralcel od - h ( 250 20 ) mm column eluting with 0.1% diethylamine in hexane ( a)/ethanol ( b ) in 36 min run time ( 010 min , 20% b ; 1020 min , 2035% b ; 2030 min , 35% b ; 30.0136 min 20% b ; flow rate 5 ml / min ) . h nmr ( 400 mhz , dmso - d6 ) : 8.24 ( d , j = 7.1 hz , 1h ) , 7.13- 7.09 ( m , 4h ) , 6.67 ( s , 1h ) , 4.164.01 ( br , 1h ) , 3.103.04 ( m , 2h ) , 3.022.85 ( m , 2h ) , 2.45 ( s , 3h ) , 2.152.06 ( m , 4h ) , 2.041.91 ( m , 1h ) . esims m / z : 344 ( mh ) . h nmr ( 500 mhz , dmso - d6 ) : 8.29 ( d , j = 8.8 hz , 1h ) , 7.287.07 ( m , 1h ) , 7.066.88 ( m , 2h ) , 6.69 ( s , 1h ) , 4.173.94 ( m , 1h ) , 3.142.92 ( m , 4h ) , 2.47 ( s , 3h ) , 2.12 ( t , j = 18.8 , 3h ) , 2.031.90 ( m , 2h ) . esims m / z : 362.1(mh ) . h nmr ( 400 mhz , meod ) : 7.177.12 ( m , 3h ) , 6.55 ( s , 1h ) , 4.154.13 ( m , 1h ) , 3.263.20 ( m , 1h ) , 3.052.94 ( m , 3h ) , 2.55 ( s , 3h ) , 2 . twenty - nine 2.25 ( m , 1h ) , 2.11 ( t , j = 18.72 hz , 3h ) , 2.011.97 ( m , 1h ) . esims m / z : 378 ( mh ) . h nmr ( 400 mhz , meod ) : 7.177.12 ( m , 3h ) , 6.55 ( s , 1h ) , 4.184.12 ( m , 1h ) , 3.263.21 ( m , 1h ) , 3.052.95 ( m , 3h ) , 2.55 ( s , 3h ) , 2.292.25 ( m , 1h ) , 2.11 ( t , j = 18.72 hz , 3h ) , 2.011.95 ( m , 1h ) . esims m / z : 378 ( mh ) . h nmr ( 400 mhz , meod ) : 7.167.13 ( m , 3h ) , 6.55 ( s , 1h ) , 4.154.12 ( m , 1h ) , 3.263.21 ( m , 1h ) , 3.012.97 ( m , 3h ) , 2.55 ( s , 3h ) , 2.292.25 ( m , 1h ) , 2.11 ( t , j = 18.75 hz , 3h ) , 2.011.94 ( m , 1h ) . esims m / z : 378(mh ) . h nmr ( 400 mhz , meod ) : 7.167.13 ( m , 3h ) , 6.55 ( s , 1h ) , 4.164.12 ( m , 1h ) , 3.263.21 ( m , 1h ) , 3.032.97 ( m , 3h ) , 2.55 ( s , 3h ) , 2.292.25 ( m , 1h ) , 2.11 ( t , j = 18.72 hz , 3h ) , 2.011.95 ( m , 1h ) . esims m / z : 378(mh ) . h nmr ( 400 mhz , cdcl3 ) : 7.327.30 ( m , 2h ) , 6.99 ( d , j = 8.04 hz,1h ) , 6.17 ( d , j = 8.32 hz , 1h ) , 6.14 ( s , 1h ) , 4.023.99 ( m , 1h ) , 3.293.23 ( m , 1h ) , 3.022.99 ( m , 2h ) , 2.912.84 ( m , 1h ) , 2.60 ( s , 3h ) , 2.312.28 ( m , 1h ) , 2.15 ( t , j = 18.76 hz , 3h ) , 1.991.94 ( m , 1h ) . h nmr ( 400 mhz , cdcl3 ) : 7.327.29 ( m , 2h ) , 6.99 ( d , j = 8.04 hz , 1h ) , 6.18 ( d , j = 7.56 hz , 1h ) , 6.15 ( s , 1h ) , 4.01 ( brs , 1h ) , 3.293.24 ( m , 1h ) , 3.022.99 ( m , 2h ) , 2.912.85 ( m , 1h ) , 2.60 ( s , 3h ) , 2.312.29 ( m , 1h ) , 2.15 ( t , j = 18.76 hz , 3h ) , 1.991.95 ( m , 1h ) . esims m / z : 424 ( m + 2 ) . h nmr ( 400 mhz , dmso - d6 ) : 8.28 ( d , j = 8.76 hz , 1h ) , 7.347.30 ( m , 2h ) , 7.11 ( d , j = 8.00 hz , 1h ) , 6.67 ( s , 1h ) , 4.07 ( brs , 1h ) , 3.093.05 ( m , 2h ) , 2.932.89 ( m , 2h ) , 2.45 ( s , 3h ) , 2.292.25 ( m , 1h ) , 2.11 ( t , j = 19.12 hz , 3h ) , 1.981.94 ( m , 1h ) . h nmr ( 400 mhz , dmso - d6 ) : 8.28 ( d , j = 8.00 hz , 1h ) , 7.347.30 ( m , 2h ) , 7.11 ( d , j = 8.16 hz , 1h ) , 6.68 ( s , 1h ) , 4.07 ( brs , 1h ) 3.093.05 ( m , 2h ) , 2.902.88 ( m , 2h ) , 2.45 ( s , 3h ) , 2.292.25 ( m , 1h ) , 2.11 ( t , j = 19.02 hz , 3h ) , 1.981.94 ( m , 1h ) . h nmr ( 300 mhz , cdcl3 ) 7.06 ( d , j = 8.3 hz , 1h ) , 6.866.63 ( m , 2h ) , 6.316.07 ( m , 2h ) , 4.153.92 ( m , 1h ) , 3.82 ( s , 3h ) , 3.27 ( dd , j = 15.7 , 4.2 hz , 1h ) , 3.132.79 ( m , 3h ) , 2.61 ( s , 3h ) , 2.442.24 ( m , 1h ) , 2.16 ( t , j = 18.8 hz , 3h ) , 2.071.88 ( m , 1h ) h nmr ( 400 mhz , meod ) : 7.05 ( d , j = 8.4 , 1h ) , 6.756.55 ( m , 2h ) , 6.55 ( s , 1h ) , 4.154.12 ( m , 1h ) , 3.76 ( s , 3h ) , 3.253.19 ( m , 1h ) , 3.012.94 ( m , 3h ) , 2.56 ( s , 3h ) , 2.282.25 ( m , 1h ) , 2.11 ( t , j = 18.75 hz , 3h ) , 2.161.94 ( m , 1h ) . h nmr ( 400 mhz , meod ) : 7.05 ( d , j = 8.4 , 1h ) , 6.756.69 ( m , 2h ) , 6.55 ( s , 1h ) , 4.144.12 ( m , 1h ) , 3.76 ( s , 3h ) , 3.253.19 ( m , 1h ) , 3.012.94 ( m , 3h ) , 2.56 ( s , 3h ) , 2.282.24 ( m , 1h ) , 2.14 ( t , j = 18.68 hz , 3h ) , 2.031.96 ( m , 1h ) . h nmr ( 500 mhz , cdcl3 ) : 7.056.92 ( m , 2h ) , 6.436.25 ( m , 2h ) , 4.133.97 ( m , 1h ) , 3.363.16 ( m , 1h ) , 3.122.85 ( m , 3h ) , 2.64 ( s , 3h ) , 2.432.28 ( m , 1h ) , 2.16 ( t , j = 18.92 hz , 3h ) , 2.081.91 ( m , 1h ) . esims m / z : 380.2 ( mh ) . h nmr ( 300 mhz , cdcl3 ) : 7.086.86 ( m , 2h ) , 6.266.13 ( s , 2h ) , 4.133.93 ( m , 1h ) , 3.333.20 ( m , 1h ) , 3.112.83 ( m , 3h ) , 2.63 ( s , 3h ) , 2.382.25 ( m , 1h ) , 2.08 ( t , j = 18.8 hz , 3h ) , 2.061.89 ( m , 1h ) . h nmr ( 500 mhz , cdcl3 ) : 7.39 ( d , j = 7.0 hz , 1h ) , 7.04 ( d , j = 10.8 hz , 1h ) , 6.366.06 ( m , 2h ) , 4.114.01 ( m , 1h ) , 3.35 ( dd , j = 16.2 , 4.7 hz , 1h ) , 3.182.92 ( m , 3h ) , 2.63 ( s , 3h ) , 2.432.31 ( m , 1h ) , 2.17 ( t , j = 18.8 hz , 3h ) , 2.061.95 ( m , 1h ) . esims m / z : 430.2 ( mh ) . h nmr ( 300 mhz , cdcl3 ) : 7.42 ( d , j = 7.0 hz , 1h ) , 6.99 ( d , j = 10.6 hz , 1h ) , 6.266.10 ( m , 2h ) , 4.103.98 ( m , 1h ) , 3.37 ( dd , j = 16.9 , 4.9 hz , 1h ) , 3.152.89 ( m , 3h ) , 2.64 ( s , 3h ) , 2.472.28 ( m , 1h ) , 2.16 ( t , j = 18.8 hz , 3h ) , 2.051.95 ( m , 1h ) . h nmr ( 300 mhz , cdcl3 ) : 7.257.09 ( m , 4h ) , 6.23 ( s , 1h ) , 6.17 ( d , j = 8.0 hz , 1h ) , 4.153.98 ( m , 1h ) , 3.35 ( dd , j = 16.4 , 5.0 hz , 1h ) , 3.152.84 ( m , 3h ) , 2.64 ( s , 3h ) , 2.412.26 ( m , 1h ) , 2.121.95 ( m , 1h ) ; esims m / z : 348.3 ( mh ) . h nmr ( 500 mhz , cdcl3 ) : 7.177.05 ( m , 1h ) , 7.06.82 ( m , 2h ) , 6.436.12 ( m , 2h ) , 4.123.99 ( m , 1h ) , 3.373.26 ( m , 1h ) , 3.102.89 ( m , 3h ) , 2.64 ( s , 3h ) , 2.402.29 ( m , 1h ) , 2.091.98 ( m , 1h ) . h nmr ( 400 mhz , meod ) : 7.177.10 ( m , 3h ) , 6.62 ( s , 1h ) , 4.194.09 ( m , 1h ) , 3.273.19 ( m , 1h ) , 3.052.90 ( m , 3h ) , 2.56 ( s , 3h ) , 2.302.20 ( m , 1h ) , 2.051.94 ( m , 1h ) . h nmr ( 400 mhz , meod ) : 7.177.10 ( m , 3h ) , 6.62 ( s , 1h ) , 4.184.10 ( m , 1h ) , 3.273.19 ( m , 1h ) , 3.052.90 ( m , 3h ) , 2.56 ( s , 3h ) , 2.302.20 ( m , 1h ) , 2.051.97 ( m , 1h ) . esims m / z : 382.0 ( mh ) . h nmr ( 400 mhz , cdcl3 ) : 7.307.24 ( m , 4h ) , 6.3 ( d , j = 7.3 hz , 1h ) , 6.18 ( s , 1h ) , 4.574.49 ( m , 1h ) , 3.51 ( dd , j = 7.1 hz , j = 16.2 hz , 2h ) , 3.133.08 ( m , 2h ) , 2.62 ( s , 3h ) , 2.13 ( t , j = 18.7 hz , 3h ) . h nmr ( 400 mhz , dmso - d6 ) : 8.558.49 ( br , 1h ) , 7.467.44 ( br , 1h ) , 7.377.35 ( m , 1h ) , 7.227.20 ( m , 1h ) , 6.63 ( s , 1h ) , 4.684.58 ( br , 1h ) , 3.323.28 ( br , 1h ) , 3.213.12 ( m , 3h ) , 2.47 ( s , 3h ) , 2.09 ( t , j = 19.2 hz , 3h ) . h nmr ( 400 mhz , dmso - d6 ) : 8.578.48 ( m , 1 h ) , 7.347.29 ( m , 1 h ) , 7.297.19 ( m , 2 h ) , 6.63 ( s , 1 h ) , 4.724.57 ( m , 1 h ) , 3.413.35 ( m , 2 h ) , 3.233.07 ( m , 2 h ) , 2.09 ( t , j = 19.1 hz , 3 h ) . esims m / z : 364 ( mh ) . h nmr ( 400 mhz , cdcl3 ) : 7.37 ( s , 2h ) , 6.28 ( d , j = 7.6 hz , 1h ) , 6.16 ( s , 1h ) , 4.604.52 ( m , 1h ) , 3.513.45 ( m , 2h ) , 3.07 ( dd , j = 4.5 hz , j = 16.4 hz , 2h ) , 2.62 ( s , 3h ) , 2.13 ( t , j = 18.7 hz , 3h ) . esims m / z : 398 ( mh ) . h nmr ( 400 mhz , dmso - d6 ) : 8.57 ( d , j = 7.6 hz , 1 h ) , 7.827.78 ( m , 1 h ) , 7.787.69 ( m , 1 h ) , 7.577.47 ( m , 1 h ) , 6.65 ( s , 1 h ) , 4.794.63 ( m , 1 h ) , 3.523.38 ( m , 2 h ) , 3.303.18 ( m , 2h ) , 3.19 ( s , 3 h ) , 2.48 ( s , 3 h ) , 2.09 ( t , j = 19.2 hz , 3 h ) ; esims m / z : 408 ( mh ) . h nmr ( 400 mhz , dmso - d6 ) : 8.59 ( d , j = 7.8 hz , 1 h ) , 7.61 ( br s , 1 h ) , 7.607.54 ( m , 1 h ) , 7.537.48 ( m , 1 h ) , 6.65 ( s , 1 h ) , 4.764.64 ( m , 1 h ) , 3.523.39 ( m , 2 h ) , 3.303.19 ( m , 2 h ) , 2.61 ( s , 6 h ) , 2.48 ( s , 3 h ) , 2.09 ( t , j = 19.2 hz , 3 h ) . h nmr ( 400 mhz , cdcl3 ) : 6.99 ( s , 2h ) , 6.31 ( d , j = 7.6 hz , 1h ) , 6.19 ( s , 1h ) , 4.584.50 ( m , 1h ) , 3.45 ( dd , j = 7.3 hz , j = 16.2 hz , 2h ) , 3.052.99 ( m , 2h ) , 2.63 ( s , 3h ) , 2.26 ( s , 6h ) , 2.14 ( t , j = 18.7 hz , 3h ) . h nmr ( 300 mhz , cdcl3 ) : 6.94 ( t , j = 5.8 hz , 2h ) , 6.42 ( brs , 1h ) , 6.21 ( s , 1h ) , 4.734.53 ( m , 1h ) , 3.703.44 ( m , 2h ) , 3.353.08 ( m , 2h ) , 2.67 ( s , 3h ) , 2.14 ( t , j = 18.8 hz , 3h ) . h nmr ( 300 mhz , cdcl3 ) : 7.24 ( dd , j = 8.1 , 5.2 hz , 1h ) , 7.046.88 ( m , 2h ) , 6.33 ( d , j = 7.3 hz , 1h ) , 6.19 ( s , 1h ) , 4.684.48 ( m , 1h ) , 3.50 ( dt , j = 15.4 , 7.7 hz , 2h ) , 3.203.00 ( m , 2h ) , 2.63 ( s , 3h ) , 2.15 ( t , j = 18.8 hz , 3h ) . h nmr ( 500 mhz , cdcl3 ) : 7.10 ( t , j = 8.6 hz , 2h ) , 6.31 ( d , j = 7.5 hz , 1h ) , 6.18 ( s , 1h ) , 4.714.48 ( m , 1h ) , 3.50 ( dd , j = 16.2 , 7.0 hz , 2h ) , 3.172.97 ( m , 2h ) , 2.64 ( s , 3h ) , 2.16 ( t , j = 18.8 hz , 3h ) . esims m / z : 366.3 ( mh ) . h nmr ( 500 mhz , cdcl3 ) : 7.56 ( d , j = 7.8 hz , 1h ) , 7.50 ( d , j = 7.5 hz , 1h ) , 7.40 ( t , j = 7.7 hz , 1h ) , 6.32 ( d , j = 7.4 hz , 1h ) , 6.22 ( s , 1h ) , 4.714.57 ( m , 1h ) , 3.71 ( dd , j = 17.2 , 7.2 hz , 1h ) , 3.60 ( dd , j = 16.5 , 7.3 hz , 1h ) , 3.30 ( dd , j = 17.1 , 4.1 hz , 1h ) , 3.19 ( dd , j = 16.5 , 4.6 hz , 1h ) , 2.66 ( s , 3h ) , 2.16 ( t , j = 18.8 hz , 3h ) . esims m / z : 398.3 ( mh ) . h nmr ( 400 mhz , meod ) : 7.17 ( t , j = 9.04 hz , 2h ) , 6.62 ( s , 1h ) , 4.784.62 ( m , 1h ) , 3.45 ( dd , j = 16.01 , 7.52 hz , 2h ) , 3.15 ( dd , j = 15.85 , 6.4 hz , 2h ) , 2.59 ( s , 3h ) . 2,3-dihydro-1h - inden-2-amine hydrochloride ( 38 ) ( 50 g , 295 mmol ) , 45 ml of acetic anhydride , and 25 g of sodium acetate were suspended in 100 ml of acetic acid and stirred overnight at rt . the mixture was extracted with chloroform , and the combined organic layers were washed with water , diluted hcl , water , dried , and evaporated . the residue was recrystallized from chcl3et2o to yield n-(2,3-dihydro-1h - inden-2-yl)acetamide ( 39 ) ( 43 g , 66% yield ) . to a stirred , cooled solution of n-(2,3-dihydro-1h - inden-2-yl)acetamide ( 39 ) ( 24.4 g , 139 mmol ) in 400 ml of chloroform was added dropwise 55 ml of chlorosulfonic acid below 0 c . the solution was stirred while cooling and then allowed to reach rt slowly by stirring overnight . the excess of chlorosulfonic acid decomposed by adding the mixture dropwise to stirred iced water . the layers were separated , and the aqueous layer was extracted several times with chloroform . the concentrate was heated and diluted with additional chloroform but was still containing some undissolved gum . it was filtered , washed with et2o , and dried to yield 2-acetamido-2,3-dihydro-1h - indene-5-sulfonyl chloride ( 40 ) as crystals ( 25.6 g , 67% yield ) . a solution of 2-acetamido-2,3-dihydro-1h - indene-5-sulfonyl chloride ( 40 ) ( 13.7 g , 50 mmol ) in 250 ml of acetic acid was stirred and warmed to 75 c whereupon a solution of 50 g of tin(ii ) chloride dihydrate in 45 ml of conc . the mixture was filtered ( became rapidly gummy when dried on a clay plate ) , washed with cold water , and dissolved in 250 ml of meoh . to the solution , under nitrogen , was added 3 g of sodium methoxide in 4 ml ( > 0.05 mol ) of methyl iodide . the mixture was concentrated and then partitioned between water and chloroform . after drying and evaporation , 8.6 g of oil were obtained , which was purified by chromatography on neutral alumina eluting with etoac . n-(5-(methylthio)-2,3-dihydro-1h - inden-2-yl)acetamide ( 41 ) was obtained as a white solid after washing with et2o and recrystallization from chloroform - et2o ( 3.7 g , 33% yield ) . a solution of n-(5-(methylthio)-2,3-dihydro-1h - inden-2-yl)acetamide ( 41 ) ( 2.2 g , 9.9 mmol ) and m - chloroperbenzoic acid ( 5 g ) in 900 ml of chloroform was stirred at rt for 2 h. the solution was washed with aq . the residue was triturated with et2o , filtered , and dried to yield the n-(5-(methylsulfonyl)-2,3-dihydro-1h - inden-2-yl)acetamide ( 42 ) as a white solid ( 1.87 g , 75% yield ) . a mixture of 1.9 g ( 7.5 mmol ) of n-(5-(methylsulfonyl)-2,3-dihydro-1h - inden-2-yl)acetamide ( 42 ) and 20 ml of 3 n hcl was stirred under reflux for 2.5 h. the solution was cooled , and the residue was azeotroped with etoh . recrystallization from meoh - etoh yielded 5-(methylsulfonyl)-2,3-dihydro-1h - inden-2-amine hydrochloride ( 43 ) as crystals ( 0.87 g , 55% yield ) . h nmr ( 500 mhz , dmso - d6 ) 8.17 ( br . s. , 2h ) , 7.84 ( s , 1h ) , 7.797.71 ( m , 1h ) , 7.54 ( d , j = 8.0 hz , 1h ) , 4.06 ( ddd , j = 2.5 , 5.0 , 7.6 hz , 1h ) , 3.403.34 ( m , 2h ) , 3.17 ( s , 3h ) , 3.102.98 ( m , 2h ) .

malaria persists as one of the most devastating global infectious diseases . the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase ( dhodh ) has been identified as a new malaria drug target , and a triazolopyrimidine - based dhodh inhibitor 1 ( dsm265 ) is in clinical development . we sought to identify compounds with higher potency against plasmodium dhodh while showing greater selectivity toward animal dhodhs . herein we describe a series of novel triazolopyrimidines wherein the p - sf5-aniline was replaced with substituted 1,2,3,4-tetrahydro-2-naphthyl or 2-indanyl amines . these compounds showed strong species selectivity , and several highly potent tetrahydro-2-naphthyl derivatives were identified . compounds with halogen substitutions displayed sustained plasma levels after oral dosing in rodents leading to efficacy in the p. falciparum scid mouse malaria model . these data suggest that tetrahydro-2-naphthyl derivatives have the potential to be efficacious for the treatment of malaria , but due to higher metabolic clearance than 1 , they most likely would need to be part of a multidose regimen .

Introduction Chemistry Results Discussion Conclusion Experimental Section

malaria is one of the most deadly infectious diseases in human history with 3.2 billion people in 97 countries at risk . human malaria , which is transmitted by the female anopheles mosquito , can be caused by five species of plasmodia ; however , plasmodium falciparum and plasmodium vivax are the most signficant.p . a large collection of drugs has been used for the treatment of malaria , but many of the most important compounds have been lost to drug resistance ( e.g. to combat drug resistance a significant effort is underway to identify new compounds that can be used for the treatment of malaria , with several new entities currently in clinical development . the triazolopyrimidine dsm265 ( 1 ) ( figure 1 ) developed by our group is the first antimalarial agent that targets dihydroorotate dehydrogenase ( dhodh ) to reach clinical development , validating this target for the treatment of malaria . we identified the triazolopyrimidine dhodh inhibitor series by a target - based high throughput screen , and the initial lead dsm1 ( 2 ) ( figure 1 ) was shown to selectively inhibit p. falciparum dhodh and to kill parasites in vitro , but it was ineffective in vivo due to poor metabolic properties . while 1 has progressed successfully to phase ii clinical trials , we sought to identify potential backup compounds if unforeseen issues arise during its clinical development . importantly , 1 has potent activity on pfdhodh and p. falciparum parasites in vivo , and its pharmacokinetic properties support its use as a single dose treatment or once weekly prophylactic . we therefore sought to identify compounds that have broad selectivity against all mammalian dhodhs , while if possible identifying compounds with higher potency against pfdhodh that could potentially reduce the dose required for treatment . we describe herein a series of triazolopyrimidines where the sf5-aniline moiety of 1 has been replaced by either substituted 1,2,3,4-tetrahydro-2-naphthyl or 2-indanyl amines . compounds from both series are inactive against a range of mammalian dhodhs , while several of the identified tetrahydronapthalenes are more potent than 1 against p. falciparum parasites in vitro and indeed are among the most potent compounds that have been identified in the series . however , these compounds are less metabolically stable than 1 in mouse liver microsomes , suggesting the likelihood of higher clearance in mice and explaining the requirement for higher doses to inhibit parasites in the in vivo mouse model of malaria . activity relationships ( sars ) of replacing the p - sf5-aniline of 1 with substituted 1,2,3,4-tetrahydro-2-naphthyl or 2-indanyl amines with the goal of eliminating activity on rodent dhodhs , while potentially improving potency against p. falciparum dhodh and thereby lowering the dose required for in vivo efficacy . because these species are required for toxicity testing in preclinical studies we sought to eliminate rodent and dog dhodh inhibitory activity for any potential candidate that might be developed as a backup to 1 . we previously reported that substitution of the p - sf5-aniline with p - cf3 aniline in the context of either mono or difluoro groups at the meta position led to greater inhibition of the mammalian enzymes . x - ray structural data suggested that increasing hydrophobicity drove the interaction of these compounds with the mammalian enzymes . we previously tested several tetrahydro-2-naphthyl and 2-indanyl amines as substitutes for the cf3-aniline of 3 but found these compounds lacked potency , and furthermore , the one analogue containing 1,2,3,4-tetrahydro-2-naphthyl also lacked metabolic stability in mouse microsomes . we therefore synthesized a series of tetrahydro-2-naphthyl or 2-indanyl analoues with halo alkyl groups at the c2 position . dhodh ic50 and p. falciparum ec50 values were determined from triplicate data points at each concentration in the dose titration . compound 1 has been previously profiled in these assays , and parasite data were previously reported . for the studies described herein , 7-chloro-2-(1,1-difluoroethyl)-5-methyltriazolo[1,5-a ] pyrimidine ( 4a ) and 7-chloro-2-(trifluoromethyl)-5-methyltriazolo[1,5-a ] pyrimidine ( 4b ) were prepared using these methods ( scheme 1 ) and were reacted with the requisite amines to afford the final products containing either the 1,2,3,4-tetrahydro-2-naphthyl ( 525 ) or 2-indanyl ( 2637 ) amines in place of the p - sf5-aniline of 1 . the 1,2,3,4-tetrahydro-2-naphthyls contained a chiral center , and for select compounds , individual enantiomers were purified using a chiral column as described in the experimental section . compounds were first analyzed for potency against p. falciparum , p. vivax , and human dhodh , and for activity on p. falciparum parasites in vitro ( table 1 ) . the most potent analogues based on the whole cell p. falciparum assay were 9 and 13 , both of which contain a halogen ( chloro or bromo , respectively ) in the 7 position . compound 13 is the most potent analogue identified in the triazolopyrimidine series that has been reported . for the tetrahydro-2-naphthyls where individual enantiomers were characterized , differences in potency between the active enantiomer ( 7 , 9 , 11 , 13 , 16 , 19 , 24 ) and the inactive enantiomer ( 8 , 10 , 12 , 14 , 17 , 18 , 25 ) ranged from 20- to 360-fold toward pfdhodh and from 30- to 2000-fold against p. falciparum 3d7 parasites . we did not isolate the individual enantiomers for compounds from the indane series ( 27 , 28 , 30 , 31 , 34 , and 36 ) , and it is likely that the purified active enantiomers would also show higher activity for these compounds as well . overall we observed a good correlation between potency on pfdhodh and potency on 3d7 p. there was a tendency for the most potent compounds on the parasite to show lower potency against pfdhodh , but this difference is likely caused by the complication of stoichiometric binding for compounds with ic50s at or below the concentration of enzyme used in the assay ( pfdhodh = 5 nm ) . in order to confirm that , for the most potent compounds , parasite killing is due to dhodh inhibition , we tested select compounds ( 9 and 13 ) for their ability to inhibit growth of a genetically engineered parasite strain that expresses yeast dhodh ( d10 ydhodh ) . species selectivity was first evaluated by testing against human and p. vivax dhodh ( table 1 ) and then for a selection of the more potent compounds , inhibitor activity was also measured for mouse , rat , and dog dhodh ( table 2 ) . however , unlike 1 , none of the compounds showed any activity against either the rodent or dog dhodhs ( table 2 ) , demonstrating that we had achieved our objective of eliminating mammalian dhodh activity . limited residues from the 4 shell around 13 are shown , and the structure has been aligned to the pfdhodh structure bound to 2 ( pdb 3i65 ) to allow comparison of the binding modes . aqueous solubility ( ph 6.5 ) was poor ( 1.66.3 g / ml ) to moderate ( 12.525 g / ml ) , and in general , compounds in this series had lower kinetic solubility than 1 ( table 3 ) . more extensive solubility studies in simulated gastric and intestinal fluids was conducted on a selection of the most potent analogues from the 2-indanyl ( 35 ) and the tetrahydro-2-naphthyl ( 7 , 9 , 11 , 13 ) series ( table 4 ) . substantial improvement in metabolic stability was obtained by moving the fluorine to the 5 and 6 positions of the ring 35 , leading to low intrinsic clearance in human and rat microsomes and moderate clearance in mouse microsomes . herg channel inhibition has been associated with qt prolongation and arrhythmias , and patch clamp assays have become a routine method to provide an initial analysis that a compound may potentially be associated with qt syndromes in humans . in order to test for the potential for drug drug interactions , select compounds were tested for their ability to inhibit cytochrome p450 isoforms in human liver microsomes ( table 5 ) . unbound clearance values for the tetrahydro-2-naphthyl compounds were somewhat lower for the two chloro - substituted compounds ( 438 and 589 ml / min / kg for 7 and 9 , respectively ) compared to the bromo compounds ( 933 and 800 ml / min / kg for 11 and 13 , respectively ) , and all were higher than for 1 ( 263 ml / min / kg ) . as a result , unbound auc values for the tetrahydro-2-naphthyls were approximately 24-fold lower than 1 after oral dosing at approximately 20 mg / kg . based on the observation of good oral exposure after dosing in mice , we tested three compounds in the scid mouse p. falciparum efficacy model , which has become the standard model for evaluating the efficacy of new antimalarial compounds . the tetrahydro-2-naphthyl compounds substituted at the 7 position ( 9 and 13 ) were more potent against p. falciparum in the in vitro parasite assay than those substituted on the 6 position ( 7 and 11 ) and showed fairly equivalent exposure in mice after oral dosing . we therefore decided to test both a 6- and 7-substituted tetrahydro-2-naphthyl ( 7 and 9 ) in the p. because 35 showed significantly higher plasma exposure in mice than the remaining compounds , we also selected this compound for an efficacy study despite the 10-fold lower potency in the p. falciparum parasite assay . because of the complicated nature of blood in the scid mouse model ( i.e. mice were infected with p. falciparum pf3d70087/n9 parasites by intravenous injection on day zero , and dosing was once daily for 4 days starting on day 3 postinfection . malaria is one of the most serious global infectious diseases , and while a number of effective drugs have been used to treat malaria , drug resistance has led to the loss of most agents that have been in widespread use . dhodh has emerged as a key new target in efforts to identify antimalarial drugs that work on clinical isolates resistant to standard therapies , and as noted , the triazolopyrimidine 1 is currently in clinical development . in order to identify potential backup compounds to 1 in case issues arise in its clinical development , we sought to identify additional compounds within the triazolopyrimidine series with the potential to advance into preclinical development . we therefore sought to identify new derivatives that showed broad selectivity against all of the key mammalian enzymes . herein we describe a series of tetrahydro-2-naphthyl and 2-indanyl triazolopyrimidine analogues , which display a range of potencies against plasmodium dhodh but importantly lack activity against human dhodh or any of the key mammalian enzymes from species that are important for toxicological profiling ( mouse , rat , and dog ) . pharmacologic profiling and in vivo efficacy assays suggest that several of the identified analogues have potential , with 9 showing the most promising profile . the most potent of the tetrahydro-2-naphthyl derivatives also showed good activity ( within 1.5-fold ) against p. vivax dhodh suggesting that they would be useful for the treatment of both p. falciparum and p. vivax . in contrast , the best of the 2-indanyl analogues ( 35 ) was 7-fold less active on p. vivax dhodh suggesting it might not have good activity against the p. vivax parasite . like the naphthyl of 2 , the tetrahydro-2-naphthyl of 13 better fills the available binding pocket than the sf5-aniline of 1 , which supports our initial hypothesis that compounds with improved potency could be identified by substitution of the sf5-aniline with bulkier aromatic groups . analogues substituted in the 7 position were more potent than 6 position analogues , and indeed 13 with subnanomolar activity against the parasite in whole cell assays is the most potent analogue that has been identified in the series . while the total plasma exposure ( auc and cmax ) in rats was higher for the 6 position analogues , correction of the data for protein binding indicated that the unbound exposure and unbound clearance were similar ( within 2-fold ) for the 6 and 7 position analogues with the chloro - substituted compounds ( 7 and 9 ) showing about 2-fold lower unbound clearance than the bromo - substituted compounds ( 11 and 13 ) . protein binding data in mouse plasma was only available for 9 , 11 , and 35 , and binding for each of these compounds was similar in each of the three species tested . mouse plasma protein binding was therefore estimated for 7 and 13 using the average of the human and rat values , suggesting that unbound concentrations for each of the compounds are likely to be within a similar range ( table 6 ) . the binding trends in human and mouse plasma would suggest that unbound concentrations of 9 in the scid mouse are likely to be considerably higher compared to those for 1 at the same total blood concentration . while all three tested compounds ( 7 , 9 , and 35 ) showed in vivo antimalarial activity , it is unlikely that any of these compounds have the necessary properties to support a single dose treatment regimen at a practical dose level in humans . cyp2d6 inhibition was modest with the most potent inhibition observed in the 36 m range , but does suggest some potential for drug drug interactions with compounds that are substrates for this enzyme . insufficient efficacy data is available for compounds 7 and 35 to determine the safety margin relative to unbound concentrations at an efficacious dose ; however , for 9 the cmax at the ed90 in the scid study ( unbound concentration of approximately 0.05 m ) is 30-fold lower than the ic50 on the herg channel , again suggesting a low likelihood the channel would be inhibited in vivo . first , only the chloro - substituted tetrahydro-2-naphthyls were tested in the scid efficacy model , leaving open the possibility that one of the bromo analogues might have shown better in vivo efficacy . decreasing lipophilicity will likely require the sf5-anline of 1 to be replaced with more hydrophilic aromatic amines , provided potency can be maintained . we have described the identification of several tetrahydro-2-naphthyl and 2-indanyl triazolopyrimidine analogues that are potent inhibitors of plasmodium dhodh and that have improved species selectivity over the mammalian enzymes in comparison to the clinical candidate 1 . the compounds have potent activity against p. falciparum in parasite assays and showed antimalarial activity in the p. falciparum scid mouse model of disease . these compounds showed good plasma exposure after oral dosing in mice and rats and were well tolerated in the in vivo studies that were performed . several of the identified compounds , most notably 9 , have the potential to be further developed for treatment of malaria . oral doses were administered as a suspension in a vehicle containing 0.5% w / v carboxymethylcellulose , 0.5% v / v benzyl alcohol ( as a preservative ) , and 0.4% v / v tween 80 , and intravenous doses were administered in cosolvent vehicle containing 40% propylene glycol , 10% ethanol , 0.4% tween 80 in water . iv doses to rats were administered as a 10 min constant rate infusion of 1 ml into an indwelling jugular vein cannula , and oral doses were administered by gavage as 10 ml / kg ( both rats and mice ) . microsomes were suspended in phosphate buffer and incubated at 37 c in the presence of probe substrates , and reactions were initiated by the addition of an nadph - regenerating system . compound levels in the formulations or in the blood of infected mice were measured by lc human biological samples were sourced ethically , and their research use was in accordance with the terms of the informed consents . the crystal of pfdhodh384413 - 13 diffracted to 2.32 and has a space group of p64 with the cell dimension of a = b = 86.1 , c = 139.1 ( table s3 ) .

metabolic syndrome ( mets ) is a constellation of interrelated metabolic risk factors that appear to directly promote the development of diabetes and cardiovascular diseases.1 identification of biomarkers associated with mets and diabetes is desirable to establish screening programs , preventive interventions , and therapeutic strategies most appropriately directed at mets and diabetes , which can reduce the increased prevalence of mets and diabetes worldwide and attenuate the morbidity and mortality of diabetes and cardiovascular disease.2,3 although several studies have analyzed the associations of mets and diabetes risk with biomarkers , including high - sensitivity c - reactive protein ( hs - crp ) , n - terminal prohormone of brain natriuretic peptide ( nt - probnp ) , and homocysteine ( hcy ) , there have been too much controversial results.414 to our knowledge , a limited amount of information is available regarding these associations in non - caucasian populations , particularly among chinese community - dwelling residents.15,16 recent studies have more and more focused on the significant role of insulin resistance ( ir ) in linking cardiovascular risk factors.17 studies about the associations of ir with biomarkers representing diverse biological roles and pathological phenomena can help us to understand the mechanisms underlying the development of mets and diabetes . notably , few studies , especially chinese community - based studies , concerning the associations of ir with biomarkers , including hs - crp , nt - probnp , and hcy , have been conducted , and it is a long way to go for the definite answer.10,1822 therefore , the aim of the current study was to perform deep analyses of the associations of biomarkers , including hs - crp , nt - probnp , and hcy , with ir , mets , and diabetes risk and evaluate the abilities of biomarkers to identify ir , mets , and diabetes risk in chinese community - dwelling middle - aged and elderly residents . the current study was conducted as part of a large health checkup program in beijing , people s republic of china , during the period from may 2007 to july 2009 . the participants were restricted to the community - dwelling residents of han origin older than 45 years at the time of health examination . there were a total of 396 participants in the current analysis after excluding 63 participants due to the diagnosis of diabetes . a stratified cluster sampling design was used in this survey . in the first stage of sampling , three districts ( fengtai , shijingshan , and daxing ) were selected from 18 districts in beijing . in the second stage of sampling , four communities were selected from these districts . in the third stage of sampling , participants were selected from these communities . the study protocol was approved by the ethics committee of chinese people s liberation army general hospital ( beijing , people s republic of china ) . the participants underwent routine clinical physical examinations , which included measurements of height , weight , waist circumference ( wc ) , and resting blood pressures ( bps ) . all data for each participant were evaluated by well - trained physicians in chinese people s liberation army general hospital . body weight was determined using a digital scale with the participants lightly clothed and without shoes . body mass index ( bmi ) was calculated as the ratio of the weight ( kg ) to the square of height ( m ) . wc was measured using a soft tape while the subjects stood balanced on both feet , with the feet touching each other and both arms hanging freely . systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) , measured using a standard mercury sphygmomanometer , were taken at the first and fifth korotkoff sounds in the seated position after participants rested for at least 5 minutes . two measurements were performed at 1-minute intervals , and the mean of two readings was used for analyses . blood samples were withdrawn between 8 am and 10 am after an overnight fast of at least 12 hours , routinely stored , and submitted to the central laboratory in the department of biochemistry , chinese people s liberation army general hospital , on the same day . fasting blood glucose ( fbg ) , triglyceride ( tg ) , low - density lipoprotein - cholesterol ( ldl - c ) , and high - density lipoprotein - cholesterol ( hdl - c ) were checked by qualified technicians blinded to clinical data using enzymatic assays ( hoffman - la roche ltd . , basel , switzerland ) on a fully automatic biochemical analyzer ( cobas c6000 ; hoffman - la roche ltd . ) . the participants attending the collection of fasting blood sample subsequently performed the standard oral glucose tolerance test and obtained the postprandial blood glucose ( pbg ) , which was done 2 hours after consumption of 75 g glucose . concentrations of hs - crp were determined by an immunoturbidimetric assay using a dimension rxl max analyzer ( siemens healthcare diagnostics inc . , munich , germany ) . concentrations of nt - probnp were measured with an electrochemiluminescence immunoassay ( hoffman - la roche ltd . ) using an analyzer ( cobas c6000 ) . fasting insulin ( fins ) levels were determined by dpc kit ( dpc cirrus inc . , los angeles , ca , usa ) on a fully automatic chemiluminescence analyzer ( dpc immulite 1000 ; dpc cirrus inc . ) . ir was assessed by the homeostasis model assessment of ir ( homa - ir ) using the following formula : homa - ir = fins ( miu / l ) fbg ( mmol / l)/22.5.23 according to the worldwide consensus on the definition of mets recommended by the international diabetes federation,24 for a person to be defined as having the mets , he or she must have central obesity plus any two of four additional factors : tg 1.7 mmol / l , hdl - c < 1.0 mmol / l in males and < 1.3 mmol / l in females ( or specific treatment for these lipid abnormalities ) , sbp 130 mmhg or dbp 85 mmhg ( or treatment of previously diagnosed hypertension ) , and fbg 5.6 general obesity , overweight , and central obesity were defined as bmi 28 kg / m , bmi 24 kg / m , and wc 85 cm for men and 80 cm for women based on the guidelines for preservation and control of overweight and obesity in chinese adults , respectively.25 more recently , our hospital set up the first risk score of the people s republic of china identifying individuals who were likely to develop diabetes in the near future , named the chinese diabetes risk score ( cdrs ) , which consisted of age ( < 66 : coded 0 ; 6675 : coded 1 ; 76 : coded 2 ) , hypertension ( coded 1 ) , history of high blood glucose ( coded 3 ) , bmi ( < 24 : coded 0 ; 24 : coded 1 ) , high fbg ( coded 2 ) , high tg ( coded 1 ) , and low hdl - c ( coded 2 ) . hypertension was defined as having the sbp 140 mmhg , dbp 90 mmhg , or the use of antihypertensive medications . the total score value ranged from 0 to 12 , and a cutoff score of 4 indicated optimum sensitivity and specificity for detecting diabetes.26 categorical variables were reported as number with percentage , normally distributed continuous variables as mean with standard deviation , and nonnormally distributed continuous variables as median with interquartile range . differences between groups were analyzed with chi - squared test for categorical variables , independent t - test for normally distributed continuous variables , and mann whitney test for nonnormally distributed continuous variables . bivariate correlations of biomarkers with each component of mets and cdrs , mets component numbers , cdrs , and homa - ir were determined using the pearson s ( normal distribution ) and spearman s ( abnormal distribution ) coefficients . receiver operating characteristic curves and area under the curve were used to evaluate whether the biomarkers were capable to identify each component of mets and cdrs , homa - ir , mets , and cdrs . the logistic regression procedures were applied to assess the risk of each component of mets and cdrs , homa - ir , mets , and cdrs according to biomarkers . all reported p - values were two tailed , and confidence intervals were calculated at the 95% level . all statistical analyses were performed using statistic package for the social sciences ( version 17.0 ; spss inc . the current study was conducted as part of a large health checkup program in beijing , people s republic of china , during the period from may 2007 to july 2009 . the participants were restricted to the community - dwelling residents of han origin older than 45 years at the time of health examination . there were a total of 396 participants in the current analysis after excluding 63 participants due to the diagnosis of diabetes . a stratified cluster sampling design was used in this survey . in the first stage of sampling , three districts ( fengtai , shijingshan , and daxing ) were selected from 18 districts in beijing . in the second stage of sampling , four communities were selected from these districts . in the third stage of sampling , participants were selected from these communities . the study protocol was approved by the ethics committee of chinese people s liberation army general hospital ( beijing , people s republic of china ) . the participants underwent routine clinical physical examinations , which included measurements of height , weight , waist circumference ( wc ) , and resting blood pressures ( bps ) . all data for each participant were evaluated by well - trained physicians in chinese people s liberation army general hospital . body weight was determined using a digital scale with the participants lightly clothed and without shoes . body mass index ( bmi ) was calculated as the ratio of the weight ( kg ) to the square of height ( m ) . wc was measured using a soft tape while the subjects stood balanced on both feet , with the feet touching each other and both arms hanging freely . systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) , measured using a standard mercury sphygmomanometer , were taken at the first and fifth korotkoff sounds in the seated position after participants rested for at least 5 minutes . two measurements were performed at 1-minute intervals , and the mean of two readings was used for analyses . blood samples were withdrawn between 8 am and 10 am after an overnight fast of at least 12 hours , routinely stored , and submitted to the central laboratory in the department of biochemistry , chinese people s liberation army general hospital , on the same day . fasting blood glucose ( fbg ) , triglyceride ( tg ) , low - density lipoprotein - cholesterol ( ldl - c ) , and high - density lipoprotein - cholesterol ( hdl - c ) were checked by qualified technicians blinded to clinical data using enzymatic assays ( hoffman - la roche ltd . , basel , switzerland ) on a fully automatic biochemical analyzer ( cobas c6000 ; hoffman - la roche ltd . ) . the participants attending the collection of fasting blood sample subsequently performed the standard oral glucose tolerance test and obtained the postprandial blood glucose ( pbg ) , which was done 2 hours after consumption of 75 g glucose . concentrations of hs - crp were determined by an immunoturbidimetric assay using a dimension rxl max analyzer ( siemens healthcare diagnostics inc . , munich , germany ) . concentrations of nt - probnp were measured with an electrochemiluminescence immunoassay ( hoffman - la roche ltd . ) using an analyzer ( cobas c6000 ) . fasting insulin ( fins ) levels were determined by dpc kit ( dpc cirrus inc . , los angeles , ca , usa ) on a fully automatic chemiluminescence analyzer ( dpc immulite 1000 ; dpc cirrus inc . ) . ir was assessed by the homeostasis model assessment of ir ( homa - ir ) using the following formula : homa - ir = fins ( miu / l ) fbg ( mmol / l)/22.5.23 according to the worldwide consensus on the definition of mets recommended by the international diabetes federation,24 for a person to be defined as having the mets , he or she must have central obesity plus any two of four additional factors : tg 1.7 mmol / l in females ( or specific treatment for these lipid abnormalities ) , sbp 130 mmhg or dbp 85 mmhg ( or treatment of previously diagnosed hypertension ) , and fbg 5.6 mmol / l ( or previously diagnosed diabetes ) . general obesity , overweight , and central obesity were defined as bmi 28 kg / m , bmi 24 kg / m , and wc 85 cm for men and 80 cm for women based on the guidelines for preservation and control of overweight and obesity in chinese adults , respectively.25 more recently , our hospital set up the first risk score of the people s republic of china identifying individuals who were likely to develop diabetes in the near future , named the chinese diabetes risk score ( cdrs ) , which consisted of age ( < 66 : coded 0 ; 6675 : coded 1 ; 76 : coded 2 ) , hypertension ( coded 1 ) , history of high blood glucose ( coded 3 ) , bmi ( < 24 : coded 0 ; 24 : coded 1 ) , high fbg ( coded 2 ) , high tg ( coded 1 ) , and low hdl - c ( coded 2 ) . hypertension was defined as having the sbp 140 mmhg , dbp 90 mmhg , or the use of antihypertensive medications . < 1.0 mmol / l in women was considered as low hdl - c . the total score value ranged from 0 to 12 , and a cutoff score of 4 indicated optimum sensitivity and specificity for detecting diabetes.26 categorical variables were reported as number with percentage , normally distributed continuous variables as mean with standard deviation , and nonnormally distributed continuous variables as median with interquartile range . differences between groups were analyzed with chi - squared test for categorical variables , independent t - test for normally distributed continuous variables , and mann whitney test for nonnormally distributed continuous variables . bivariate correlations of biomarkers with each component of mets and cdrs , mets component numbers , cdrs , and homa - ir were determined using the pearson s ( normal distribution ) and spearman s ( abnormal distribution ) coefficients . receiver operating characteristic curves and area under the curve were used to evaluate whether the biomarkers were capable to identify each component of mets and cdrs , homa - ir , mets , and cdrs . the logistic regression procedures were applied to assess the risk of each component of mets and cdrs , homa - ir , mets , and cdrs according to biomarkers . all reported p - values were two tailed , and confidence intervals were calculated at the 95% level . all statistical analyses were performed using statistic package for the social sciences ( version 17.0 ; spss inc . , chicago , il , usa ) software . for the total population , the median age was 66 years ( 5871 years ) , and 53% were male , with median hs - crp , nt - probnp , and hcy concentrations of 0.29 mg / dl ( 0.170.38 mg / dl ) , 41.42 pg / ml ( 17.3480.72 pg / ml ) , and 17.20 mol / l ( 14.1021.58 mol / l ) , respectively . the median level of homa - ir was 1.5 ( 1.02.1 ) , and 25% of participants had cdrs 4 . the characteristics of the whole cohort grouped by the levels of homa - ir and the presence of mets are provided in table 1 . the individuals with homa - ir > 1.5 and mets were more likely to have higher levels of bmi , wc , dbp , fins , pbg , tg , and hypertension and lower hdl - c levels than others ( p<0.05 for all ) . the levels of sbp in the individuals with mets and the levels of fbg in the individuals with homa - ir > 1.5 were higher than those of others ( p<0.05 for all ) . in spite of no difference in sex ratio between the individuals with and without homa - ir > 1.5 ( p>0.05 ) , there were more males with mets ( p<0.05 ) . the levels of ldl - c were similar between the individuals with and without mets and homa - ir > 1.5 ( p>0.05 for all ) . both groups were similar with regard to history of high blood glucose ( p>0.05 for all ) . serum hs - crp concentrations in the individuals with mets were higher than those of others ( p<0.05 ) , but they were similar between the individuals with and without homa - ir > 1.5 ( p>0.05 ) . serum nt - probnp concentrations in the individuals with homa - ir > 1.5 were lower than those of others ( p<0.05 ) , but they were similar between the individuals with and without mets ( p>0.05 ) . there was no difference in serum hcy concentrations between the individuals with and without homa - ir > 1.5 and mets ( p>0.05 for all ) . the characteristics of the whole cohort based on the levels of cdrs and the presence of general obesity are described in table 2 . the individuals with cdrs 4 and general obesity tended to have higher levels of bmi , wc , sbp , fins , pbg , tg , and hypertension and lower levels of hdl - c than others ( p<0.05 for all ) . both groups were similar with regard to sex ratio and ldl - c levels ( p>0.05 for all ) . the levels of dbp in the individuals with general obesity were higher than those of others ( p<0.05 ) , but they were similar between the individuals with and without cdrs 4 ( p>0.05 ) . the levels of serum fbg and history of high blood glucose in the individuals with cdrs 4 were higher and more than those of others ( p<0.05 for all ) , but they were similar between the individuals with and without general obesity ( p>0.05 ) . serum hs - crp concentrations in the individuals with cdrs 4 were higher than those of others ( p<0.05 ) . serum nt - probnp concentrations in the individuals with cdrs 4 were higher than those of others ( p<0.05 ) , but they were similar between the individuals with and without general obesity ( p>0.05 ) . individuals who did not have cdrs 4 and general obesity were likely to have higher serum hcy concentrations compared with those who had cdrs 4 and general obesity ( p<0.05 for all ) . correlation coefficients for biomarkers in relation to the components of mets and cdrs , mets component numbers , cdrs , and homa - ir are presented in table 3 . serum hs - crp concentrations were related to the levels of bmi , wc , fbg , hdl - c , mets component numbers , and cdrs ( p<0.05 for all ) . serum nt - probnp concentrations were correlated with the levels of sbp , dbp , tg , cdrs , and homa - ir ( p<0.05 for all ) . serum hcy concentrations were linked to the levels of bmi , wc , sbp , fbg , hdl - c , and cdrs ( p<0.05 for all ) . however , no other significant relationship was observed in the correlation analyses ( p>0.05 for all ) . area under the curve values of biomarkers for identifying the components of mets and cdrs , homa - ir , mets , and cdrs are shown in table 4 . serum hs - crp concentrations were able to identify mets , cdrs 4 , hdl - c < 0.9/1.0 mmol / l , and hdl - c < 1.0/1.3 mmol / l ( p<0.05 for all ) . serum nt - probnp concentrations were able to identify homa - ir > 1.5 , cdrs 4 , overweight , and bp 140/90 mmhg ( p<0.05 for all ) . serum hcy concentrations were able to identify cdrs 4 , general obesity , overweight , and bp 140/90 mmhg ( p<0.05 for all ) . the results from logistic analyses for biomarkers with the components of mets and cdrs , homa - ir , mets , and cdrs are displayed in table 5 . serum hs - crp concentrations were independently associated with mets as well as hdl - c < 1.0/1.3 mmol / l and hdl - c < 0.9/1.0 mmol / l after adjustment shown in table 5 ( p<0.05 for all ) . serum nt - probnp concentrations were independently associated with bp 140/90 mmhg after adjustment shown in table 5 ( p<0.05 ) . serum hcy concentrations were independently associated with cdrs 4 after adjustment shown in table 5 ( p<0.05 ) . however , other associations for biomarkers did not reach statistical significance with adjustment ( p>0.05 for all ) . for the total population , the median age was 66 years ( 5871 years ) , and 53% were male , with median hs - crp , nt - probnp , and hcy concentrations of 0.29 mg / dl ( 0.170.38 mg / dl ) , 41.42 pg / ml ( 17.3480.72 pg / ml ) , and 17.20 mol / l ( 14.1021.58 mol / l ) , respectively . the median level of homa - ir was 1.5 ( 1.02.1 ) , and 25% of participants had cdrs 4 . the characteristics of the whole cohort grouped by the levels of homa - ir and the presence of mets are provided in table 1 . the individuals with homa - ir > 1.5 and mets were more likely to have higher levels of bmi , wc , dbp , fins , pbg , tg , and hypertension and lower hdl - c levels than others ( p<0.05 for all ) . the levels of sbp in the individuals with mets and the levels of fbg in the individuals with homa - ir > 1.5 were higher than those of others ( p<0.05 for all ) . in spite of no difference in sex ratio between the individuals with and without homa - ir > 1.5 ( p>0.05 ) , there were more males with mets ( p<0.05 ) . the levels of ldl - c were similar between the individuals with and without mets and homa - ir > 1.5 ( p>0.05 for all ) . both groups were similar with regard to history of high blood glucose ( p>0.05 for all ) . serum hs - crp concentrations in the individuals with mets were higher than those of others ( p<0.05 ) , but they were similar between the individuals with and without homa - ir > 1.5 ( p>0.05 ) . serum nt - probnp concentrations in the individuals with homa - ir > 1.5 were lower than those of others ( p<0.05 ) , but they were similar between the individuals with and without mets ( p>0.05 ) . there was no difference in serum hcy concentrations between the individuals with and without homa - ir > 1.5 and mets ( p>0.05 for all ) . the characteristics of the whole cohort based on the levels of cdrs and the presence of general obesity are described in table 2 . the individuals with cdrs 4 and general obesity tended to have higher levels of bmi , wc , sbp , fins , pbg , tg , and hypertension and lower levels of hdl - c than others ( p<0.05 for all ) . both groups were similar with regard to sex ratio and ldl - c levels ( p>0.05 for all ) . the levels of dbp in the individuals with general obesity were higher than those of others ( p<0.05 ) , but they were similar between the individuals with and without cdrs 4 ( p>0.05 ) . the levels of serum fbg and history of high blood glucose in the individuals with cdrs 4 were higher and more than those of others ( p<0.05 for all ) , but they were similar between the individuals with and without general obesity ( p>0.05 ) . serum hs - crp concentrations in the individuals with cdrs 4 were higher than those of others ( p<0.05 ) . serum nt - probnp concentrations in the individuals with cdrs 4 were higher than those of others ( p<0.05 ) , but they were similar between the individuals with and without general obesity ( p>0.05 ) . individuals who did not have cdrs 4 and general obesity were likely to have higher serum hcy concentrations compared with those who had cdrs 4 and general obesity ( p<0.05 for all ) . correlation coefficients for biomarkers in relation to the components of mets and cdrs , mets component numbers , cdrs , and homa - ir are presented in table 3 . serum hs - crp concentrations were related to the levels of bmi , wc , fbg , hdl - c , mets component numbers , and cdrs ( p<0.05 for all ) . serum nt - probnp concentrations were correlated with the levels of sbp , dbp , tg , cdrs , and homa - ir ( p<0.05 for all ) . serum hcy concentrations were linked to the levels of bmi , wc , sbp , fbg , hdl - c , and cdrs ( p<0.05 for all ) . however , no other significant relationship was observed in the correlation analyses ( p>0.05 for all ) . area under the curve values of biomarkers for identifying the components of mets and cdrs , homa - ir , mets , and cdrs are shown in table 4 . serum hs - crp concentrations were able to identify mets , cdrs 4 , hdl - c < 0.9/1.0 mmol / l , and hdl - c < 1.0/1.3 mmol / l ( p<0.05 for all ) . serum nt - probnp concentrations were able to identify homa - ir > 1.5 , cdrs 4 , overweight , and bp 140/90 mmhg ( p<0.05 for all ) . serum hcy concentrations were able to identify cdrs 4 , general obesity , overweight , and bp 140/90 mmhg ( p<0.05 for all ) . the results from logistic analyses for biomarkers with the components of mets and cdrs , homa - ir , mets , and cdrs are displayed in table 5 . serum hs - crp concentrations were independently associated with mets as well as hdl - c < 1.0/1.3 mmol / l and hdl - c < 0.9/1.0 mmol / l after adjustment shown in table 5 ( p<0.05 for all ) . serum nt - probnp concentrations were independently associated with bp 140/90 mmhg after adjustment shown in table 5 ( p<0.05 ) . serum hcy concentrations were independently associated with cdrs 4 after adjustment shown in table 5 ( p<0.05 ) . however , other associations for biomarkers did not reach statistical significance with adjustment ( p>0.05 for all ) . mets is a cluster of metabolic risk factors associated with an increased risk for developing diabetes and cardiovascular diseases.1 the prevalence of mets and diabetes is increasing worldwide , thus creating an urgent need for evaluating biomarkers that can be used to further identify and treat individuals at greatest risk for mets and diabetes and reduce morbidity and mortality associated with diabetes and cardiovascular diseases.2,3 crp is a protein that is synthesized by liver and then secreted into circulation . it is a critical component of immune system and one of acute phase proteins that increase during systemic inflammation . previous studies have revealed that serum hs - crp levels were strongly related to mets.4,5 on the contrary , other studies have discovered that serum hs - crp levels were associated with mets due to the influence of other variables.6 in the current study , serum hs - crp levels were not only able to identify the presence of mets but also independently associated with mets after adjusting for all components of mets and cdrs except serum hdl - c levels . moreover , we found that serum hdl - c levels were the only component of mets and cdrs that was independently associated with and identified by serum hs - crp levels . thus , serum hdl - c levels were the major determinant of the association between serum hs - crp levels and mets and even the key link between inflammation and mets . it gave us some significant enlightenment on how to further understand the underlying mechanisms of inflammation and mets , which can lead to better prevention and treatment of diabetes and cardiovascular diseases . serum hs - crp levels have been reported to be a risk factor of diabetes and able to identify the development of diabetes.7 but studies from lee et al8 have observed that there was no independent association between serum crp levels and diabetes risk . in the current study , although serum hs - crp levels had the ability to identify diabetes , serum hs - crp levels had insignificant association with the risk of diabetes in fully adjusted analysis . additionally , some studies have found a relationship between serum hs - crp levels and ir,18 while other studies have revealed that serum hs - crp levels were not closely related to ir.19 the current study indicated that serum hs - crp levels had no chance to detect ir and were not related to ir after controlling for all components of mets and cdrs.20,27 conflicting data have existed on the associations of biomarkers , including serum nt - probnp and hcy levels , with ir , mets , and diabetes risk . in some studies , serum nt - probnp and hcy levels have been significantly related to ir , mets , and diabetes risk.912,21 however , there has been no relationship of serum nt - probnp and hcy levels with ir , mets , and diabetes risk in other studies.13,14,22 the current study showed that despite the identification of one or more of ir , mets , and diabetes risk by serum nt - probnp and hcy levels , there was no independent association of serum nt - probnp and hcy levels with ir , mets , and diabetes risk after full adjustment covering all components of mets and cdrs . previous studies have reported a positive association between serum nt - probnp levels and bp.28 in conformity with these data , we found that bp was not only able to be detected by serum nt - probnp levels but also the only component of mets and cdrs independently associated with serum nt - probnp levels after eliminating the influence of all other components of mets and cdrs . the relationships of serum nt - probnp levels with other components of mets and cdrs have been described recently,2931 but they lost independence after full adjustment in the current study . meanwhile , although serum hcy levels were able to identify several components of mets , they were not independently related to them in the current study , which was in accordance with the results from previous studies.32,33 the current study performed deep analyses of the associations of biomarkers , including serum hs - crp , nt - probnp , and hcy levels , with ir , mets , and diabetes risk in non - diabetic chinese community - dwelling middle - aged and elderly population , which possess significant clinical implications . in the current study , serum hdl - c levels were the major determinant of the association between serum hs - crp levels and mets and the key link between inflammation and mets . there was no other association of these biomarkers with ir , mets , and diabetes risk after full adjustment .

objectivethe current study was designed to perform deep analyses of the associations of biomarkers , including high - sensitivity c - reactive protein ( hs - crp ) , n - terminal prohormone of brain natriuretic peptide ( nt - probnp ) , and homocysteine ( hcy ) , with insulin resistance ( ir ) , metabolic syndrome ( mets ) , and diabetes risk and evaluate the abilities of biomarkers to identify ir , mets , and diabetes risk in chinese community - dwelling middle - aged and elderly residents.participants and methodsa total of 396 participants older than 45 years underwent physical examinations and laboratory analyses following standardized protocol.resultsserum hs - crp concentrations were able to identify mets , chinese diabetes risk score ( cdrs ) 4 , high - density lipoprotein - cholesterol ( hdl - c ) < 0.9/1.0 mmol / l , and hdl - c < 1.0/1.3 mmol / l ( p<0.05 for all ) . serum nt - probnp concentrations were able to identify homeostasis model assessment of ir > 1.5 , cdrs 4 , overweight , and blood pressure ( bp ) 140/90 mmhg ( p<0.05 for all ) . serum hcy concentrations were able to identify cdrs 4 , general obesity , overweight , and bp 140/90 mmhg ( p<0.05 for all ) . serum hs - crp concentrations were independently associated with mets as well as hdl - c < 1.0/1.3 mmol / l and hdl - c < 0.9/1.0 mmol / l ( p<0.05 for all ) . serum nt - probnp concentrations were independently associated with bp 140/90 mmhg ( p<0.05 ) . serum hcy concentrations were independently associated with cdrs 4 ( p<0.05).conclusionserum hdl - c levels were the major determinant of the associations between serum hs - crp levels and mets and the key link between inflammation and mets . there was no other association of these biomarkers with ir , mets , and diabetes risk after full adjustment .

Introduction Participants and methods Study participants and information collection Laboratory analyses Variable definitions Statistical analyses Results Baseline characteristics Simple comparisons Correlation analyses Receiver operating characteristic curve analyses Logistic regression analyses Discussion Conclusion

metabolic syndrome ( mets ) is a constellation of interrelated metabolic risk factors that appear to directly promote the development of diabetes and cardiovascular diseases.1 identification of biomarkers associated with mets and diabetes is desirable to establish screening programs , preventive interventions , and therapeutic strategies most appropriately directed at mets and diabetes , which can reduce the increased prevalence of mets and diabetes worldwide and attenuate the morbidity and mortality of diabetes and cardiovascular disease.2,3 although several studies have analyzed the associations of mets and diabetes risk with biomarkers , including high - sensitivity c - reactive protein ( hs - crp ) , n - terminal prohormone of brain natriuretic peptide ( nt - probnp ) , and homocysteine ( hcy ) , there have been too much controversial results.414 to our knowledge , a limited amount of information is available regarding these associations in non - caucasian populations , particularly among chinese community - dwelling residents.15,16 recent studies have more and more focused on the significant role of insulin resistance ( ir ) in linking cardiovascular risk factors.17 studies about the associations of ir with biomarkers representing diverse biological roles and pathological phenomena can help us to understand the mechanisms underlying the development of mets and diabetes . notably , few studies , especially chinese community - based studies , concerning the associations of ir with biomarkers , including hs - crp , nt - probnp , and hcy , have been conducted , and it is a long way to go for the definite answer.10,1822 therefore , the aim of the current study was to perform deep analyses of the associations of biomarkers , including hs - crp , nt - probnp , and hcy , with ir , mets , and diabetes risk and evaluate the abilities of biomarkers to identify ir , mets , and diabetes risk in chinese community - dwelling middle - aged and elderly residents . fasting blood glucose ( fbg ) , triglyceride ( tg ) , low - density lipoprotein - cholesterol ( ldl - c ) , and high - density lipoprotein - cholesterol ( hdl - c ) were checked by qualified technicians blinded to clinical data using enzymatic assays ( hoffman - la roche ltd . ir was assessed by the homeostasis model assessment of ir ( homa - ir ) using the following formula : homa - ir = fins ( miu / l ) fbg ( mmol / l)/22.5.23 according to the worldwide consensus on the definition of mets recommended by the international diabetes federation,24 for a person to be defined as having the mets , he or she must have central obesity plus any two of four additional factors : tg 1.7 mmol / l , hdl - c < 1.0 mmol / l in males and < 1.3 mmol / l in females ( or specific treatment for these lipid abnormalities ) , sbp 130 mmhg or dbp 85 mmhg ( or treatment of previously diagnosed hypertension ) , and fbg 5.6 general obesity , overweight , and central obesity were defined as bmi 28 kg / m , bmi 24 kg / m , and wc 85 cm for men and 80 cm for women based on the guidelines for preservation and control of overweight and obesity in chinese adults , respectively.25 more recently , our hospital set up the first risk score of the people s republic of china identifying individuals who were likely to develop diabetes in the near future , named the chinese diabetes risk score ( cdrs ) , which consisted of age ( < 66 : coded 0 ; 6675 : coded 1 ; 76 : coded 2 ) , hypertension ( coded 1 ) , history of high blood glucose ( coded 3 ) , bmi ( < 24 : coded 0 ; 24 : coded 1 ) , high fbg ( coded 2 ) , high tg ( coded 1 ) , and low hdl - c ( coded 2 ) . fasting blood glucose ( fbg ) , triglyceride ( tg ) , low - density lipoprotein - cholesterol ( ldl - c ) , and high - density lipoprotein - cholesterol ( hdl - c ) were checked by qualified technicians blinded to clinical data using enzymatic assays ( hoffman - la roche ltd . ir was assessed by the homeostasis model assessment of ir ( homa - ir ) using the following formula : homa - ir = fins ( miu / l ) fbg ( mmol / l)/22.5.23 according to the worldwide consensus on the definition of mets recommended by the international diabetes federation,24 for a person to be defined as having the mets , he or she must have central obesity plus any two of four additional factors : tg 1.7 mmol / l in females ( or specific treatment for these lipid abnormalities ) , sbp 130 mmhg or dbp 85 mmhg ( or treatment of previously diagnosed hypertension ) , and fbg 5.6 mmol / l ( or previously diagnosed diabetes ) . general obesity , overweight , and central obesity were defined as bmi 28 kg / m , bmi 24 kg / m , and wc 85 cm for men and 80 cm for women based on the guidelines for preservation and control of overweight and obesity in chinese adults , respectively.25 more recently , our hospital set up the first risk score of the people s republic of china identifying individuals who were likely to develop diabetes in the near future , named the chinese diabetes risk score ( cdrs ) , which consisted of age ( < 66 : coded 0 ; 6675 : coded 1 ; 76 : coded 2 ) , hypertension ( coded 1 ) , history of high blood glucose ( coded 3 ) , bmi ( < 24 : coded 0 ; 24 : coded 1 ) , high fbg ( coded 2 ) , high tg ( coded 1 ) , and low hdl - c ( coded 2 ) . for the total population , the median age was 66 years ( 5871 years ) , and 53% were male , with median hs - crp , nt - probnp , and hcy concentrations of 0.29 mg / dl ( 0.170.38 mg / dl ) , 41.42 pg / ml ( 17.3480.72 pg / ml ) , and 17.20 mol / l ( 14.1021.58 mol / l ) , respectively . the individuals with homa - ir > 1.5 and mets were more likely to have higher levels of bmi , wc , dbp , fins , pbg , tg , and hypertension and lower hdl - c levels than others ( p<0.05 for all ) . the levels of sbp in the individuals with mets and the levels of fbg in the individuals with homa - ir > 1.5 were higher than those of others ( p<0.05 for all ) . in spite of no difference in sex ratio between the individuals with and without homa - ir > 1.5 ( p>0.05 ) , there were more males with mets ( p<0.05 ) . the levels of ldl - c were similar between the individuals with and without mets and homa - ir > 1.5 ( p>0.05 for all ) . serum hs - crp concentrations in the individuals with mets were higher than those of others ( p<0.05 ) , but they were similar between the individuals with and without homa - ir > 1.5 ( p>0.05 ) . serum nt - probnp concentrations in the individuals with homa - ir > 1.5 were lower than those of others ( p<0.05 ) , but they were similar between the individuals with and without mets ( p>0.05 ) . there was no difference in serum hcy concentrations between the individuals with and without homa - ir > 1.5 and mets ( p>0.05 for all ) . the individuals with cdrs 4 and general obesity tended to have higher levels of bmi , wc , sbp , fins , pbg , tg , and hypertension and lower levels of hdl - c than others ( p<0.05 for all ) . the levels of dbp in the individuals with general obesity were higher than those of others ( p<0.05 ) , but they were similar between the individuals with and without cdrs 4 ( p>0.05 ) . the levels of serum fbg and history of high blood glucose in the individuals with cdrs 4 were higher and more than those of others ( p<0.05 for all ) , but they were similar between the individuals with and without general obesity ( p>0.05 ) . serum hs - crp concentrations in the individuals with cdrs 4 were higher than those of others ( p<0.05 ) . serum nt - probnp concentrations in the individuals with cdrs 4 were higher than those of others ( p<0.05 ) , but they were similar between the individuals with and without general obesity ( p>0.05 ) . individuals who did not have cdrs 4 and general obesity were likely to have higher serum hcy concentrations compared with those who had cdrs 4 and general obesity ( p<0.05 for all ) . serum hs - crp concentrations were related to the levels of bmi , wc , fbg , hdl - c , mets component numbers , and cdrs ( p<0.05 for all ) . serum nt - probnp concentrations were correlated with the levels of sbp , dbp , tg , cdrs , and homa - ir ( p<0.05 for all ) . serum hcy concentrations were linked to the levels of bmi , wc , sbp , fbg , hdl - c , and cdrs ( p<0.05 for all ) . serum hs - crp concentrations were able to identify mets , cdrs 4 , hdl - c < 0.9/1.0 mmol / l , and hdl - c < 1.0/1.3 mmol / l ( p<0.05 for all ) . serum nt - probnp concentrations were able to identify homa - ir > 1.5 , cdrs 4 , overweight , and bp 140/90 mmhg ( p<0.05 for all ) . serum hcy concentrations were able to identify cdrs 4 , general obesity , overweight , and bp 140/90 mmhg ( p<0.05 for all ) . serum hs - crp concentrations were independently associated with mets as well as hdl - c < 1.0/1.3 mmol / l and hdl - c < 0.9/1.0 mmol / l after adjustment shown in table 5 ( p<0.05 for all ) . serum nt - probnp concentrations were independently associated with bp 140/90 mmhg after adjustment shown in table 5 ( p<0.05 ) . serum hcy concentrations were independently associated with cdrs 4 after adjustment shown in table 5 ( p<0.05 ) . for the total population , the median age was 66 years ( 5871 years ) , and 53% were male , with median hs - crp , nt - probnp , and hcy concentrations of 0.29 mg / dl ( 0.170.38 mg / dl ) , 41.42 pg / ml ( 17.3480.72 pg / ml ) , and 17.20 mol / l ( 14.1021.58 mol / l ) , respectively . the individuals with homa - ir > 1.5 and mets were more likely to have higher levels of bmi , wc , dbp , fins , pbg , tg , and hypertension and lower hdl - c levels than others ( p<0.05 for all ) . the levels of sbp in the individuals with mets and the levels of fbg in the individuals with homa - ir > 1.5 were higher than those of others ( p<0.05 for all ) . in spite of no difference in sex ratio between the individuals with and without homa - ir > 1.5 ( p>0.05 ) , there were more males with mets ( p<0.05 ) . the levels of ldl - c were similar between the individuals with and without mets and homa - ir > 1.5 ( p>0.05 for all ) . serum hs - crp concentrations in the individuals with mets were higher than those of others ( p<0.05 ) , but they were similar between the individuals with and without homa - ir > 1.5 ( p>0.05 ) . serum nt - probnp concentrations in the individuals with homa - ir > 1.5 were lower than those of others ( p<0.05 ) , but they were similar between the individuals with and without mets ( p>0.05 ) . there was no difference in serum hcy concentrations between the individuals with and without homa - ir > 1.5 and mets ( p>0.05 for all ) . the individuals with cdrs 4 and general obesity tended to have higher levels of bmi , wc , sbp , fins , pbg , tg , and hypertension and lower levels of hdl - c than others ( p<0.05 for all ) . the levels of serum fbg and history of high blood glucose in the individuals with cdrs 4 were higher and more than those of others ( p<0.05 for all ) , but they were similar between the individuals with and without general obesity ( p>0.05 ) . serum hs - crp concentrations in the individuals with cdrs 4 were higher than those of others ( p<0.05 ) . serum nt - probnp concentrations in the individuals with cdrs 4 were higher than those of others ( p<0.05 ) , but they were similar between the individuals with and without general obesity ( p>0.05 ) . individuals who did not have cdrs 4 and general obesity were likely to have higher serum hcy concentrations compared with those who had cdrs 4 and general obesity ( p<0.05 for all ) . serum hs - crp concentrations were related to the levels of bmi , wc , fbg , hdl - c , mets component numbers , and cdrs ( p<0.05 for all ) . serum nt - probnp concentrations were correlated with the levels of sbp , dbp , tg , cdrs , and homa - ir ( p<0.05 for all ) . serum hcy concentrations were linked to the levels of bmi , wc , sbp , fbg , hdl - c , and cdrs ( p<0.05 for all ) . serum hs - crp concentrations were able to identify mets , cdrs 4 , hdl - c < 0.9/1.0 mmol / l , and hdl - c < 1.0/1.3 mmol / l ( p<0.05 for all ) . serum nt - probnp concentrations were able to identify homa - ir > 1.5 , cdrs 4 , overweight , and bp 140/90 mmhg ( p<0.05 for all ) . serum hcy concentrations were able to identify cdrs 4 , general obesity , overweight , and bp 140/90 mmhg ( p<0.05 for all ) . serum hs - crp concentrations were independently associated with mets as well as hdl - c < 1.0/1.3 mmol / l and hdl - c < 0.9/1.0 mmol / l after adjustment shown in table 5 ( p<0.05 for all ) . serum nt - probnp concentrations were independently associated with bp 140/90 mmhg after adjustment shown in table 5 ( p<0.05 ) . serum hcy concentrations were independently associated with cdrs 4 after adjustment shown in table 5 ( p<0.05 ) . previous studies have revealed that serum hs - crp levels were strongly related to mets.4,5 on the contrary , other studies have discovered that serum hs - crp levels were associated with mets due to the influence of other variables.6 in the current study , serum hs - crp levels were not only able to identify the presence of mets but also independently associated with mets after adjusting for all components of mets and cdrs except serum hdl - c levels . moreover , we found that serum hdl - c levels were the only component of mets and cdrs that was independently associated with and identified by serum hs - crp levels . thus , serum hdl - c levels were the major determinant of the association between serum hs - crp levels and mets and even the key link between inflammation and mets . serum hs - crp levels have been reported to be a risk factor of diabetes and able to identify the development of diabetes.7 but studies from lee et al8 have observed that there was no independent association between serum crp levels and diabetes risk . additionally , some studies have found a relationship between serum hs - crp levels and ir,18 while other studies have revealed that serum hs - crp levels were not closely related to ir.19 the current study indicated that serum hs - crp levels had no chance to detect ir and were not related to ir after controlling for all components of mets and cdrs.20,27 conflicting data have existed on the associations of biomarkers , including serum nt - probnp and hcy levels , with ir , mets , and diabetes risk . in some studies , serum nt - probnp and hcy levels have been significantly related to ir , mets , and diabetes risk.912,21 however , there has been no relationship of serum nt - probnp and hcy levels with ir , mets , and diabetes risk in other studies.13,14,22 the current study showed that despite the identification of one or more of ir , mets , and diabetes risk by serum nt - probnp and hcy levels , there was no independent association of serum nt - probnp and hcy levels with ir , mets , and diabetes risk after full adjustment covering all components of mets and cdrs . previous studies have reported a positive association between serum nt - probnp levels and bp.28 in conformity with these data , we found that bp was not only able to be detected by serum nt - probnp levels but also the only component of mets and cdrs independently associated with serum nt - probnp levels after eliminating the influence of all other components of mets and cdrs . meanwhile , although serum hcy levels were able to identify several components of mets , they were not independently related to them in the current study , which was in accordance with the results from previous studies.32,33 the current study performed deep analyses of the associations of biomarkers , including serum hs - crp , nt - probnp , and hcy levels , with ir , mets , and diabetes risk in non - diabetic chinese community - dwelling middle - aged and elderly population , which possess significant clinical implications . in the current study , serum hdl - c levels were the major determinant of the association between serum hs - crp levels and mets and the key link between inflammation and mets . there was no other association of these biomarkers with ir , mets , and diabetes risk after full adjustment .

glutamate ( glu ) is one of the key neurotransmitters in the mammalian central nervous system responsible for fast excitatory synaptic signaling in the brain . high levels of glu resulting in activation of respective receptors lead to neuronal cell death called excitotoxicity that is involved in pathophysiology of various neurodegenerative diseases , epilepsy , hypoxia or stroke ( peng et al . 2011 ) . to exert its function their structure and function were recently reviewed in detail ( lau and tymianski 2010 ) , thereby we only want to stress that glutamate receptors constitute two main groups : metabotropic glutamate receptors ( mglurs ) , which belong to the superfamily of g - protein coupled receptors , and ionotropic receptors ( iglurs ) , which form ion channels . based on the sequence homology and intracellular signal transduction mechanisms , mglurs have been classified into three subgroups : mglur1 and mglur5 , coupled to the phospholipase c , belong to group i ; group ii consists of mglur2 and mglur3 , whereas group iii contains mglur4 , mglur6 , mglur7 and mglur8 , which are all negatively coupled to adenylate cyclase . metabotropic glurs function as dimers , with two glutamate molecules being required for full receptor activation . likewise , the iglurs are divided into three groups based on structural similarities and named according to the type of synthetic agonist that activates them : n - methyl - d - aspartate ( nmda ) receptors ( nmdars ) , amino-3-hydroxy-5-methyl-4-isoxazolepropionate ( ampa ) receptors ( ampars ) , and 2-carboxy-3-carboxymethyl-4-isopropenylpyrrolidine ( kainate , ka ) receptors ( kars ) . nmdars are assembled from seven subunits : nr1 , nr2a / b / c / d , nr3a / b , each one being a product of a separate gene . nmdars consist of two obligatory nr1 subunits and two of four types of regulatory subunits , nr2a , b , c and d , which assemble as a dimer of dimers . the resulting complex might eventually join with either nr3a or nr3b , which , in such a case , replaces one of the nr2 subunits . moreover , glycine is a requisite natural agonist for nmdars ( kew and kemp 2005 ; mayer 2005 ) . for the nmdars , the nr1 subunit is necessary for calcium conductivity of the channel , while nr2 and nr3 subunits determine electrophysiological and pharmacological properties of the receptor ( traynelis et al . ampa receptors are composed of four subunits : glur1/2/3/4 , that show 70 % of homology while being products of separate genes . the glur2 subunit plays a critical role in the determination of the ca permeability of the ampars . interestingly , the genomic dna of the glur2 subunit contains a code for a glutamine ( q ) residue at amino acid 607 , but during the process of nuclear rna - editing glutamine is replaced by arginine ( r ) in the vast majority of neuronal cells , which results in a very low calcium permeability for receptors containing the glur2 subunit ( kew and kemp 2005 ; lau and tymianski 2010 ; palmer et al . 2005 ) . kainate receptors comprise two types of subunits : glur5/6/7 and ka1 and ka2 , which form homo or heterotetramers . ka1/2 homotetramers , even though they combine with the agonist , remain inactive . functional receptors that consist of ka1/2 subunits interestingly , glur2 , glur5 and glur6 undergo glutamine / arginine editing ( contractor et al . therefore , an opportunity of receptors formation with distinct physicochemical and pharmacological properties exists due to the diversity of ionotropic receptor subunits . glutamate has been proven to regulate proliferation , migration , and survival of neuronal progenitor cells and immature neurons during brain development ( ikonomidou et al . the ability of uncontrollable propagation and migration characterizes neoplastic cells as well ; therefore , glutamate was suggested as a potential growth factor in tumor development . support for this hypothesis was provided by studies that showed the generation of neurotoxic quantities of glutamate by glial tumor cells ex vivo ( in surgical specimens ) ( takano et al . 2001 ) as well as in vitro in glioma cell lines , where extracellular glutamate concentrations up to 500 mol / l were demonstrated ( ye and sontheimer 1999 ) . moreover , genetic modifications of c6 rat glioma cells facilitated creation of cell lines that release excessive amounts of glu along with non - secreting lines . subsequent xenograft studies proved that experimental tumors created from cells that release high amounts of glu grow more aggressively than those from non - modified cells ( which release moderate amounts of glu ) or from non - secreting cells ; this phenomenon was closely related to animals overall survival times ( takano et al . 2001 ) . recent studies showed also that excessive glutamate concentrations ( in the range of 100 m ) might be found in the extracellular space at the tumor margin in glioblastoma - bearing patients ( de groot and sontheimer 2011 ) resulting in neuronal cell death , which in turn facilitates tumor growth ( rothstein and brem 2001 ) and possibly relates to epileptic seizures in glioma patients ( oberndorfer et al . interestingly , the latter observation has recently been supported by a study that proved the relationship between increased levels of an excitatory neuropeptide , dynorphin 117 that comprises glutamate , and cell death or surgery - related tissue injury in vivo in epilepsy patients ( broderick et al . such levels of glu might arise from glutamate uptake / release systems that are aberrantly expressed and/or activated in glioma cells . it has also been proven that glutamate release in gliomas is , at least in part , mediated by a na - independent cystine glutamate exchanger xc system , expressed in glioma cell lines and patient - derived glioma cells ( lyons et al . pharmacologic inhibition of this system slows tumor growth and extends survival of tumor - bearing animals ( chung et al . importantly , xc system seems to be up - regulated in glioma cells acting in concert with the lack of functional na - dependent transport systems of the excitatory amino acid transporters ( eaats ) , which are responsible for glutamate uptake and are amply expressed in nonmalignant glial cells ( lyons et al . in glioma cells , eaat1/2 transporters ( especially eaat2 ) seem to inversely correlate with the degree of malignancy with almost no expression in gbms ( de groot and sontheimer 2011 ) . accordingly , glioma cells not only release glu to enhance their highly malignant behavior but also are incapable of its reuptake . more recently , it has also been demonstrated that cells from non - cns cancers may secrete glu . mda - mb-231 ( human breast ) , b16f1 ( mouse melanoma ) and matlylu ( rat prostate ) cancer cell lines release significant quantities of glutamate into their extracellular environment ( seidlitz et al . a very recent study reported that prostate cancer - bearing patients have serum glutamate levels directly correlating with gleason score ( 6 vs. 8 ) and primary prostate cancer aggressiveness ( koochekpour 2013 ) . glutamate tissue levels in fresh frozen human normal pancreatic tissue , chronic pancreatitis ( cp ) and pancreatic ductal adenocarcinoma ( pdac ) tissues demonstrated a striking increase of glutamate in cp and pdac samples , suggesting that glu might serve as a molecular switch that decreases the threshold of k - ras - induced oncogenic signaling and increases the chance of malignant transformation of pancreatic cancer precursor lesions ( herner et al . glutamate and synthetic glur agonists stimulated proliferation of a549 lung cancer cells in vitro ( rzeski et al . 2001 ; stepulak et al . 2005 , 2007 ) and invasion of pdac ( herner et al . 2011 ) . thereby , released glu can act as a growth factor and a signal mediator in non - neuronal tumor tissues , in both , autocrine and paracrine fashions . a long - standing paradigm suggested that glutamate signaling is limited to the central nervous system . recently , however this opinion has been changed owing to the studies that proved glutamate receptors existence in peripheral organs and neoplastic cells ( hinoi et al . 2004 first studies in the mid - nineties of the last century provided evidence that labeled nmda agonist ( [ 3h]mk801 ) couples with cns tumor cells ; moreover , in vitro glutamate application resulted in ca influx into those cells ( ohkuma et al . follow - up studies proved the existence of ionotropic glur1 and glur4 ( korczak et al . 1995 ) , glur2/3/4/6/7 , ka1 ( yoshioka et al . 1996 ) ] , nr1 and nr2c ( casado et al . 1996 ) , or glur1/2/3/4 in surgical samples of glioblastomas ( ishiuchi et al . 2002 ) along with various other combinations of subunits in cell lines and cns tumor samples ( aronica et al . 2001 ; de groot et al . it has been also demonstrated that glutamate receptor subunits are expressed in a variety of cancer cell lines and tumors , originating outside of cns such as colorectal ( chang et al . 2011 ) , hepatocellular ( li et al . 2012 ) or gastric cancer ( liu et al . 2007 ) , breast cancer ( north et al . 2010b ) , ovarian cancer ( choi et al . 2012 ) , lung cancers ( north et al . 2010a ) , thyroid cancer ( stepulak et al . 2009 ) , oral squamous cell carcinoma ( scc ) ( park et al . 2007 ) , larynx cancer ( stepulak et al . 2011 ) , prostate cancer ( abdul and hoosein 2005 ) , melanoma ( marin and chen 2004 ; pollock et al . 2003 ) or osteosarcoma ( kalariti et al . 2004 ) , as well as blood neoplasms such as leukemia and lymphoma ( ganor et al . 2009 ) . still , the majority of the existing studies describe single receptor or its subunit expression in selected types of cancers while a more detailed analysis of glutamate receptor subunit expression in cancer cell lines or solid tumors remains scarce . hence , we decided to analyze the presence of iglurs and mglurs receptor subunits in several cancer cell lines of neuronal and non - neuronal origin . these studies revealed differential patterns of receptor subunit expression , with some specificity for particular neoplasms ( stepulak et al . interestingly , the nr1 subunit of the nmda receptor , although present in other cell lines derived from cns neoplasms , was lacking in glioma cell lines ( stepulak et al . this observation is supported by previous studies , which showed that four analyzed glioma cell lines and patient - derived gliomas were devoid of nr1 subunit ( lyons et al . 2007 ) or that its expression was very rare in pediatric gbms ( brocke et al . 2010 ) . in contrast to nmdars , ampa receptors are abundantly expressed in glioma cells , where they play an important role in glutamate - mediated proliferative signals thus enhancing its malignant phenotype ( de groot and sontheimer 2011 ; sontheimer 2008 ) . as a matter of fact , all ampa receptor subunits were found in cns - derived tumors ( aronica et al . furthermore , the presence or lack of glur2 subunit seems to be crucial for glioma cells invasion potential . it was observed that the majority of invasive gliomas either lack glur2 expression ( lyons et al . 2007 ) or glur2 is expressed at significantly lower levels , as demonstrated in highly malignant pediatric glioblastomas , ependymomas or medulloblastomas ( all who iii or iv ) in contrast to low - grade astrocytomas ( who i or ii ) ( brocke et al . 2010 ) . additionally , rnai experiments in a low - grade glioma cell line demonstrated that down - regulation of glur2 expression caused a significant increase of cell proliferation ( beretta et al . 2009 ) . on the other hand , over - expression of edited glur2 subunit by adenovirus - mediated transfer inhibited migration of glioma cells both in vitro and in vivo ( ishiuchi et al . 2002 ) , proving that ca - permeable ampa receptors are crucial for glioma invasion . importantly , the presence of unedited ( q ) or edited ( r ) glur2 ( q / r site ) subunit is critical for calcium permeability of ampa receptor . developmentally controlled replacement of the arginine with glutamine at this critical site ( q / r site ) during an rna - editing process renders ampa receptors permeable ( unedited glur2 ) or non - permeable to ca ( hollmann and heinemann 1994 ; seeburg 1993 ) . edited form of glur2 exists exclusively in the adult brain while unedited forms are present in the fetal brain ( burnashev et al . 1992 ) and in some cancer cells or tumors ( brocke et al . 2010 ; maas et al . 2001 ; stepulak et al . when analyzing glur2 q / r - editing status in different cancer cell lines , it has been shown that some of them exclusively express the unedited form of glur2 ( sk - na - s , neuroblastoma ) or both , the edited and the unedited glur2 forms ( moggccm , astrocytoma ) . interestingly , unedited glur2 rna was found also in sk - lu-1 lung cancer cells ( stepulak et al . 2009 ) , forming highly malignant tumors with a tendency to set distant metastases . this shows that cancer cells could express ampa receptor forms characteristic for ca - permeable fetal cells , which could in turn contribute to cancer cells invasion potential , as it has been demonstrated for glioma cells ( ishiuchi et al . 2002 ) . of particular interest is the fact that glioblastoma - tumor initiating cells express high levels of functional , calcium - permeable ampa receptors containing glur1 and glur4 subunits , when compared with the differentiated tumor cultures consisting of non - stem cells derived from the same tumor tissues ( oh et al . these finding suggests that functional ampa receptors can be formed in specific areas of the tumor . as mentioned above , in contrast to ampars , moderate to high expression of nr1 subunit of nmdar has been demonstrated in prostate cancer samples , whereas its expression in normal prostate tissue and benign prostate hyperplasia was very low or absent . similar expression pattern was found in normal colon or cancer specimens ( abdul and hoosein 2005 ) . nr1 subunit immunohistochemical reactivity was observed in the majority of small - cell lung ( north et al . 2010a ) or breast cancer samples , where nr2b subunit was also detected ( north et al . 2010b ) . different combinations or single subunits of nmdars were demonstrated in cell lines derived from colon cancer ( stepulak et al . 2004 ) , laryngeal carcinoma ( stepulak et al . 2011 ) , lung cancers ( north et al . 2005 , 2007 ) , prostate cancer ( abdul and hoosein 2005 ) , thyroid cancer ( stepulak et al . watanabe et al . 2008 ) , esophageal ( kim et al . 2006 ) , and hepatocellular carcinomas ( yamaguchi et al . 2013 ) . interestingly , the expression of kainate receptors in cancer cells has not been extensively studied . the presence of glur57 subunits in human glioneuronal tumors ( aronica et al . 2001 ) , glur57 and ka1 in medulloblastomas , and additionally ka2 subunit in neuroblastoma cell lines ( yoshioka et al . 1996 ) or genome - wide association studies have recently identified glur5 expression in hepatocellular carcinoma samples ( li et al . 2012 ) , whereas glur6 subunits were detected in gastric cancer tissue and gastric cancer cell lines ( wu et al . likewise , our earlier study demonstrated the presence of glur5 in u343 glioma cells , whereas glur6 , glur7 , ka1 and ka2 subunits were found in all 12 analyzed cancer cell lines , which suggests a role of kainate receptors in metabolism and proliferation of cancer cells ( stepulak et al . however , when analyzing relative glutamate receptor subunit amounts in cancer cells in comparison to their expression in normal human brain on mrna level , significant differences were observed . as measured by means of real - time pcr technique , the majority of cancer cell lines expressed either nmda or ampa / kainate receptor subunits at much lower levels than the normal human brain ( hb ) . nmdar nr2b subunit expression level in cancer cell lines was very low compared to hb , with the exception of the human colon adenocarcinoma cell line ls180 , which showed an expression level approximating 50 % of the estimated level of nr2b subunit expression in hb . similarly , a strong expression of glur4 ampa receptor subunit was detected in two cell lines : te671 ( rhabdomyosarcoma / medulloblastoma ) and rpmi ( plasmocytoma ) , and of glur6 kainate receptor subunit in sk - na - s ( neuroblastoma ) and moggccm ( astrocytoma ) cell lines at levels comparable to those in the hb . similar levels of expression of ka2 were found also in human te671 and the hb ( stepulak et al . expression of nr2d , nr3a , ka1 , glur4 , mglur1 , mglur4 , mglur5 and mglur6 was higher in the high - grade tumors compared to human brain . in low - grade astrocytomas , expression of these glutamate receptor subunits was comparable or lower than in hb ( brocke et al . aforementioned studies provide compelling evidence that glutamate receptors are expressed at higher level in the tumors and neoplastic cell lines of brain origin than in those derived from peripheral cancers . the observation that glurs subunits , which are poorly represented in the adult brain , are expressed in cancer cells ( stepulak et al . 2009 ) is of interest as well . on the other hand , it has been shown that the expression of nmda receptor subunits in the brain varies during development ; especially nr2d subunits are present at high levels prenatally in rapidly dividing cns cells with subsequent decrease postnatally . in adults , nr2d presence is limited to small numbers of cells in selected regions of the brain ( cull - candy et al . thus , expression of nr2d subunits in all the virtually analyzed cancer cell lines ( stepulak et al . 2009 ) suggests that the re - expression of nr2d in cancer cells may correlate with their proliferative potential . interestingly , silencing of nr2d subunit did not influence cancer cells phenotypes . the same was observed for ka2 subunit , also present in all cancer cell lines analyzed ( luksch et al . it has been demonstrated with patch - clamp electrophysiological recordings that glutamate might evoke whole - cell currents in human hypothalamic hamartoma slices immediately after surgical resection ( wu et al . similarly , glutamate and nmdars agonists in the presence of glycine increased membrane - depolarization currents in neuroblastoma cells ( north et al . 1997 ) , and glioblastoma , astrocytoma , and oligodendroglioma cells responded to kainate by depolarization of tumor cells in culture or tissue slices ( labrakakis et al . it was also demonstrated that iglurs are active and functional in cancer cells derived from peripheral tumors . an analysis of whole - cell patch - clamp recordings of membrane currents proved that in a549 lung cancer and te671 ( rhabdomyosarcoma / medulloblastoma ) cell lines application of glutamate ( 10 mm ) resulted in inward currents that were almost completely blocked by application of nmda and ampa receptor antagonists . interestingly , the evoked currents were small , which is consistent with the low expression of these receptors in examined cancer cells ( stepulak et al . importantly , experimental data have also implicated important role of mglurs in malignant tumor metabolism and progression . likewise iglurs , metabotropic receptors were first detected in tumors of cns origin such as gliomas ( albasanz et al . 1997 ) , gangliogliomas and dysembryoplastic neuroepithelial tumors ( aronica et al . 2001 ) . interestingly , mglur3 receptors were present in almost all of the glioma tumor samples ( nicoletti et al . 2007 ) , including glioma initiating cells ( ciceroni et al . 2008 ) and glioma cell lines ( nicoletti et al . 2007 ) with exception of the u343 cell line ( stepulak et al . 2009 ) . in contrast , mglur1 and mglur5 were highly represented in the neuronal components of brain tumors ( aronica et al . 2001 ) . in pediatric cns tumors , the metabotropic glutamate receptor subtypes mglur1 , mglur2 , mglur4 , mglur5 and mglur6 were expressed at higher levels in the malignant tumors than in low - grade astrocytomas . glioblastoma , ependymoma and low - grade astrocytoma all showed low expression levels of mglur8 , whereas expression of mglur8 was firmly up - regulated in medullo - blastomas ( brocke et al . similarly , most of the analyzed medulloblastoma tissue samples and medulloblastoma cell lines displayed the presence of mglur4 receptors , which inversely correlated with tumor growth ( iacovelli et al . parallel to cns tumors , mglurs have been shown to be over - expressed in some types of peripheral cancers and neoplasms . high expression of mglur1 was reported in primary and metastatic prostate cancers , in contrast to non - cancerous prostate tissues in immunohistochemical analysis ( koochekpour et al . 2012 ) . moreover , mglur1 expression displayed a cell type - dependent pattern , being higher in androgen - independent and metastatic cell lines rather than in androgen - sensitive or primary prostate cancer cell lines ( koochekpour et al . mglur1/2/3/4/5 expression was demonstrated in both androgen - dependent pc-3 and androgen - independent lncap prostate cancer cell lines , whereas mglur6/8 were present in lncap cells only ( pissimissis et al . mglur4 was reported to be more specifically expressed in colorectal cancers than in normal tissues ( chang et al . 2005 ) ; different mglur combinations were also demonstrated in colon cancer derived cell lines , including ls180 cell line , where all mglurs types were detected ( stepulak et al . given the fact that mglur4 mediates 5-fluorouracil resistance in human colon cancer cells , which is a major obstacle in chemotherapy of this cancer type ( yoo et al . 2004 ) , it seems that the presence of mglur4 in some cancers could have functional significance . mglur4 was also present in the 50 % of immunohistologically analyzed laryngeal carcinomas , with lower expression in stomach , gall bladder and pancreas adenocarcinomas ( 1733 % ) . very low presence of mglur4 was demonstrated in thyroid , adrenal glands , and kidney cancers ( 813 % ) , whereas it was not detected in esophageal , endometrial and prostate cancers , as well as in neoplasms derived from salivary glands and testis ( chang et al . single studies presented an expression of different mglurs in several cancer cell lines , including those originating from thyroid and breast cancers and blood malignancies ( stepulak et al . recent studies proved that mglur1 are responsible for cell growth regulation in breast ( speyer et al . 2012 ) and renal cancer cells ( martino et al . 2013 ) , both in vitro and in vivo ; simultaneously their presence was not found in oral cancer tissues and cell lines , in contrast to mglur5 , which was present in the majority of oral cancer specimens and weakly in adjacent dysplastic oral mucosa ( park et al . moreover , melanoma development was connected with the presence of mglur1 receptors ( ohtani et al . 2008 ) , a finding that was supported by observations showing its expression in melanoma cell lines and melanoma samples , but not in normal melanocytes and benign nevi ( pollock et al . , considerable evidence exists for glutamate receptors expression in a variety of tumors and cancer cell lines along with proofs that they are functional , thereby might play an important role in neoplastic transformation and cancer progression . to substantiate the hypothesis that glutamate receptors are functionally important for tumor growth , several studies evaluated their involvement in tumorigenesis and subsequently proved that at least some of the glurs might have oncogenic properties . the impact of metabotropic glutamate receptors on tumor growth was highlighted by a series of experiments which showed causal relationship between mglur1 expression and melanoma development . chen s group was the first to demonstrate that ectopic expression of mglur1 in melanocytes , which normally lack this receptor , was sufficient to induce transformation to malignant melanoma in vivo ( pollock et al . , they were able to show that mglur1 was indispensable for the maintenance of transformation of immortalized melanocytes into tumors with short 35 days latency . their tumorigenic potential in both immunodeficient nude and syngenic mice suggested that the immune system does not influence either tumors formation or distant intestine and muscle metastases formation ( shin et al . direct evidences for mglur1-driven melanoma formation and progression were provided in the same year by ohtani and coworkers who showed that mglur1 conditionally expressed in melanocyte - induced pigmented lesions at the first stage , followed by appearance of melanoma tumors 52 weeks after transgene activation . when the transgene was inactivated , melanoma growth was inhibited as compared to animals bearing tumors with persistent mglur1 expression ( ohtani et al . similar findings were reported for mglur5 transgene activation in mice , resulting in skin hyperpigmentation , seconded by melanoma tumor formation with metastases or primary melanoma lesions detected in lymph nodes , lungs , spleen , liver , uvea and meninges that eventually penetrated into the skull bones ( choi et al . mouse kidney epithelial cells displayed in vivo tumorigenicity , when transfected to ectopically express functional mglur1 , resulting in tumor formation in nude mice . parallel , sirna - mediated inhibition of mglur1 expression in renal cancer cells impaired tumor growth in vivo , thus suggesting that sustained expression of mglur1 is necessary for neoplastic transformation and tumor progression ( martino et al . 2013 ) , whereas targeting mglur1 gene using shrna - expressing lentiviral construct reduced growth of breast cancer cells both in vitro and in vivo ( speyer et al . changes in the levels of expression of ionotropic glutamate receptors or their single subunits in experimental conditions were also demonstrated to be important for cancer cells proliferation and invasion , which suggests the involvement of glurs in cancer progression . as demonstrated in knockout experiments , diminished expression of glur1 ( ampar ) subunit at mrna and protein levels inhibited proliferation of glioma cells in vitro and in vivo ( de groot et al . another study presented that rnai - mediated suppression of glur1 or glur2 did not affect pancreatic cancer cell growth , however significantly decreased invasion in vitro , and inhibited tumor cell settling in a mouse model in vivo ( herner et al . similar , knock - down of the glur3 gene reduced proliferation and migration , as well as enhanced apoptosis of pancreatic cancer cells , while over - expression of this gene was reported to have opposite effect in vitro and in a subcutaneous xenograft model ( ripka et al . in contrast , the silencing of glur2 by sirna transfection increased glioma cell proliferation ( beretta et al . 2009 ) , whereas gene silencing of glur4 modulated the mrna expression of various tumor - suppressor genes , oncogenes and other genes involved in invasion , adhesion and metastatic capabilities , which resulted in significant increase of cell viability of human rhabdomyosarcoma / medulloblastoma ( te671 ) and human multiple myeloma rpmi8226 cells . 2011 ) . similarly to ampar subunits , modulation of expression of genes for nmdar subunits influenced behavior of cancer cells . silencing the nr2a subunit - targeted gene inhibited gastric cancer cells proliferation and cell cycle progression resulting in increased proportion of cells in g1 phase ( watanabe et al . te671 and a549 lung cancer cells demonstrated reduced cell viability after transfection and specific knockdown of nr1 gene ( luksch et al . importantly , the expression of glurs as well as their function in cancer development and progression is influenced by genomic and epigenetic modifications resulting in aberrant posttranscriptional processing . causative for the aberrant cellular function of glutamate receptors in cancer are changes in genomic sequences for mglurs and selected subunits of iglurs . of particular importance is the notion that the presence of rearranged or mutated forms of glutamate receptor subunits might activate cancer cell growth . as mentioned above , insertional mutagenesis of an ectopically expressed mglur1 in mouse results in melanoma development ( pollock et al . 2003 ) . likewise , somatic mutations within mglur3 gene result in an activation of gpcr - mediated mitogen - activated protein kinase 1/2 signaling that results in a transformed cells phenotype , which renders an increased migration of melanoma cells along with a loss of anchorage dependency in growth regulation ( prickett et al moreover , a very recent study highlighted the importance of naturally occurring grm1 somatic mutations for mglur1 surface expression , altered basal and agonist - dependent activity , and disruption of intracellular signaling pathways downstream of the receptor , including inositol phosphate ( ip ) formation , and altered erk1/2 kinases activity ( esseltine et al . 2013 ) . since these mutations were identified in different types of neoplasms including lung adeno- and scc ( kan et al . 2008 ) , it has been hypothesized that they are relevant and contribute to a cancer phenotype ( esseltine et al . 2013 ) . clinical genetic analysis of grm1 showed that single nucleotide polymorphism of the c allele of rs362962 ( coding mglur1 ) contributes to human melanoma susceptibility , especially in a subgroup of patients with a low level of sun exposure and tumors located on the trunk and extremities ( ortiz et al . a similar study performed in women carrying breast cancer revealed a significant correlation between the grm1 cc genotype of rs362962 and the development of hormone receptor - negative breast cancer and association of rs6923492 and rs362962 genotypes with age at diagnosis ( mehta et al . in contrast to metabotropic receptors , somatic mutations of iglur subunits in cancers were scarcely investigated . whole - exome sequencing analysis revealed moderate to high prevalence of somatic mutations in genes coding nr2a , and nr1 subunits of nmda receptors in melanoma ( wei et al . 2011 ) ; however , their possible consequences are not known ( prickett and samuels 2012 ) . nonetheless , in addition to genetic rearrangements , epigenetic alterations seem to play an important role in cancer development and progression . human cancers are characterized by a global impairment of dna methylation . still , hypermethylation of some dna regions , especially at the promoter cpg islands of tumor - suppressor genes , is observed ( virani et al . , considerable interests were demonstrated regarding methylation status of nmdar subunits : nr2a and nr2b promoters . aberrant promoter cpg islands hypermethylation of grin2b ( nr2b coding gene ) during breast cancer progression was reported , showing higher methylation levels and frequencies in ductal carcinoma in situ when compared with preinvasive lesions such as flat epithelial atypia or atypical ductal hyperplasia ; significantly higher methylation frequencies in grade iii than in grade i of invasive ductal carcinoma have also been shown which suggests that cpg island methylation of grin2b might be an early event in breast cancer progression ( park et al . other groups , on the other hand , found that nr2b promoter methylation exhibits tumor - suppressive activity in human esophageal ( kim et al . 2007 ) , as well as in non - small cell lung carcinoma ( tamura et al . 2011 ) . aberrant methylation status of nr2b promoter was present in more than 60 % of human gastric and non - small cell lung carcinoma samples , whereas the grin2b methylation status alterations were found in no more than 5 % of corresponding normal tissues ( liu et al . interestingly , gene methylation of nr2b displayed an inverse correlation with gene ( kim et al . 2011 ) expression , suggesting that nr2b inactivation occurs mainly through epigenetic events ( kim et al . moreover , reintroduction of this gene in esophageal cancer or forced expression in gastric cancer cell lines was accompanied by apoptosis or inhibited cell colony formation , respectively , suggesting tumor - suppressor activity for nr2b ( kim et al . , the same research group demonstrated similar results for nr2a subunit in colorectal cancers ( kim et al . nr2b methylation was significantly associated with a better prognosis regarding survival of patients with scc rather than those with adenocarcinoma ( tamura et al . therefore , rearrangements of glutamate receptors at different genetic and epigenetic levels seem to play a distinct role in their expression and function . despite the fact that the issue at hand requires more extensive studies , one may already hypothesize that different modifications of glurs and their respective genes exist in cancer cells , as demonstrated recently by the discovery of new spliced variants of human grm1 gene in melanoma cells ( diraddo et al . the unequivocally proven role of glurs in oncogenesis turned attention towards their potential clinical significance in different types of tumors . thus , in the clinical settings the expression of glutamate receptors might influence histological differentiation , clinical tumor staging , the presence of metastases and/or overall patient survival rate . one of the key features that distinguish various tumors relates to their histological differentiation and histological signs of malignancy , classified as tumor grading that influences tumor s malignancy potential and its clinical course . it has been reported that glutamate receptors expression is associated with differentiation status in a variety of tumor subtypes . in pancreatic cancer , precursor lesions as well as pancreatic intraepithelial neoplasia ampar glur1 subunit levels were increased in a step - wise manner , suggesting glutamate involvement in a malignant transformation . on the other hand , however , the expression of glur1 , glur2 and glur4 subunits was down - regulated in pdac ( herner et al . 2011 ) . moreover , in other tumor types a direct relationship between the degree of malignancy and glurs expression was found . in brain tumors , glur1 subunit was differentially expressed according to the tumor grading , being elevated in glioblastomas when compared with anaplastic astrocytomas and low - grade astrocytomas , hence correlating with tumor aggressiveness ( de groot et al . opposite association was observed when expression of glur2 was analyzed , which proved to be present in slow - growing gbm - derived tumor stem cells ( gbm tscs ) and low - grade tumor samples but not in fast - growing gliomas or high - grade tumor specimens , indicating that glur2 expression is associated with a low degree of malignancy ( beretta et al . likewise , immunohistochemistry with an anti - glur2 antibody showed a significant difference in the positivity of staining that was uniformly present in virtually 100 % of benign secretory prostatic epithelium when compared with a high - grade prostatic intraepithelial neoplasia and low gleason - patterned carcinomas where scarcely any or very low immunoreactivity of glur2 was observed . this suggests that the presence of glur2 in benign glands , including post - atrophic and adenosis - type ones , readily distinguishes them from prostate cancer ( hechtman et al . an opposite pattern was observed in normal oral mucosa showing very weak expression of nr1 subunit of nmdar , whereas majority of analyzed oral scc specimens expressed this subunit ; albeit the presence of nr1 did not correlate with histological grading of this cancer type ( choi et al . another study that implemented human tissue microarrays revealed that nmdar subunit nr2b expression was associated with the her2-positive breast cancer subtype , in contrast to the luminal subtype , where nr2b expression was not observed ( li and hanahan 2013 ) . in medulloblastoma , mglu4 receptor immunoreactivity highly correlated with the histological features showing decreasing expression levels of this receptor in the following rank order : nodular desmoplastic > classic large - cell anaplastic tumors ( iacovelli et al . 2006 ) . the relationship between glurs expression and aforementioned clinical features of cancers has been scarcely investigated though . only a few reports demonstrated that the tumor size , presence of lymph node metastases and cancer stage were significantly related to high nr1 ( choi et al . 2007 ) expression in oral scc , whereas significantly lower expression levels of mglu4 receptors were correlated with spinal cord metastases , csf spreading , and recurrence of medulloblastoma ( iacovelli et al . . tumor progression along with its dissemination and recurrence are closely related to patients prognosis and overall survival . it has been demonstrated that nr1 subunit expression was associated with unfavorable outcome in patients with oral scc ( choi et al . 2004 ) , whereas mglur5 expression showed positive correlation with an overall survival of patients with the same malignancy ( park et al . similarly , in medulloblastoma the expression of mglur4 was higher in patients , who survived 5-year after surgery ( iacovelli et al . , overexpression of mglur4 is associated with a poor prognosis in colorectal carcinoma ( chang et al . interestingly , glur2 expression showed a significant correlation with longer progression - free and overall survivals and was down - regulated in chemoresistant tumors , proving to be a positive prognostic factor for patients with advanced serous papillary ovarian adenocarcinoma ( choi et al . gene expression analysis of several hundred glioblastoma samples revealed that a loss of gria2 ( gene for glur2 ) expression was 1 of the 38 gene changes that predict a poor prognosis in glioblastoma ( colman et al . whenever high nr2b expression levels correlated with high vglut2 vesicular glutamate transporter expression , the survival of patients bearing glioblastoma was significantly shorter , when compared with the patients groups that expressed low levels of nr2b / vglut2 ( li and hanahan 2013 ) . research into the role of glu signaling in cancer development and progression is still in its infancy ; however , important progress has been made in recent years . considerable evidence exists and indicates that glutamate plays an important role in tumor development , acting as a growth factor and a signal mediator in neural as well as non - neuronal tumors tissues , in both autocrine and paracrine fashions . it has been proven that its actions involve mainly a family of receptors consisting of metabotropic glutamate receptors and ionotropic glutamate receptors whose presence has been proven in a variety of benign and malignant lesions throughout the body . their actions , however , might differ significantly due to the fact that glurs are combined from various subunits , which result in a large diversity of intracellular signaling and distinct pharmacological properties . moreover , receptor subunits such as the nr1 subunit of the nmda receptor are prone to post - transcriptional events ( rna editing and alternative splicing of rna ) , which may result in distinct isoforms ( for example eight in case of nr1 subunit ) due to the presence of independent sites of alternative splicing . on top of that receptor subunits might undergo further posttranslational and epigenetic modifications resulting in even more complex glutamate signal transduction . thus , the clinical significance of glutamate receptor expression may differ among tumor entities and it is difficult to predict how the expression of a particular subunit will influence cancer behavior . still , it is tempting to speculate that glurs and their signaling pathways render promising targets for therapeutic interventions . as a matter of fact , in recent years , there have been multiple attempts to implement iglur and mglur antagonists , in particular in malignant glioma treatment . drugs such as ampar / kr inhibitors , zk 200775 and gyki 52466 , despite having little effect on glioma growth in vitro , have been shown to exert anti - proliferative and anti - excitotoxic effects in rat hippocampal glioma models . similar results were obtained for the nmdar antagonists , norketamine 72 and mk801 ( memantine ) . in fact , memantine has even been employed in a phase ii clinical trial to determine its safety / efficacy in glioma patients ; however , results are not yet available ( http://clinicaltrials.gov # nct01260467 ) . similarly , the effectiveness of talampanel ( an ampar antagonist ) against glioma has been explored in the clinic . this study was completed with results that were not encouraging though ( de groot and sontheimer 2011 ) . interestingly , despite all of the preclinical work and clinical trials ( in progress or completed ) involving iglur antagonists , studies targeting mglur in glioma treatment are lacking . mglurs , being able to form functional homodimers as typical gpcrs , are considered more susceptible to anti - cancer drug design ( willard and koochekpour 2013 ) and may constitute better drug targets than iglurs ( teh and chen 2012 ) . yet , no clinical trials are currently ongoing . future studies in this field are clearly needed to determine the efficacy of mglur antagonists and glu release inhibitors such as riluzole against tumors . it is clear that more research is needed to define the clinical significance of glu glur expression and signaling in various cancers . given existing preliminary studies , it will be extremely interesting to follow the field of glutamatergic signaling in cancer in future years .

glutamate , a nonessential amino acid , is a major bioenergetic substrate for proliferating normal and neoplastic cells on one hand and an excitatory neurotransmitter that is actively involved in biosynthetic , bioenergetic , metabolic , and oncogenic signaling pathways on the other . it exerts its action through a family of receptors consisting of metabotropic glutamate receptors ( mglurs ) and ionotropic glutamate receptors ( iglurs ) , both of which have been implicated previously in a broad spectrum of acute and chronic neurodegenerative diseases . in this review , we discuss existing data on the role of glutamate as a growth factor for neoplastic cells , the expression of glutamate receptors in various types of benign and malignant neoplasms , and the potential roles that glurs play in cancer development and progression along with their clinical significance . we conclude that glutamate - related receptors and their signaling pathways may provide novel therapeutic opportunities for a variety of malignant human diseases .

Introduction Glutamate as a growth factor for cancer cells Expression of glutamate receptors in cancer cells The role of GluRs in cancer Potential clinical significance of GluRs in cancer Concluding remarks

high levels of glu resulting in activation of respective receptors lead to neuronal cell death called excitotoxicity that is involved in pathophysiology of various neurodegenerative diseases , epilepsy , hypoxia or stroke ( peng et al . to exert its function their structure and function were recently reviewed in detail ( lau and tymianski 2010 ) , thereby we only want to stress that glutamate receptors constitute two main groups : metabotropic glutamate receptors ( mglurs ) , which belong to the superfamily of g - protein coupled receptors , and ionotropic receptors ( iglurs ) , which form ion channels . based on the sequence homology and intracellular signal transduction mechanisms , mglurs have been classified into three subgroups : mglur1 and mglur5 , coupled to the phospholipase c , belong to group i ; group ii consists of mglur2 and mglur3 , whereas group iii contains mglur4 , mglur6 , mglur7 and mglur8 , which are all negatively coupled to adenylate cyclase . likewise , the iglurs are divided into three groups based on structural similarities and named according to the type of synthetic agonist that activates them : n - methyl - d - aspartate ( nmda ) receptors ( nmdars ) , amino-3-hydroxy-5-methyl-4-isoxazolepropionate ( ampa ) receptors ( ampars ) , and 2-carboxy-3-carboxymethyl-4-isopropenylpyrrolidine ( kainate , ka ) receptors ( kars ) . nmdars consist of two obligatory nr1 subunits and two of four types of regulatory subunits , nr2a , b , c and d , which assemble as a dimer of dimers . interestingly , the genomic dna of the glur2 subunit contains a code for a glutamine ( q ) residue at amino acid 607 , but during the process of nuclear rna - editing glutamine is replaced by arginine ( r ) in the vast majority of neuronal cells , which results in a very low calcium permeability for receptors containing the glur2 subunit ( kew and kemp 2005 ; lau and tymianski 2010 ; palmer et al . the ability of uncontrollable propagation and migration characterizes neoplastic cells as well ; therefore , glutamate was suggested as a potential growth factor in tumor development . interestingly , the latter observation has recently been supported by a study that proved the relationship between increased levels of an excitatory neuropeptide , dynorphin 117 that comprises glutamate , and cell death or surgery - related tissue injury in vivo in epilepsy patients ( broderick et al . it has also been proven that glutamate release in gliomas is , at least in part , mediated by a na - independent cystine glutamate exchanger xc system , expressed in glioma cell lines and patient - derived glioma cells ( lyons et al . importantly , xc system seems to be up - regulated in glioma cells acting in concert with the lack of functional na - dependent transport systems of the excitatory amino acid transporters ( eaats ) , which are responsible for glutamate uptake and are amply expressed in nonmalignant glial cells ( lyons et al . mda - mb-231 ( human breast ) , b16f1 ( mouse melanoma ) and matlylu ( rat prostate ) cancer cell lines release significant quantities of glutamate into their extracellular environment ( seidlitz et al . a very recent study reported that prostate cancer - bearing patients have serum glutamate levels directly correlating with gleason score ( 6 vs. 8 ) and primary prostate cancer aggressiveness ( koochekpour 2013 ) . glutamate tissue levels in fresh frozen human normal pancreatic tissue , chronic pancreatitis ( cp ) and pancreatic ductal adenocarcinoma ( pdac ) tissues demonstrated a striking increase of glutamate in cp and pdac samples , suggesting that glu might serve as a molecular switch that decreases the threshold of k - ras - induced oncogenic signaling and increases the chance of malignant transformation of pancreatic cancer precursor lesions ( herner et al . thereby , released glu can act as a growth factor and a signal mediator in non - neuronal tumor tissues , in both , autocrine and paracrine fashions . a long - standing paradigm suggested that glutamate signaling is limited to the central nervous system . recently , however this opinion has been changed owing to the studies that proved glutamate receptors existence in peripheral organs and neoplastic cells ( hinoi et al . 1995 ) , glur2/3/4/6/7 , ka1 ( yoshioka et al . it has been also demonstrated that glutamate receptor subunits are expressed in a variety of cancer cell lines and tumors , originating outside of cns such as colorectal ( chang et al . 2009 ) , oral squamous cell carcinoma ( scc ) ( park et al . 2004 ) , as well as blood neoplasms such as leukemia and lymphoma ( ganor et al . still , the majority of the existing studies describe single receptor or its subunit expression in selected types of cancers while a more detailed analysis of glutamate receptor subunit expression in cancer cell lines or solid tumors remains scarce . interestingly , the nr1 subunit of the nmda receptor , although present in other cell lines derived from cns neoplasms , was lacking in glioma cell lines ( stepulak et al . in contrast to nmdars , ampa receptors are abundantly expressed in glioma cells , where they play an important role in glutamate - mediated proliferative signals thus enhancing its malignant phenotype ( de groot and sontheimer 2011 ; sontheimer 2008 ) . additionally , rnai experiments in a low - grade glioma cell line demonstrated that down - regulation of glur2 expression caused a significant increase of cell proliferation ( beretta et al . on the other hand , over - expression of edited glur2 subunit by adenovirus - mediated transfer inhibited migration of glioma cells both in vitro and in vivo ( ishiuchi et al . 2002 ) , proving that ca - permeable ampa receptors are crucial for glioma invasion . when analyzing glur2 q / r - editing status in different cancer cell lines , it has been shown that some of them exclusively express the unedited form of glur2 ( sk - na - s , neuroblastoma ) or both , the edited and the unedited glur2 forms ( moggccm , astrocytoma ) . this shows that cancer cells could express ampa receptor forms characteristic for ca - permeable fetal cells , which could in turn contribute to cancer cells invasion potential , as it has been demonstrated for glioma cells ( ishiuchi et al . of particular interest is the fact that glioblastoma - tumor initiating cells express high levels of functional , calcium - permeable ampa receptors containing glur1 and glur4 subunits , when compared with the differentiated tumor cultures consisting of non - stem cells derived from the same tumor tissues ( oh et al . 2006 ) , and hepatocellular carcinomas ( yamaguchi et al . interestingly , the expression of kainate receptors in cancer cells has not been extensively studied . 2001 ) , glur57 and ka1 in medulloblastomas , and additionally ka2 subunit in neuroblastoma cell lines ( yoshioka et al . likewise , our earlier study demonstrated the presence of glur5 in u343 glioma cells , whereas glur6 , glur7 , ka1 and ka2 subunits were found in all 12 analyzed cancer cell lines , which suggests a role of kainate receptors in metabolism and proliferation of cancer cells ( stepulak et al . similarly , a strong expression of glur4 ampa receptor subunit was detected in two cell lines : te671 ( rhabdomyosarcoma / medulloblastoma ) and rpmi ( plasmocytoma ) , and of glur6 kainate receptor subunit in sk - na - s ( neuroblastoma ) and moggccm ( astrocytoma ) cell lines at levels comparable to those in the hb . similar levels of expression of ka2 were found also in human te671 and the hb ( stepulak et al . aforementioned studies provide compelling evidence that glutamate receptors are expressed at higher level in the tumors and neoplastic cell lines of brain origin than in those derived from peripheral cancers . the observation that glurs subunits , which are poorly represented in the adult brain , are expressed in cancer cells ( stepulak et al . on the other hand , it has been shown that the expression of nmda receptor subunits in the brain varies during development ; especially nr2d subunits are present at high levels prenatally in rapidly dividing cns cells with subsequent decrease postnatally . 2009 ) suggests that the re - expression of nr2d in cancer cells may correlate with their proliferative potential . 1997 ) , and glioblastoma , astrocytoma , and oligodendroglioma cells responded to kainate by depolarization of tumor cells in culture or tissue slices ( labrakakis et al . interestingly , the evoked currents were small , which is consistent with the low expression of these receptors in examined cancer cells ( stepulak et al . importantly , experimental data have also implicated important role of mglurs in malignant tumor metabolism and progression . in pediatric cns tumors , the metabotropic glutamate receptor subtypes mglur1 , mglur2 , mglur4 , mglur5 and mglur6 were expressed at higher levels in the malignant tumors than in low - grade astrocytomas . parallel to cns tumors , mglurs have been shown to be over - expressed in some types of peripheral cancers and neoplasms . given the fact that mglur4 mediates 5-fluorouracil resistance in human colon cancer cells , which is a major obstacle in chemotherapy of this cancer type ( yoo et al . very low presence of mglur4 was demonstrated in thyroid , adrenal glands , and kidney cancers ( 813 % ) , whereas it was not detected in esophageal , endometrial and prostate cancers , as well as in neoplasms derived from salivary glands and testis ( chang et al . 2012 ) and renal cancer cells ( martino et al . 2013 ) , both in vitro and in vivo ; simultaneously their presence was not found in oral cancer tissues and cell lines , in contrast to mglur5 , which was present in the majority of oral cancer specimens and weakly in adjacent dysplastic oral mucosa ( park et al . moreover , melanoma development was connected with the presence of mglur1 receptors ( ohtani et al . 2008 ) , a finding that was supported by observations showing its expression in melanoma cell lines and melanoma samples , but not in normal melanocytes and benign nevi ( pollock et al . , considerable evidence exists for glutamate receptors expression in a variety of tumors and cancer cell lines along with proofs that they are functional , thereby might play an important role in neoplastic transformation and cancer progression . to substantiate the hypothesis that glutamate receptors are functionally important for tumor growth , several studies evaluated their involvement in tumorigenesis and subsequently proved that at least some of the glurs might have oncogenic properties . the impact of metabotropic glutamate receptors on tumor growth was highlighted by a series of experiments which showed causal relationship between mglur1 expression and melanoma development . parallel , sirna - mediated inhibition of mglur1 expression in renal cancer cells impaired tumor growth in vivo , thus suggesting that sustained expression of mglur1 is necessary for neoplastic transformation and tumor progression ( martino et al . changes in the levels of expression of ionotropic glutamate receptors or their single subunits in experimental conditions were also demonstrated to be important for cancer cells proliferation and invasion , which suggests the involvement of glurs in cancer progression . another study presented that rnai - mediated suppression of glur1 or glur2 did not affect pancreatic cancer cell growth , however significantly decreased invasion in vitro , and inhibited tumor cell settling in a mouse model in vivo ( herner et al . similar , knock - down of the glur3 gene reduced proliferation and migration , as well as enhanced apoptosis of pancreatic cancer cells , while over - expression of this gene was reported to have opposite effect in vitro and in a subcutaneous xenograft model ( ripka et al . 2009 ) , whereas gene silencing of glur4 modulated the mrna expression of various tumor - suppressor genes , oncogenes and other genes involved in invasion , adhesion and metastatic capabilities , which resulted in significant increase of cell viability of human rhabdomyosarcoma / medulloblastoma ( te671 ) and human multiple myeloma rpmi8226 cells . importantly , the expression of glurs as well as their function in cancer development and progression is influenced by genomic and epigenetic modifications resulting in aberrant posttranscriptional processing . causative for the aberrant cellular function of glutamate receptors in cancer are changes in genomic sequences for mglurs and selected subunits of iglurs . likewise , somatic mutations within mglur3 gene result in an activation of gpcr - mediated mitogen - activated protein kinase 1/2 signaling that results in a transformed cells phenotype , which renders an increased migration of melanoma cells along with a loss of anchorage dependency in growth regulation ( prickett et al moreover , a very recent study highlighted the importance of naturally occurring grm1 somatic mutations for mglur1 surface expression , altered basal and agonist - dependent activity , and disruption of intracellular signaling pathways downstream of the receptor , including inositol phosphate ( ip ) formation , and altered erk1/2 kinases activity ( esseltine et al . since these mutations were identified in different types of neoplasms including lung adeno- and scc ( kan et al . clinical genetic analysis of grm1 showed that single nucleotide polymorphism of the c allele of rs362962 ( coding mglur1 ) contributes to human melanoma susceptibility , especially in a subgroup of patients with a low level of sun exposure and tumors located on the trunk and extremities ( ortiz et al . a similar study performed in women carrying breast cancer revealed a significant correlation between the grm1 cc genotype of rs362962 and the development of hormone receptor - negative breast cancer and association of rs6923492 and rs362962 genotypes with age at diagnosis ( mehta et al . whole - exome sequencing analysis revealed moderate to high prevalence of somatic mutations in genes coding nr2a , and nr1 subunits of nmda receptors in melanoma ( wei et al . nonetheless , in addition to genetic rearrangements , epigenetic alterations seem to play an important role in cancer development and progression . other groups , on the other hand , found that nr2b promoter methylation exhibits tumor - suppressive activity in human esophageal ( kim et al . 2007 ) , as well as in non - small cell lung carcinoma ( tamura et al . therefore , rearrangements of glutamate receptors at different genetic and epigenetic levels seem to play a distinct role in their expression and function . despite the fact that the issue at hand requires more extensive studies , one may already hypothesize that different modifications of glurs and their respective genes exist in cancer cells , as demonstrated recently by the discovery of new spliced variants of human grm1 gene in melanoma cells ( diraddo et al . the unequivocally proven role of glurs in oncogenesis turned attention towards their potential clinical significance in different types of tumors . thus , in the clinical settings the expression of glutamate receptors might influence histological differentiation , clinical tumor staging , the presence of metastases and/or overall patient survival rate . it has been reported that glutamate receptors expression is associated with differentiation status in a variety of tumor subtypes . on the other hand , however , the expression of glur1 , glur2 and glur4 subunits was down - regulated in pdac ( herner et al . opposite association was observed when expression of glur2 was analyzed , which proved to be present in slow - growing gbm - derived tumor stem cells ( gbm tscs ) and low - grade tumor samples but not in fast - growing gliomas or high - grade tumor specimens , indicating that glur2 expression is associated with a low degree of malignancy ( beretta et al . an opposite pattern was observed in normal oral mucosa showing very weak expression of nr1 subunit of nmdar , whereas majority of analyzed oral scc specimens expressed this subunit ; albeit the presence of nr1 did not correlate with histological grading of this cancer type ( choi et al . 2007 ) expression in oral scc , whereas significantly lower expression levels of mglu4 receptors were correlated with spinal cord metastases , csf spreading , and recurrence of medulloblastoma ( iacovelli et al . tumor progression along with its dissemination and recurrence are closely related to patients prognosis and overall survival . similarly , in medulloblastoma the expression of mglur4 was higher in patients , who survived 5-year after surgery ( iacovelli et al . interestingly , glur2 expression showed a significant correlation with longer progression - free and overall survivals and was down - regulated in chemoresistant tumors , proving to be a positive prognostic factor for patients with advanced serous papillary ovarian adenocarcinoma ( choi et al . research into the role of glu signaling in cancer development and progression is still in its infancy ; however , important progress has been made in recent years . considerable evidence exists and indicates that glutamate plays an important role in tumor development , acting as a growth factor and a signal mediator in neural as well as non - neuronal tumors tissues , in both autocrine and paracrine fashions . it has been proven that its actions involve mainly a family of receptors consisting of metabotropic glutamate receptors and ionotropic glutamate receptors whose presence has been proven in a variety of benign and malignant lesions throughout the body . their actions , however , might differ significantly due to the fact that glurs are combined from various subunits , which result in a large diversity of intracellular signaling and distinct pharmacological properties . thus , the clinical significance of glutamate receptor expression may differ among tumor entities and it is difficult to predict how the expression of a particular subunit will influence cancer behavior . still , it is tempting to speculate that glurs and their signaling pathways render promising targets for therapeutic interventions . as a matter of fact , in recent years , there have been multiple attempts to implement iglur and mglur antagonists , in particular in malignant glioma treatment . in fact , memantine has even been employed in a phase ii clinical trial to determine its safety / efficacy in glioma patients ; however , results are not yet available ( http://clinicaltrials.gov # nct01260467 ) . future studies in this field are clearly needed to determine the efficacy of mglur antagonists and glu release inhibitors such as riluzole against tumors . it is clear that more research is needed to define the clinical significance of glu glur expression and signaling in various cancers .

subjective tinnitus is the phantom perception of sound in the absence of an external stimulus . in 13% of the general population it constitutes a significant impairment of the quality of life . despite significant research efforts , one major obstacle arises from the fact that by its very nature tinnitus is a subjective phenomenon , and the only possible diagnosis relies on self - reports of the subjects . this fact poses a problem not only for diagnosing tinnitus and identifying subtypes in human patients but also in animal models of tinnitus . at present , however , only research on animal models can provide us with the necessary understanding of the peripheral and central mechanisms that lead to the aberrant neuronal activity ultimately perceived as tinnitus . one proposed mechanism is that the pathological activity originates from plastic changes of the central auditory system following damages to the periphery . in a healthy system , this plasticity is essential for adjusting neuronal activity to changing acoustic environments . an acoustic trauma damaging the cochlea leads to a loss of input to the central stages of the auditory processing hierarchy . the lack of input is then overcompensated by increasing the spontaneous activity and neuronal synchrony . results from animal models of tinnitus are an essential element in the combined efforts of different audiological specializations for developing new tinnitus therapies . the irreplaceable advantage of animal models lies in the possibility to study small neuronal networks and individual nerve cells through invasive methods such as extra- and intracellular recordings in potentially genetically engineered or sound exposed subjects . these means provide high spatial and temporal resolution ( i.e. , micrometer and millisecond range , resp . ) which is impossible in human studies applying electroencephalography or functional magnetic resonance imaging ( exceptions are recordings during brain surgery ) . in fact , current hypotheses about the pathogenesis of tinnitus are mostly based on results from animal models , in particular from studies on tinnitus following noise - induced hearing loss . however , since tinnitus is a conscious percept , many aspects have to be studied and characterized in laboratory animals through behavioral means . furthermore , physiological measurements of tinnitus - related neuronal activity should ideally be sampled in awake animals in order to exclude artefacts from anesthesia and to facilitate a comparison with human subjects who only perceive tinnitus when awake . in summary , any behavioral assessment of tinnitus in the animal model should try to mimic as closely as possible conditions under which tinnitus develops in humans . the first section of this review provides an overview of the different species used as animal models in tinnitus research . finally , the competing behavioral paradigms used for assessing subjective tinnitus in the animal model are discussed . this sequence reflects a natural order of the main decisions to be made when designing animal experiments . which species mimics the human condition and pathology best ? what is the most appropriate way to induce tinnitus ? the first behavioral test for tinnitus in an animal model was established by jastreboff et al . [ 6 , 7 ] in 1988 using rats . since then a number of different laboratory animal species and various strains have been used for the behavioral assessment of tinnitus . besides the laboratory rat ( rattus norvegicus ) [ 851 ] , these include the domestic house mouse ( mus musculus ) [ 5258 ] , the chinchilla ( chinchilla laniger ) [ 59 , 60 ] , the syrian golden hamster ( mesocricetus auratus ) [ 6164 ] , the guinea pig ( cavia porcellus ) [ 6567 ] , and the mongolian gerbil ( meriones unguiculatus ) [ 68 , 69 ] . since the early studies by jastreboff et al . , the laboratory rat remains the most prominent species used for investigating tinnitus at the behavioral level . however , an increasing number of studies are being performed on mice since the wide range of genetically modified strains is not available for rats at present . comparing the hearing abilities and the suitability of different species for tinnitus assessment reveals advantages and drawbacks of the different approaches . compared to research on other sensory systems ( e.g. , the somatosensory modality which is usually investigated in the rat or mouse barrel cortex ) , investigation on hearing in mammals is characterized by a larger variety of established animal models . usually , the criteria for selecting one species over another are not documented in the literature , even though this choice has serious consequences for the interpretation of results and their transferability to human subjects . despite the fact that all species mentioned above belong to the same systematic order ( rodentia ) , their acoustical and behavioral ecology and physiology varies significantly from one another , and more importantly from the final subject of interest , the homo sapiens . the hearing range mostly covers the high frequency range beyond the upper human limit ( highest audible frequency at 60 db spl for human is 17.6 khz , rat : 58 to 70 khz [ 71 , 72 ] , mouse : 85.5 khz , chinchilla : 33 khz , hamster : 46.5 khz , guinea pig : 50 khz , and gerbil : 58 khz ) . the same applies to the low frequency hearing limit ( at 60 db spl in humans : 0.03 khz , rat : 0.52 khz , mouse : 2.3 khz , chinchilla : 0.05 khz , hamster : 0.096 khz , guinea pig : 0.05 khz , and gerbil : 0.032 khz ) . it has been proposed that mammals that do not hear below 0.5 khz do not use temporal encoding for pitch perception , with the exact frequency of this boundary being discussed . this suggests that the two most widely used behavioral models of tinnitus , rat , and mouse employ neuronal mechanisms for pitch perception that fundamentally differ from those of humans . it has been argued that this difference applies only to the lower frequency range ( < 5 khz ) . nevertheless , interpretation of animal studies in relation to a human disease would be more directed in species with audiograms similar to humans ( e.g. , gerbil or chinchilla ) . however , gerbils are prone to a certain degenerative disorder of the auditory system , at least when supplied by a commercial manufacturer . this caveat has to be taken into account when considering the gerbil as a potential model for subjective tinnitus . furthermore , choosing an animal model with human - like audiograms would facilitate the comparability of tinnitus pitch . many studies cited above induced tinnitus through a noise trauma centered at 16 khz . this treatment is presumed to give rise to a phantom percept that has a higher frequency than the region of highest sensitivity in the rat ( around 8 khz [ 71 , 72 ] ) . in the mouse studies mentioned above the tinnitus inducing noise in contrast to the rat , the mouse has its highest sensitivity at 16 khz . in humans , the average tinnitus pitch is in the range of 58 khz and the highest sensitivity lies around 3 to 4 khz . independent of the species this means that the tinnitus - inducing stimuli have to be carefully matched to the hearing range of subjects in order to achieve comparability with the human pathology . in recent years one of the reasons why mice entered the scene so late could be their presumed limited cooperation in behavioral training paradigms assumed from the larger variability in the effects found in acoustic startle experiments . characteristically , all mouse behavioral studies of tinnitus mentioned above use a paradigm ( gap - startle paradigm , introduced in 2006 by turner et al . ) that does not necessarily require a functional auditory cortex and does not require any behavioral training beyond adaptation to the setup . however , so far no evidence has been published that substantiates the cognitive difference between rats and mice . on the contrary , in the somatosensory modality , rats and mice exhibit similar performance levels and learning curves when facing a complex 2-alternative forced choice task , which requires the discrimination of simultaneously presented whisker deflections at different frequencies . the big advantage of the mouse model is the almost infinite range of genetically modified strains available . this allows the recording and manipulation of specific types of neurons ( e.g. , excitatory pyramidal neurons or inhibitory neurons of a certain cortical layer ) . however , so far no one has taken advantage of this feature of the mouse model . the downside , however , is that some mouse strains exhibit elevated auditory thresholds ( measured as auditory brainstem responses ) within 2 months after birth , a problem that may be aggravated in genetically modified lines . rats do not exhibit this early onset of age - dependent hearing loss [ 84 , 85 ] . comparable to the diversity of species used in behavioral testing of tinnitus , there is a number of different ways of inducing tinnitus in animal models . in principle , there are two ways of inducing tinnitus . alternatively , tinnitus is induced by presenting high level stimuli for one hald to two hours . both approaches try to mimic the etiology of tinnitus in humans even though the pathogenesis of subjective tinnitus remains poorly understood . however , it is commonly accepted that in many cases it commences with noise - induced damage to the hair cells of the inner ear , followed by deafferentation and hearing loss . such a trauma leads then to the initiation of compensatory processes in the central nervous system . in the healthy system , these processes warrant an activity level that is optimal for encoding the present acoustic environment . however , after a trauma and consequential deafferentation , this beneficial plasticity of the auditory system goes astray and overcompensates the missing input from the damaged region of the cochlea , leading to a permanently present phantom percept . the first study assessing subjective tinnitus in an animal model used a pharmacological method for induction . the main advantages of a pharmacological tinnitus induction are its potential reversibility and its previous use in human subjects for inducing tinnitus as well ( i.e. , 3.9 g salicylate / day for 5 days [ 89 , 90 ] ) . the two most commonly used substances where tinnitus is assessed by behavioral means in animal models are salicylate [ 9 , 1113 , 1620 , 22 , 32 , 33 , 35 , 36 , 38 , 39 , 4345 , 47 , 50 , 57 , 58 , 67 ] and quinine [ 10 , 12 , 18 ] , an antimalarial drug . other ototoxic drugs that have been investigated in animal studies are cisplatin ( cis - diammine - dichloroplatinum ( ii ) ) and carboplatin . both are chemotherapeutics , with cisplatin predominantly targeting the outer hair cells and carboplatin most likely affecting the inner hair cells . salicylate , the active component of aspirin , is the most commonly used drug in animal models . therapeutically it is administered usually as a mild analgesic or in anti - inflammatory therapy ( e.g. , against rheumatic arthritis ) . salicylate has the advantage of fast induction within minutes and its effects reverse within 72 hours of the last administration [ 94 , 95 ] . in most studies cited above , salicylate was applied locally to the inner ear [ 39 , 50 ] or central structures ( e.g. , auditory cortex ) as well . salicylate most likely exerts its effects on hearing at high doses , both in the sensory periphery and in the central nervous system . in the auditory periphery it mainly targets outer hair cells , inhibiting their electromotility most likely by partitioning into the membrane and blocking the prestin protein . the consequence is a reduced cochlear sensitivity which manifests itself in a reduction of otoacoustic emissions ( spontaneous and evoked ) , a decreased neural output , and ultimately a temporary hearing loss . long - term application of salicylate , however , leads to an increased expression of prestin , most likely as a compensatory reaction . parallel to these effects on the sensory epithelium , there is strong evidence that salicylate affects the central nervous system as well . different levels of the auditory pathway have been identified as being modulated by salicylate . amongst others these are the cochlear nucleus ( cn ) , the inferior colliculus ( ic ) , the medial geniculate body ( mgb ) , and the auditory cortex ( ac ) . the observed effects can either originate from changes of the input ( i.e. , altered cochlear output ) or from direct action on the neuronal activity . in particular , it has been shown that different parts of the inhibitory gabaergic neurotransmission can be modulated by salicylate and that a modulation of the gabaergic inhibition reduces salicylate - induced ototoxicity . after chronic systemic administration , salicylate causes an increase in the expression of the gaba - synthesizing enzyme gad . in slice preparations , salicylate decreases gabaergic inhibition of auditory cortical pyramidal neurons , potentially facilitating hyperactivity . these pieces of evidence indicate that acute salicylate administration reduces the gabaergic inhibition in the network , which is then compensated by an increased gaba synthesis . other effects of salicylate are a reduced spontaneous firing rate in the inferior colliculus , adjustments in the tonotopy of the auditory cortex , and changes in the cochlear nucleus . however , gabaergic transmission is most likely not the only target of salicylate . another very likely target is the nmda receptor ( n - methyl - d - aspartate ) [ 38 , 104 ] . finally , it has been proposed that salicylate acts on the extralemniscal pathway while noise trauma induces tinnitus in the lemniscal pathway . however , it seems that with the right dosage ( 100 mg sodium salicylate / kg / day for two consecutive days ) there is a reliable tinnitus induction , as shown with a behavioral test in rats . how such a dosage in rats translates to a comparably critical serum level in humans is a source of uncertainty . in humans injection of 350 mg / kg sodium salicylate ( corresponding to 300 mg / kg salicylic acid ) , the salicylate serum level in the rat was 625 mg / l . for the dosage of 100 mg / kg inducing reliable tinnitus in the rat , . these differences ( 56 mg salicylate / l in rat vs 300 mg salicylate / l in human serum concentration ) might indicate a higher sensitivity of the rat , differences in underlying clearance mechanisms , or different threshold criteria and administration schedules . the second established method for inducing tinnitus in behavioral models is through acoustic trauma [ 8 , 15 , 20 , 21 , 23 , 2527 , 30 , 31 , 37 , 3942 , 4649 , 5156 , 5966 , 68 , 69 , 107 ] . it is assumed that a cochlear damage is in most cases the trigger for a sequence of events leading to the development of tinnitus in humans . however , not every hearing loss resulting from a trauma gives rise to tinnitus and a subset of patients exhibit normal audiogram indicating that hidden hearing losses play a role as well . acoustic trauma and subsequent hearing loss induces a number of acute and chronic changes in the periphery and the central nervous system . at the periphery , an acoustic trauma results in outer hair cell damage , cochlear dead regions ( no functional inner hair cells ) , damaged stereocilia in both types of hair cells , and deafferentation of auditory nerve fibers . typically , the hearing loss accompanying tinnitus is located in the high - frequency range . the tinnitus pitch itself is either near the edge of the hearing loss or in the frequency range of the damaged region itself . the parameters for inducing tinnitus through acoustic trauma in the animal model are quite variable . typically , a high level noise stimulus is applied for 1 to 2 hours under anesthesia , either to one or both ears . for the rats , a widely used stimulation paradigm consists of an octave - band noise with a peak intensity of 116 db sound - pressure level centered at 16 khz for 1 hour . however , sound level ( 80 db spl to 130 db spl , ) , duration ( 2 min to 7 hours ) , frequency ( 2 khz to 22 khz ) , frequency range ( pure tones to broadband noise ) , and concerned ear ( uni- or bilateral ) vary a lot between studies . in rats , binaural exposure to a 10 khz tone for 1 - 2 h leads to significant tinnitus when the sound level was 120 db but not at 80 , 100 , or 110 db spl . the primary criteria for selecting the stimulus parameters are usually the hearing range of the species , the targeted tinnitus pitch , and time course ( temporary versus chronic ) . mice exposed to noise centered at 16 khz at 116 db spl for 1 hour exhibited signs of tinnitus for 25 months afterwards , while in rats exposed to 17 khz pure tones at 115 db spl for 2 minutes tinnitus lasted only 13 min ( induction under isoflurane anesthesia ) . in gerbils , a reliable and chronically induced tinnitus can be achieved by noise stimuli with an exposure time of at least 1 hour and narrow bandwidth leading to a temporary threshold shift and ultimately to self - sustaining activity perceived as phantom sound . such a protocol leads to a hearing loss that disappears after 3 to 6 weeks and a tinnitus percept centered at the center - trauma frequency appearing 5 to 7 weeks after induction . hamsters exposed to a 10 khz tone at 110 db spl for 4 h exhibited tinnitus symptoms within one day after exposure indicating the possibility of an almost immediately tinnitus onset after acoustic trauma . the changes after acoustic trauma at the different stages of the ascending auditory pathway are manifold and complex . within hours after an acoustic trauma , the spontaneous neuronal activity in the primary auditory cortex ( a1 ) of the cat increases in the frequency region below the damage . this increase presumably originates from a loss of inhibition from the cortical regions representing frequencies of the cochlear damage . weeks after an acoustic trauma , the tonotopic map of a1 reorganizes so that there are no neurons with characteristic frequencies above the frequency of the traumatizing stimulus . in parallel , neurons in the inferior colliculus exhibit increased spontaneous firing rates after an acoustic trauma . in the dorsal cochlear nucleus ( dcn ) an acoustic trauma induces an increase in spontaneous activity which correlated with the strength of the behavioral tinnitus evidence and specifically in fusiform cells . while salicylate can be administered for tinnitus induction in awake animals , it is usually anesthetized for tinnitus induction through acoustic trauma . the anesthesia is either injectable ( very often a combination of ketamine and xylazine , or pentobarbital ) or an inhalable one ( usually isoflurane ) . how different anesthetics influence the development of hearing loss and tinnitus after acoustic trauma is largely unknown . however , isoflurane has been shown to diminish the amplitude and duration of temporary tinnitus after a short exposure to loud sounds . under pentobarbital , isoflurane , or halothane anesthesia noise - induced hearing loss in mice is less ( 62.5 db , 45.5 db , 39.3 db threshold increase , respectively ) compared to the unanesthetized control group ( 77.5 db threshold increase ) . in addition , the influence of anesthesia on any electrophysiological recordings has to be taken into account , as anesthesia influences the receptive fields and the spontaneous activity of the rat auditory cortex . salicylate administration can be locally confined either to the cochlea [ 50 , 117 ] or to specific brain structures and systemic administration is possible without anesthesia . the drawbacks are a presumed multitude of mechanisms giving rise to tinnitus , a lackof specificity interms of the locus of action , tinnitus pitch ( 0.9 to 14.5 khz ) , and relevance for the human pathology since in humans it is usually triggered by noise trauma . furthermore , salicylate does not induce chronic tinnitus as it recedes when the intake is stopped . these aspects hinder the identification of neuronal substrates involved in the pathogenesis and maintenance of human tinnitus by means of salicylate . one advantage of inducing tinnitus through acoustic trauma is the possibility to induce unilateral tinnitus , allowing the animal to serve potentially as its own control as done in some studies ( e.g. , turner et al . ) . however , one has to keep in mind that the ascending auditory pathway is characterized by significant binaural projections on every stage . even if the tinnitus is perceived unilaterally , it is manifest in contra- and ipsilateral instances . therefore , real controls ( i.e. , animals not exposed to noise as done by turner et al . ) are required as well . another advantage of tinnitus induction by acoustic trauma is the fact that this is most likely the most common form observed in human patients . one of the biggest uncertainties when inducing tinnitus through an acoustic trauma is the resulting percentage of animals exhibiting tinnitus in behavioral tests . these numbers vary significantly in the literature , according to knipper et al . from 30% to 80% . ultimately , the choice of how to induce tinnitus in a behavioral study depends on the research question and which form of tinnitus will be studied . it has to be kept in mind that an acoustic trauma and drugs induce tinnitus through different mechanisms and that both methods have certain methodological constrains ( e.g. , that an acoustic trauma very often has to be applied under anesthesia , depending on local animal welfare regulations ) . diagnosis of subjective tinnitus in human patients relies almost exclusively on the self - report as there is no external sound source present and it manifests itself only in the neuronal activity of subject 's brain . there are some noninvasive approaches that provide potentially objective measures for subjective tinnitus by means of functional magnetic resonance tomography , electroencephalography , magnetoencephalography , and positron emission tomography . however , at present none of these methods is applied routinely for diagnosing tinnitus and it is unknown whether the observed effects are directly caused by tinnitus or by the emotional stress usually accompanying severe tinnitus . this challenge of diagnosing tinnitus poses a supreme obstacle for developing an animal model with behavioral evidence of tinnitus . nevertheless , a reliable behavioral assessment of tinnitus in the animal model is essential for understanding the pathology and the development of therapies . in typical behavioral tests performed in sensory physiology , the presence of a stimulus has to be detected or stimuli have to be discriminated and the animal 's decision is indicated by a nose poke or a lever press . the absence of a stimulus usually requires no specific response , as seen in go / nogo paradigms . a continuous phantom percept like tinnitus hardly fits into such a framework of psychophysical experiments , as it is assumed to abolish the notion of silence . since the first publications by jastreboff et al . [ 6 , 7 ] 25 years ago , a number of different behavioral paradigms for addressing this issue have been developed . any behavioral assessment of tinnitus has to consider the confounding influences of possible hearing loss ( after noise trauma ) and hyperacusis accompanying tinnitus induction . furthermore , an ideal test for tinnitus in animals would be closely modeled on tinnitus tests performed in humans and might even be applicable to humans as well . [ 6 , 7 ] used a standard learning technique , the pavlovian conditioned response suppression by the induction of fear . water - restricted animals were exposed to a constant background noise ( approximately 40 db spl ) during which they were allowed to collect water from a drinking tube . the conditioned stimulus ( cs ) was the offset of the background noise for 30 s. the behavioral readout was the ratio of licks during the cs compared to the number of licks in the period preceding the silent gap ( suppression ratio ) . during suppression training the cs periods were terminated with an inevitable foot shock as unconditioned stimulus ( us ) . next , animals were injected with salicylate in order to induce a phantom sound that was assumed to fill out the silent gap of the cs . during the testing there was no foot shock ( us ) and the response suppression extinguished over time . in salicylate - treated animals the response suppression extinguished within 2 days , while it took saline - injected animals 4 days until the response suppression was extinguished . the faster extinction time course in salicylate - treated animals has been interpreted as an indicator of tinnitus as the animals did not perceive the silent gaps ( cs ) anymore . the most important control of this study was a group of animals that received salicylate before the suppression training . consequently , during the testing sessions , when no foot shock was given , the animals stopped licking during the silent gaps as they associated the tinnitus with punishment and the extinction took longer . this rules out the possibility that salicylate by itself changed the behavior in some ways ( e.g. , increased thirst , altered impulsivity ) . hearing loss after salicylate administration as an explanation for the faster extinction was ruled out since reducing the amplitude of the continuous noise by 20 db did not lead to a faster extinction . they aimed at a protocol that allows to measure behavioral indicators of tinnitus in individual animals . the basic paradigm again consisted of a broadband noise during which the animals were allowed to drink ( safe signal ) and silence during which the animals had to stop drinking . in training , performance was calculated as the average percentage of time the animal contacted the spout during noise and was not in contact during silence . the tinnitus was induced by a pure tone acoustic trauma ( 10 khz , 124 db spl for 4 h ) applied to the left ear . during test sessions ( 5 days after acoustic trauma ) [ 6 , 7 ] the time course of the extinction of the response suppression during silent periods was indicative of the perceived phantom sound . animals receiving a pure tone trauma were more likely to drink during silent periods compared to a control group . however , the variability was quite big and there was a certain overlap in the distributions of performance scores of the control group and the one that received a trauma . similar conditioned suppression paradigms have been used in other studies as well ( e.g. , zheng et al . ) . the main advantage of their approach is that it can be applied easily to larger numbers of animals since the training period is quite short . different tinnitus induction protocols have been proven to be effective with such paradigms which allow pitch and amplitude of the tinnitus to be characterized . since the indication for tinnitus is the time course of suppression extinction ( no foot shock ) , only short time spans ( days ) can be monitored and a more detailed analysis of the tinnitus over time is impossible . bauer and brozoski published an operant conditioning approach for measuring tinnitus in the animal model ( rats ) . here , subjects were trained to lever - press in order to receive a food reward when an auditory test stimulus was present ( 60 db spl broadband noise or pure tones ) . during silent periods , a running index of lever press behavior was computed for windows of 1 min length . if the animals kept lever pressing in the silent periods , they were punished with a foot shock if they met or exceeded a certain criterion of the running index . in the testing sessions , pure tones of different frequencies and amplitudes were presented as well as silent gaps . lever pressing during pure tone presentation was not punished ; however , pressing during silent gaps was still punished . the discrimination functions ( pure tones and silence ) of animals receiving an acoustic trauma ( noise centered at 16 khz , 1 octave bandwidth ) and unexposed control animals ( or animals with a simulated hearing loss through ear plugs ) differed significantly . this has been interpreted as an indicator for tinnitus as the traumatized animals could not differentiate between test tones and real silent gaps which were filled with the phantom sound . since the behavioral contingencies were the same during testing and training , it was possible to measure the tinnitus induced by noise trauma over extended periods ( up to 17 months ) . additionally , the tinnitus properties ( pitch , loudness ) could be measured in detail , as bauer and brozoski identified the tinnitus pitch at 20 khz . the downside of this approach is that it requires careful training and can take extended periods of time for the animals to reach criterion before the actual testing takes place . rats were put on a food restriction schedule and received a food pellet in regular intervals . this scheduled food intake induced polydipsia leading to a constant licking for water between the food deliveries . animals perceiving a phantom sound are expected to lick less during quiet periods as they try to avoid a foot shock . the motivation to develop such a schedule - induced polydipsia avoidance conditioning paradigm was to assess tinnitus in individual animals and over extended periods of time . iin order to achieve a performance of > 90% of licks during quite periods the animals were trained for 2 - 3 weeks . another study confirmed the sensitivity of this test for tinnitus by measuring it with different paradigms as well . an operant paradigm with positive reinforcement has been proposed by rttiger et al . which reduced the need for punishment through foot shocks to a minimum . again , a continuous noise was a safe signal for the rat to access one of two water spouts in order to receive a reward ( 3% sucrose in water ) . the rat had to switch from one spout to the other in order to collect a reward . if the animal accessed one spout during a silent period , no reward was delivered and a foot shock is applied . during testing for tinnitus , there was no reward and no punishment during the silent gaps . in order to still get useful behavioral responses , even before testing for tinnitus , this prolonged the time to extinction of the discriminative behavior between noise and silent gaps . it should be emphasized that the foot shock in this study was quite weak and avoidable and the behavior of the animals was most likely driven by the reward value of the sugar water itself . tinnitus was induced with an injection of salicylate ( 350 mg / kg bodyweight ) after the animals achieved a certain performance level ( 12 to 15 sessions before administration ) . testing took place immediately after tinnitus induction in order to characterize the immediate effects of salicylate . the behavioral indicator was the ratio between number of reward spout access during noise and during silence , divided by the ratio between noise duration and silence duration . after salicylate treatment , the number of access to the reward spouts during silent periods increased relative to the access during noise presentation . this paradigm has been used in a couple of follow - up studies , where the tinnitus was induced through an acoustic trauma , emphasizing its robustness and applicability to a wider range of tinnitus models [ 42 , 51 , 119 ] . another paradigm using only mild electric shocks and positive reinforcement was published by heffner and koay . here , hamsters received a unilateral acoustic trauma and were trained to localize a sound source ( left or right ) in order to collect a reward at that side . , sound trials were interleaved with a few silent trials ( catch trials ) which were not punished or rewarded . after the acoustic trauma , the side preference shifted to the side where the trauma was applied . this was interpreted as a result of a phantom sound perceived by the animals , as they were trained to go to the side where a stimulus was localized . in summary , the operant conditioning paradigms described here usually require a very careful and time - consuming training of the animals . however , this is compensated by the possibility to test animals repeatedly and over extended periods . one very recently published paradigm does not apply any aversive stimulus at all but only positive reinforcement through food pellets . here , the rats had to press one lever in the presence of a sound ( tone lever ) and press another lever in the absence of sound ( 0 hz lever ) . after treatment with salicylate ( 75 , 150 , 300 , or 450 mg / kg body weight ) or exposure to intense sounds ( 140 db spl at 4 khz for 4 hours ) the animals exhibited an increased number of tone lever presses in the absence of any sound . again , the extensive training required ( 2 - 3 months ) by this paradigm is balanced by the possibility to test animals over extended periods . a navigation approach was pursued by guitton and dudai . here , the rats had to swim in a water t - maze and find a hidden platform . the platform was in one of the two arms of the maze if a tone was presented and in the other arm , when no tone was presented . two measures were taken for quantifying the sound perception of the animal : time spend in one arm of the maze and percentage of correct choices . after 3 days of training the animals reached the correct arm in 80% of the cases within an average time of 4 s. after an acoustic trauma approximately half of the rats ( 12 out of 26 ) behaved as if they perceived in tone even when there was no sound present ( measured as an increased time spent in the arm associated with the tone ) . during the last years , a completely different and objective paradigm was established for measuring tinnitus in laboratory animals . it is based on the acoustic startle reflex or response ( asr ) which is a very rapid contraction of skeletal muscles following the presentation of acoustic stimulus with high intensity . the central pathway for this startle response is well described and involves only three synapses . the cochlear input is relayed through the brainstem to the pedunculopontine tegmental nucleus and the nucleus reticularis pontis caudalis which initiates the startle response . the amplitude of this response is modulated by many factors like fear potentiation and sensitization . in particular it can be reduced by a preceding stimulus or silent gap in a continuous background noise . the basic idea for tinnitus detection is that a phantom sound can mask these gaps . in animals experiencing tinnitus , the acoustic startle reflex this concept was first tested and published by turner et al . as a new approach to efficiently test for tinnitus in the animal model . to this end rats received an acoustic trauma ( unilateral 16 khz octave - band noise at 116 db spl , under anesthesia ) . next , animals were placed in a testing chamber where a continuous background noise was presented ( centered at 10 or 16 khz or broadband noise , 60 db spl ) . the animal 's response was measured as force applied to a piezo transducer in the floor of the chamber . the startle stimulus was a 115 db spl noise burst for 20 ms . half of the startle stimuli were preceded by a 50 ms gap in background noise which would reduce the startle amplitude in nave animals . animals receiving an acoustic trauma exhibit less inhibition of the startle response when it was preceded by a gap compared to controls . however , this was only the case when the background noise was centered at 10 khz and not at 16 khz or for broad band noise . this result confirmed the previously characterized tinnitus pitch at 10 khz which was determined by an operant conditioning paradigm . hearing loss was ruled out as possible explanation for this effect as a simulated unilateral hearing loss ( ear plugs ) did not change the inhibition of the startle response by a preceding gap . this paradigm or some derivatives ( e.g. , measuring the preyer reflex in guinea pigs by berger et al . ) were adopted by many research groups [ 11 , 20 , 23 , 46 , 48 , 49 , 52 , 53 , 64 , 65 , 68 , 69 ] because they offer a number of advantages . the main benefit for experimentalists is that it is a fast method in terms of training and testing . no training beyond test chamber adaptation is required and testing can take place in less than one hour , allowing high - throughput screening which is not possible with more complex conditioned behavioral paradigms . additionally , the animals do not have to be on a restricted food or water schedule and the neuronal circuitry giving rise to the startle response is well described . finally , this is a fairly objective measurement as the reflex is only to a certain degree modulated by top - down processes . however , a number of issues have to be taken into account when considering a gap startle paradigm for assessing tinnitus in animal models . first , it is unknown whether in human tinnitus patients gaps are filled with the phantom percept . in the light of transferability of results from the animal model to humans , this is a major drawback and has been only very recently addressed by fournier and hbert . this study explicitly tested gap inhibition of a startle response ( eye blink ) in tinnitus patients ( high - pitched ) in order to compare it to animal studies . the key finding was that tinnitus patients exhibited a similar change of startle response amplitude when preceded by a gap as the traumatized animals did in the studies mentioned above . despite some differences in the results compared to the study by turner et al . ( e.g. , gap deficits occurred at high- and low - frequency background noise in humans but not in the animal study ) this is evidence that the gap startle paradigm could be a valid model for studying tinnitus and that it measures manifestations of a phantom sound comparable to the one observed humans . one objection put forward regarding the gap startle paradigm is its reflex nature and that it does not necessarily involve the auditory cortex . it has been shown that ablation of auditory cortex in mice does not change the gap startle response after one month compared to a control group . however , one day after cortex ablation there were differences , indicating a temporary modulatory effect of auditory cortex on activity in the brain stem circuitry that gives rise to the startle response . other studies in rats [ 127 , 128 ] lesioning or deactivating the auditory cortex found changes for certain gap durations . thus , the role of auditory cortex in the gap startle paradigm still remains to be elucidated . it has been hypothesized that the neural substrate of tinnitus involves an increase in spontaneous activity , an increase in neuronal synchrony , and a reorganization of the tonotopic map in auditory cortex [ 105 , 120 ] . testing this hypothesis ideally requires a behavioral paradigm , which necessarily involves the auditory cortex and not only a brain stem circuit . it has been shown that tinnitus patients and healthy subjects can detect gaps typically used in gap startle paradigms with similar performance . this result indicates that changes in gap startle paradigms do not automatically mean that higher processing of these stimuli is impaired in tinnitus patients . put forward the potential influence of hearing loss on the gap startle response and tackle this concern twofold in a dedicated study : first , by optimizing the startle stimulus so that it was outside the range of the hearing loss and second , by substituting the broad band noise startle stimulus with a rapid air puff to the animal 's back which can not be subject to hearing loss . in particular , the air puff approach preserved the startle response , even after conductive hearing loss . the ultimate benchmark for any animal model measuring subjective tinnitus is comparability to the human patient . any researcher starting to model tinnitus in laboratory animals has to make a decision regarding the species , the method of tinnitus induction , and the behavioral test . the most important criteria for choosing a certain species is its hearing range , its aptitude for behavioral studies and the availability of genetically modified strains . these strains allow the recording and manipulation of specific types of neurons revealing their role in tinnitus . the majority of studies discussed here were done in rats , considered to be well suited for behavioral testing even with more difficult sensory decision making paradigms . another advantage of the rat as an experimental model for studying the neuronal circuitry underlying tinnitus is the possibility to implant electrode arrays with high channel counts and perform chronic recordings in awake and behaving animals ( e.g. , otazu and zador ) . the disadvantage of the rat as a model is its high - frequency hearing range , which differs significantly from the human one . still , it remains unclear so far if these differences in hearing rage are significant for the pathogenesis , perception , and potential therapy of tinnitus . however , this last factor is certainly changing in the future as more and more recombinase - driver rat lines are developed ( e.g. , ) and the establishment of the potentially universally applicable crispr genome - editing technique , which has already been applied successfully in cynomolgus monkey ( macaca fascicularis ) . the tinnitus induction protocol should model the human pathogenesis . for the majority of human cases , this favors a tinnitus induction through acoustic trauma over a pharmacological induction . on the other hand , an induction through salicylate has the advantage of fast onset of tinnitus and its reversibility . this allows a behavioral setting that can be controlled for tinnitus related behavioral peculiarities of individual animals . furthermore , salicylate can be applied locally which allows to study tinnitus - related changes at different stages of the auditory processing hierarchy . whichever method is used , the accompanying hearing loss and hyperacusis have to be taken into account for interpreting the results . however , to disentangle tinnitus and hyperacusis is very challenging as they are comorbid . very recently , it has been demonstrated that mice exposed to neuropathic noise displayed a hyperresponsivity to acoustic startle stimuli . at the same time the gap detection deficits ( measured as prepulse inhibition of the startle response ) were limited to certain gap - stimulus latencies which can not be explained by the presence of a phantom sound which should fill the gap for all latencies and which therefore has be interpreted as a potential indicator of hyperacusis . the behavioral approaches testing for subjective tinnitus presented here include paradigms using reflexes , pavlovian conditioning , and operant conditioning . it is usually measured through sensory decision making tests which can be applied over extended periods . a behavioral test for laboratory animals should be shaped along these aspects , in particular the cortical involvement and extended testing period . additionally , such a test should only require limited training periods in order to achieve a high throughput . for conditioned responses the auditory cortex is not essential , as a cortical ablation does not prevent an animal from a classical conditioning response to simple tones . however , more complex tones ( e.g. , frequency modulated tones ) necessarily require a functional auditory cortex for discrimination . this has to be balanced with the usually more time consuming training protocols required for operant conditioning paradigms . for the conditioning paradigms introduced here , an involvement of the auditory cortex has not been shown yet , leaving an explanatory gap between the observed behavior and its neuronal substrate . furthermore , modulation of the tinnitus percept through higher cognitive functions as demonstrated in humans ( e.g. , attention ) has been ignored in animal studies so far , most likely due to a lack of behavioral paradigms allowing the manipulation of these functions . however

tinnitus is one of the major audiological diseases , affecting a significant portion of the ageing society . despite its huge personal and presumed economic impact there are only limited therapeutic options available . the reason for this deficiency lies in the very nature of the disease as it is deeply connected to elementary plasticity of auditory processing in the central nervous system . understanding these mechanisms is essential for developing a therapy that reverses the plastic changes underlying the pathogenesis of tinnitus . this requires experiments that address individual neurons and small networks , something usually not feasible in human patients . however , in animals such invasive experiments on the level of single neurons with high spatial and temporal resolution are possible . therefore , animal models are a very critical element in the combined efforts for engineering new therapies . this review provides an overview over the most important features of animal models of tinnitus : which laboratory species are suitable , how to induce tinnitus , and how to characterize the perceived tinnitus by behavioral means . in particular , these aspects of tinnitus animal models are discussed in the light of transferability to the human patients .

1. Introduction 2. Species Used for Behavioral Testing of Tinnitus 3. Established Ways of Tinnitus Induction in Animal Research 4. Behavioral Models for Assessing Tinnitus in Animals 5. Summary

subjective tinnitus is the phantom perception of sound in the absence of an external stimulus . in 13% of the general population it constitutes a significant impairment of the quality of life . despite significant research efforts , one major obstacle arises from the fact that by its very nature tinnitus is a subjective phenomenon , and the only possible diagnosis relies on self - reports of the subjects . this fact poses a problem not only for diagnosing tinnitus and identifying subtypes in human patients but also in animal models of tinnitus . at present , however , only research on animal models can provide us with the necessary understanding of the peripheral and central mechanisms that lead to the aberrant neuronal activity ultimately perceived as tinnitus . one proposed mechanism is that the pathological activity originates from plastic changes of the central auditory system following damages to the periphery . in a healthy system , this plasticity is essential for adjusting neuronal activity to changing acoustic environments . an acoustic trauma damaging the cochlea leads to a loss of input to the central stages of the auditory processing hierarchy . results from animal models of tinnitus are an essential element in the combined efforts of different audiological specializations for developing new tinnitus therapies . the irreplaceable advantage of animal models lies in the possibility to study small neuronal networks and individual nerve cells through invasive methods such as extra- and intracellular recordings in potentially genetically engineered or sound exposed subjects . these means provide high spatial and temporal resolution ( i.e. which is impossible in human studies applying electroencephalography or functional magnetic resonance imaging ( exceptions are recordings during brain surgery ) . in fact , current hypotheses about the pathogenesis of tinnitus are mostly based on results from animal models , in particular from studies on tinnitus following noise - induced hearing loss . however , since tinnitus is a conscious percept , many aspects have to be studied and characterized in laboratory animals through behavioral means . in summary , any behavioral assessment of tinnitus in the animal model should try to mimic as closely as possible conditions under which tinnitus develops in humans . the first section of this review provides an overview of the different species used as animal models in tinnitus research . finally , the competing behavioral paradigms used for assessing subjective tinnitus in the animal model are discussed . which species mimics the human condition and pathology best ? what is the most appropriate way to induce tinnitus ? besides the laboratory rat ( rattus norvegicus ) [ 851 ] , these include the domestic house mouse ( mus musculus ) [ 5258 ] , the chinchilla ( chinchilla laniger ) [ 59 , 60 ] , the syrian golden hamster ( mesocricetus auratus ) [ 6164 ] , the guinea pig ( cavia porcellus ) [ 6567 ] , and the mongolian gerbil ( meriones unguiculatus ) [ 68 , 69 ] . , the somatosensory modality which is usually investigated in the rat or mouse barrel cortex ) , investigation on hearing in mammals is characterized by a larger variety of established animal models . usually , the criteria for selecting one species over another are not documented in the literature , even though this choice has serious consequences for the interpretation of results and their transferability to human subjects . despite the fact that all species mentioned above belong to the same systematic order ( rodentia ) , their acoustical and behavioral ecology and physiology varies significantly from one another , and more importantly from the final subject of interest , the homo sapiens . the same applies to the low frequency hearing limit ( at 60 db spl in humans : 0.03 khz , rat : 0.52 khz , mouse : 2.3 khz , chinchilla : 0.05 khz , hamster : 0.096 khz , guinea pig : 0.05 khz , and gerbil : 0.032 khz ) . this suggests that the two most widely used behavioral models of tinnitus , rat , and mouse employ neuronal mechanisms for pitch perception that fundamentally differ from those of humans . however , gerbils are prone to a certain degenerative disorder of the auditory system , at least when supplied by a commercial manufacturer . in the mouse studies mentioned above the tinnitus inducing noise in contrast to the rat , the mouse has its highest sensitivity at 16 khz . independent of the species this means that the tinnitus - inducing stimuli have to be carefully matched to the hearing range of subjects in order to achieve comparability with the human pathology . in recent years one of the reasons why mice entered the scene so late could be their presumed limited cooperation in behavioral training paradigms assumed from the larger variability in the effects found in acoustic startle experiments . on the contrary , in the somatosensory modality , rats and mice exhibit similar performance levels and learning curves when facing a complex 2-alternative forced choice task , which requires the discrimination of simultaneously presented whisker deflections at different frequencies . however , so far no one has taken advantage of this feature of the mouse model . comparable to the diversity of species used in behavioral testing of tinnitus , there is a number of different ways of inducing tinnitus in animal models . alternatively , tinnitus is induced by presenting high level stimuli for one hald to two hours . both approaches try to mimic the etiology of tinnitus in humans even though the pathogenesis of subjective tinnitus remains poorly understood . however , it is commonly accepted that in many cases it commences with noise - induced damage to the hair cells of the inner ear , followed by deafferentation and hearing loss . such a trauma leads then to the initiation of compensatory processes in the central nervous system . in the healthy system , these processes warrant an activity level that is optimal for encoding the present acoustic environment . however , after a trauma and consequential deafferentation , this beneficial plasticity of the auditory system goes astray and overcompensates the missing input from the damaged region of the cochlea , leading to a permanently present phantom percept . the main advantages of a pharmacological tinnitus induction are its potential reversibility and its previous use in human subjects for inducing tinnitus as well ( i.e. the two most commonly used substances where tinnitus is assessed by behavioral means in animal models are salicylate [ 9 , 1113 , 1620 , 22 , 32 , 33 , 35 , 36 , 38 , 39 , 4345 , 47 , 50 , 57 , 58 , 67 ] and quinine [ 10 , 12 , 18 ] , an antimalarial drug . salicylate , the active component of aspirin , is the most commonly used drug in animal models . in most studies cited above , salicylate was applied locally to the inner ear [ 39 , 50 ] or central structures ( e.g. salicylate most likely exerts its effects on hearing at high doses , both in the sensory periphery and in the central nervous system . parallel to these effects on the sensory epithelium , there is strong evidence that salicylate affects the central nervous system as well . different levels of the auditory pathway have been identified as being modulated by salicylate . in particular , it has been shown that different parts of the inhibitory gabaergic neurotransmission can be modulated by salicylate and that a modulation of the gabaergic inhibition reduces salicylate - induced ototoxicity . after chronic systemic administration , salicylate causes an increase in the expression of the gaba - synthesizing enzyme gad . other effects of salicylate are a reduced spontaneous firing rate in the inferior colliculus , adjustments in the tonotopy of the auditory cortex , and changes in the cochlear nucleus . finally , it has been proposed that salicylate acts on the extralemniscal pathway while noise trauma induces tinnitus in the lemniscal pathway . these differences ( 56 mg salicylate / l in rat vs 300 mg salicylate / l in human serum concentration ) might indicate a higher sensitivity of the rat , differences in underlying clearance mechanisms , or different threshold criteria and administration schedules . it is assumed that a cochlear damage is in most cases the trigger for a sequence of events leading to the development of tinnitus in humans . acoustic trauma and subsequent hearing loss induces a number of acute and chronic changes in the periphery and the central nervous system . at the periphery , an acoustic trauma results in outer hair cell damage , cochlear dead regions ( no functional inner hair cells ) , damaged stereocilia in both types of hair cells , and deafferentation of auditory nerve fibers . typically , the hearing loss accompanying tinnitus is located in the high - frequency range . the tinnitus pitch itself is either near the edge of the hearing loss or in the frequency range of the damaged region itself . however , sound level ( 80 db spl to 130 db spl , ) , duration ( 2 min to 7 hours ) , frequency ( 2 khz to 22 khz ) , frequency range ( pure tones to broadband noise ) , and concerned ear ( uni- or bilateral ) vary a lot between studies . the primary criteria for selecting the stimulus parameters are usually the hearing range of the species , the targeted tinnitus pitch , and time course ( temporary versus chronic ) . within hours after an acoustic trauma , the spontaneous neuronal activity in the primary auditory cortex ( a1 ) of the cat increases in the frequency region below the damage . weeks after an acoustic trauma , the tonotopic map of a1 reorganizes so that there are no neurons with characteristic frequencies above the frequency of the traumatizing stimulus . in parallel , neurons in the inferior colliculus exhibit increased spontaneous firing rates after an acoustic trauma . in the dorsal cochlear nucleus ( dcn ) an acoustic trauma induces an increase in spontaneous activity which correlated with the strength of the behavioral tinnitus evidence and specifically in fusiform cells . while salicylate can be administered for tinnitus induction in awake animals , it is usually anesthetized for tinnitus induction through acoustic trauma . however , isoflurane has been shown to diminish the amplitude and duration of temporary tinnitus after a short exposure to loud sounds . salicylate administration can be locally confined either to the cochlea [ 50 , 117 ] or to specific brain structures and systemic administration is possible without anesthesia . the drawbacks are a presumed multitude of mechanisms giving rise to tinnitus , a lackof specificity interms of the locus of action , tinnitus pitch ( 0.9 to 14.5 khz ) , and relevance for the human pathology since in humans it is usually triggered by noise trauma . furthermore , salicylate does not induce chronic tinnitus as it recedes when the intake is stopped . these aspects hinder the identification of neuronal substrates involved in the pathogenesis and maintenance of human tinnitus by means of salicylate . one advantage of inducing tinnitus through acoustic trauma is the possibility to induce unilateral tinnitus , allowing the animal to serve potentially as its own control as done in some studies ( e.g. however , one has to keep in mind that the ascending auditory pathway is characterized by significant binaural projections on every stage . even if the tinnitus is perceived unilaterally , it is manifest in contra- and ipsilateral instances . another advantage of tinnitus induction by acoustic trauma is the fact that this is most likely the most common form observed in human patients . one of the biggest uncertainties when inducing tinnitus through an acoustic trauma is the resulting percentage of animals exhibiting tinnitus in behavioral tests . these numbers vary significantly in the literature , according to knipper et al . ultimately , the choice of how to induce tinnitus in a behavioral study depends on the research question and which form of tinnitus will be studied . diagnosis of subjective tinnitus in human patients relies almost exclusively on the self - report as there is no external sound source present and it manifests itself only in the neuronal activity of subject 's brain . there are some noninvasive approaches that provide potentially objective measures for subjective tinnitus by means of functional magnetic resonance tomography , electroencephalography , magnetoencephalography , and positron emission tomography . however , at present none of these methods is applied routinely for diagnosing tinnitus and it is unknown whether the observed effects are directly caused by tinnitus or by the emotional stress usually accompanying severe tinnitus . this challenge of diagnosing tinnitus poses a supreme obstacle for developing an animal model with behavioral evidence of tinnitus . nevertheless , a reliable behavioral assessment of tinnitus in the animal model is essential for understanding the pathology and the development of therapies . a continuous phantom percept like tinnitus hardly fits into such a framework of psychophysical experiments , as it is assumed to abolish the notion of silence . the conditioned stimulus ( cs ) was the offset of the background noise for 30 s. the behavioral readout was the ratio of licks during the cs compared to the number of licks in the period preceding the silent gap ( suppression ratio ) . next , animals were injected with salicylate in order to induce a phantom sound that was assumed to fill out the silent gap of the cs . the most important control of this study was a group of animals that received salicylate before the suppression training . during test sessions ( 5 days after acoustic trauma ) [ 6 , 7 ] the time course of the extinction of the response suppression during silent periods was indicative of the perceived phantom sound . however , the variability was quite big and there was a certain overlap in the distributions of performance scores of the control group and the one that received a trauma . since the indication for tinnitus is the time course of suppression extinction ( no foot shock ) , only short time spans ( days ) can be monitored and a more detailed analysis of the tinnitus over time is impossible . if the animals kept lever pressing in the silent periods , they were punished with a foot shock if they met or exceeded a certain criterion of the running index . in the testing sessions , pure tones of different frequencies and amplitudes were presented as well as silent gaps . lever pressing during pure tone presentation was not punished ; however , pressing during silent gaps was still punished . in order to still get useful behavioral responses , even before testing for tinnitus , this prolonged the time to extinction of the discriminative behavior between noise and silent gaps . testing took place immediately after tinnitus induction in order to characterize the immediate effects of salicylate . this paradigm has been used in a couple of follow - up studies , where the tinnitus was induced through an acoustic trauma , emphasizing its robustness and applicability to a wider range of tinnitus models [ 42 , 51 , 119 ] . this was interpreted as a result of a phantom sound perceived by the animals , as they were trained to go to the side where a stimulus was localized . in summary , the operant conditioning paradigms described here usually require a very careful and time - consuming training of the animals . the platform was in one of the two arms of the maze if a tone was presented and in the other arm , when no tone was presented . two measures were taken for quantifying the sound perception of the animal : time spend in one arm of the maze and percentage of correct choices . after 3 days of training the animals reached the correct arm in 80% of the cases within an average time of 4 s. after an acoustic trauma approximately half of the rats ( 12 out of 26 ) behaved as if they perceived in tone even when there was no sound present ( measured as an increased time spent in the arm associated with the tone ) . it is based on the acoustic startle reflex or response ( asr ) which is a very rapid contraction of skeletal muscles following the presentation of acoustic stimulus with high intensity . the central pathway for this startle response is well described and involves only three synapses . in animals experiencing tinnitus , the acoustic startle reflex this concept was first tested and published by turner et al . the animal 's response was measured as force applied to a piezo transducer in the floor of the chamber . half of the startle stimuli were preceded by a 50 ms gap in background noise which would reduce the startle amplitude in nave animals . hearing loss was ruled out as possible explanation for this effect as a simulated unilateral hearing loss ( ear plugs ) did not change the inhibition of the startle response by a preceding gap . however , a number of issues have to be taken into account when considering a gap startle paradigm for assessing tinnitus in animal models . first , it is unknown whether in human tinnitus patients gaps are filled with the phantom percept . in the light of transferability of results from the animal model to humans , this is a major drawback and has been only very recently addressed by fournier and hbert . despite some differences in the results compared to the study by turner et al . , gap deficits occurred at high- and low - frequency background noise in humans but not in the animal study ) this is evidence that the gap startle paradigm could be a valid model for studying tinnitus and that it measures manifestations of a phantom sound comparable to the one observed humans . however , one day after cortex ablation there were differences , indicating a temporary modulatory effect of auditory cortex on activity in the brain stem circuitry that gives rise to the startle response . thus , the role of auditory cortex in the gap startle paradigm still remains to be elucidated . it has been hypothesized that the neural substrate of tinnitus involves an increase in spontaneous activity , an increase in neuronal synchrony , and a reorganization of the tonotopic map in auditory cortex [ 105 , 120 ] . put forward the potential influence of hearing loss on the gap startle response and tackle this concern twofold in a dedicated study : first , by optimizing the startle stimulus so that it was outside the range of the hearing loss and second , by substituting the broad band noise startle stimulus with a rapid air puff to the animal 's back which can not be subject to hearing loss . in particular , the air puff approach preserved the startle response , even after conductive hearing loss . the ultimate benchmark for any animal model measuring subjective tinnitus is comparability to the human patient . any researcher starting to model tinnitus in laboratory animals has to make a decision regarding the species , the method of tinnitus induction , and the behavioral test . the most important criteria for choosing a certain species is its hearing range , its aptitude for behavioral studies and the availability of genetically modified strains . another advantage of the rat as an experimental model for studying the neuronal circuitry underlying tinnitus is the possibility to implant electrode arrays with high channel counts and perform chronic recordings in awake and behaving animals ( e.g. the disadvantage of the rat as a model is its high - frequency hearing range , which differs significantly from the human one . still , it remains unclear so far if these differences in hearing rage are significant for the pathogenesis , perception , and potential therapy of tinnitus . however , this last factor is certainly changing in the future as more and more recombinase - driver rat lines are developed ( e.g. on the other hand , an induction through salicylate has the advantage of fast onset of tinnitus and its reversibility . furthermore , salicylate can be applied locally which allows to study tinnitus - related changes at different stages of the auditory processing hierarchy . a behavioral test for laboratory animals should be shaped along these aspects , in particular the cortical involvement and extended testing period .

cl - transporting membrane proteins of the clc family fall into two distinctly different mechanistic subclasses : cl - selective ion channels and h - coupled secondary active cl transporters ( accardi and miller , 2004 ; jentsch et al . , 2005 ; picollo and pusch , 2005 ; scheel et al . , 2005 ) . the former catalyze transmembrane movement of small inorganic anions via passive electrodiffusion through an aqueous pore , while the latter mediate stoichiometric exchange of cl for h. of the nine clcs in the human genome , four ( clc-1 , -2 , -ka , and -kb ) are plasma membrane ion channels , and three ( clc-3 , -4 , and -5 ) are exchangers residing in intracellular membranes that bound acidified compartments ; the remaining two homologues , clc-6 and -7 , have not been characterized functionally , but are likely to be exchange transporters as well since they are found in acid - transporting intracellular membranes ( kida et al . , 2001 ; the structure of clc - ec1 , a clc homologue from escherichia coli , has been determined at high resolution by x - ray crystallography ( dutzler et al . , 2002 , 2003 ) . this protein belongs to the cl / h exchanger subclass ( accardi and miller , 2004 ) and participates in the extreme acid - resistance response that enables the organism to survive in the acid environment of the stomach ( iyer et al . , 2002 ) . the x - ray structure has proven useful in guiding electrophysiological studies of eukaryotic clc channels , for which no direct structural information exists , but its greatest potential utility lies in gaining understanding of the exchange transport mechanism . we know very little about the molecular details by which this protein brings about transmembrane cl movement coupled to h transport in the opposite direction , beyond an initial estimate of the exchange stoichiometry , 2 cl/1 h ( accardi and miller , 2004 ) . one key mechanistic insight is that an extracellular - facing glutamate residue , e148 , specifically mediates the transfer of h between external solution and protein interior . removal of this carboxylate group by mutation leads to complete loss of h movement while preserving cl permeation ( accardi et al . , 2004 ) . moreover , the strong stimulation of cl flux rates by low ph , a characteristic feature of the wild - type protein , is totally absent in e148 mutants , for which cl flux takes on maximal rates across the entire ph range of 37 ( iyer et al . , 2002 ; accardi et al . , thus , in the absence of a carboxyl group at this position , protons are no longer coupled to the transport of cl nor do they control the anion 's flux rate . substitution of neutral residues for the e148 equivalents in several eukaryotic clc channels abolishes extracellular ph dependence of gating and produces constitutively open channels ( dutzler et al . , 2003 ; estevez et al . , 2003 ; niemeyer et al . , 2003 ) , mimicking the opening of wild - type channels at low extracellular ph ( chen and chen , 2001 ) . these phenomena are readily comprehensible from the crystal structure of clc - ec1 ( dutzler et al . , 2003 ) . in wild - type protein , the e148 side chain lies close to a bound cl ion and blocks the anion 's access to the extracellular solution . in the e148q mutant , the side chain rotates away from the bound cl and thereby opens an extracellular aqueous pathway . for this reason , e148 is thought to act as a proton - linked gate in clc channels ; in the transporter subclass , such a movement provides an obvious means for linking h and cl binding at some point in the transport cycle . the existence of a specific extracellular proton transfer group suggests that a similar site might be present on the intracellular side of this protein . in this work we search for such a residue by mutating inward - facing glutamate and aspartate side chains in clc - ec1 , identifying e203 as a likely inward - facing proton transfer group clc - ec1 and variants were expressed in e. coli , purified , and crystallized after complexation with a fab fragment of antibody 10ec3/g4 ( national cell culture center ) , as previously described ( dutzler et al . , 2003 ) . crystallographic datasets were collected at the swiss light source or the advanced photon source , using radiation at the br absorption edge ( 0.919 ) . diffraction images were indexed and integrated in the hkl program , and electron density and anomalous difference maps were calculated in the ccp4 suite , after molecular replacement with phaser and refinement with refmac5 , using the wild - type clc - ec1-fab model ( accession no . the refined model was also minimally rebuilt using sigmaa - weighted 2fo - fc and fo - fc maps in the coot program . model bias was suppressed by prime - and - switch density modification in the solve / resolve program within the phenix suite ( terwilliger , 2000 ) . clc - ec1 ion transport functions were assayed by cl and h fluxes in liposomes and voltage - clamped currents in planar lipid bilayers as previously described ( accardi et al . , 2004 ; accardi and miller , 2004 ) ; in preparations used for lipid bilayer experiments , an anion - exchange chromatography step was used in lieu of gel filtration to remove outer membrane porins ( accardi and miller , 2004 ) . we set out to search for an intracellular proton transfer residue by mutagenesis of clc - ec1 , seeking a dissociable side chain whose neutralization would specifically abolish h transport coupled to cl movement . to make this task tractable in a protein containing 55 protonatable side chains ( 16 glutamates , 11 aspartates , 6 histidines , 13 lysines , and 9 tyrosines ) this transporter operates physiologically under acid conditions , < ph 3.5 extracellular , < ph 5.0 intracellular ( iyer et al . , 2002 ; foster , 2004 ; richard and foster , 2004 ) , and in vitro attains maximal rates < ph 4.5 ( iyer et al . , 2002 ) , and so low - pka carboxylate residues are likely to be involved in the h transport mechanism . most of the glutamates and aspartates of clc - ec1 are found in two loci , intracellular and extracellular ( fig . 1 ) , and we accordingly focus mainly on the former set of residues . with these constraints , the initial search reduces to nine candidates , seven glutamates and two aspartates , which we substitute with the isosteric analogues of the protonated side chains , glutamine and asparagine , respectively . the positions of all carboxyl - bearing residues in a single subunit of the homodimeric protein are shown in fig . 1 , accompanied by a clc alignment in a strongly conserved region commonly known as the pigg - pen . the alignment reveals a glutamate residue , e203 in clc - ec1 , that is uniformly present in the cl / h exchangers but absent in the cl channels , where valine takes its place . this residue is located in an intracellular - facing cluster of glutamates that computations ( yin et al . , 2004 ) have suggested could plausibly contribute to an electronegative h pathway through the protein . ( a ) ribbon representation of the clc - ec1 dimer , view from within the membrane , with extracellular side up . carbons of the carboxylate residues mutated here are shown for a single subunit as yellow spheres , except for e148 and e203 ( red ) , and e113 ( black ) ; gray spheres mark carboxyl residues that are not studied here , and green spheres designate cl ions . also shown ( b ) sequence alignment of the pigg - pen region , with e203 equivalent indicated in red . genes listed are of human origin except for clc-0 ( torpedo marmorata ) , clc - ec1 ( e. coli ) , clc - st1 ( salmonella typhimurium ) , and gef1 ( saccharomyces cerevisiae ) . the obvious suggestion leaping out of the sequence alignment is difficult to resist . consequently , we first examined the ion transport behavior of the mutant e203q , which resembles an always - protonated glutamate side chain . the results meet expectations : removing the carboxylate group at e203 completely abolishes h coupling to cl transport , as can be seen from several functional indicators . 2 a illustrates e203q currents across lipid bilayers separating symmetrical cl solutions , with or without a ph gradient . with wild - type protein , imposition of a 4-unit ph gradient across the bilayer produces a large shift in reversal potential ( 62 mv , fig . 2 b ) , a direct manifestation of h transport ( accardi and miller , 2004 ) ; in contrast , e203q shows no ph - induced shift whatsoever ( fig . if this loss in h transport reflects specific abolition of h coupling rather than merely protein damage , a gain in cl selectivity should appear in the mutant , since the altered clc would catalyze permeation of only a single ionic species , cl . 2 c , where reversal potentials are plotted against cl gradient at symmetrical ph . for both wild - type and e203q , reversal potential varies logarithmically with cl gradient , and the mutation raises the subnernstian slope observed for wild type ( accardi and miller , 2004 ) close to the ideally cl - selective value ( 53 mv / decade ) . this result directly demonstrates that the mutant protein is competent in cl transport but has lost its ability to move h. loss of h coupling in e203q . ( a ) currents mediated by e203q in symmetrical 300 mm cl , with or without a 4-unit ph gradient . a 3-s voltage pulse ( 100 to + 100 mv in 10-mv steps ) was applied from a holding potential of 0 mv , followed by a 1-s tail pulse to 100 mv . dashed line marks zero current . ( b ) i - v curves , normalized to the current in the same bilayer at 100 mv in symmetrical ph 3.0 and 300 mm cl . open circles , wild type , ph 3.0/ph 7 ; black circles , e203 ph 3/ph 3 ; red triangles , e203 ph 3.0/ph 7 ; green squares , e203 ph 7/ph 3 ; yellow triangles , gradients are indicated by convention cis / trans sides of planar bilayer , with the trans side defined as zero voltage . ( c ) reversal potential , vrev , as a function of [ cl ] for wild type ( black circles ) and e203q ( red triangles ) ; dashed line is nernst potential predicted for ideal cl selectivity . an additional indication of the loss of h coupling can be seen in a direct proton - pumping assay . here , liposomes reconstituted with clc protein and loaded with high cl are exposed to low external cl ; in a coupled exchanger , cl flowing outward down its imposed gradient drives h uphill into the liposomes , as is readily seen with wild - type protein ( fig . 3 ) . in the same assay , however , e203q produces no observable proton flux , while still fully competent to mediate cl uptake ( fig . it is notable that cl influx time courses at ph 4.5 are similar for mutant and wild - type protein , a result implying that the maximal cl turnover rate is not greatly altered by the mutation . these experiments demonstrate that e203q conducts cl but has completely lost coupling of anion movement to h. this behavior is similar to that displayed upon mutation of the extracellular h transfer residue e148 ( accardi et al . , 2004 ) . we also note that substitution by aspartate here does not undermine the transport mechanism , since e203d supports h coupling ( fig . 4 ) as well as cl - driven proton pumping ( not depicted ) ; this result is somewhat surprising in light of the conservation of glutamate at this position in the exchange transporter subclass ( fig fluxes of cl and h. liposomes reconstituted with clc - ec1 ( 15 g / mg lipid ) were assayed for either cl uptake or cl - driven h pumping . top panels , cl uptake at ph 7.0 ( circles ) and ph 4.5 ( triangles ) for wt ( a ) , e203q ( b ) , and e148a / e203q ( c ) . lower traces show h flux assays , upward deflection indicating increase in ph of the liposome suspension . additions of 1 m valinomycin or fccp are denoted by * and + , respectively . reversal potentials of indicated mutants in presence of ( a ) a cl gradient ( 300 mm/45 mm ) at symmetrical ph 3.0 or ( b ) a ph gradient ( 3/7 ) at symmetrical 300 mm cl . colored bars indicate mutants with h transport inhibited completely ( red ) or partially ( blue ) . since neutralization of either e148 or e203 abolishes h transport , these two residues appear to have identical functions . however , this is not strictly true . while mutation of e148 leads to complete loss of the ph dependence of cl transport rates ( accardi et al . , 2004 ) , neutralization of e203 merely weakens it , as shown by both fluxes and currents ( fig . 2 b and fig . for example , while wild type and e203q have similar influx time courses at ph 4.5 , the mutant influx is well over 10-fold faster than wild type at ph 7 . thus , if e148 is neutralized , cl flux rates become insensitive to the protonation state of e203 ; in contrast , if e203 is neutralized , cl flux rates still respond to ph , presumably through protonation of e148 , which appears to be an absolute requirement for cl transport . to verify that this residual ph dependence arises from e148 in the e203q background , we examined the double mutant e148a / e203q , and found that it displays ph - independent cl uptake and no active h transport ( figs . 3 and 4 ) , as expected . to assess whether the loss of h transport upon mutation is specific to e203 , we performed similar experiments at the seven other carboxylates indicated in fig . 1 a. for each mutant ( except for e113q , which failed to express protein ) , proton coupling was gauged electrically , using reversal potential shifts induced by ph or cl gradients . h coupling is similar to wild type except with e202q and d278n , which display distinctly lower coupling efficiency , as indicated by the smaller shifts in reversal potential induced by a ph gradient and the greater shift in a cl gradient . the former , which is strictly conserved , has been proposed to electrostatically influence the two cl ions bound to wild - type clc - ec1 ( faraldo - gomez and roux , 2004 ) , while the latter forms an intersubunit salt bridge with e203 . however , r28l shows fully wild - type transport behavior , while k131 m still mediates robust proton transport , though with slightly altered characteristics . thus , e203 is unique among all residues tested in leading to complete loss of proton transport . we crystallized e203q and determined its structure at 3.3 resolution by molecular replacement ( fig . the mutant crystallized isomorphically to wild type with a virtually identical structure ( backbone rmsd = 0.4 ) except close to the site of mutation . in wild - type protein , e203 forms a salt bridge with r28 from the other subunit of the homodimer and an h bond with e113 in the same subunit . in the mutant structure , the q203 side chain occupies the same position as e203 in the wild type , but r28 lacks side chain density and so is most likely disordered . this result raises the intriguing possibility that crosstalk between the two subunits is involved in the proton - coupling mechanism ; however , the wild - type behavior shown by r28l ( fig . 4 ) eliminates this cross - subunit salt bridge as a necessary element of the transport cycle . ( a ) wild type ; ( b ) e203q . top panel , 2fo - fc electron density maps ( blue ) contoured at 1.2 , with protein model shown in stick representation . wild - type map was calculated to 3.3 resolution from deposited structure factors ( accession no . bottom panel , anomalous difference density maps ( green ) of crystals grown in nabr , determined from data collected at the br absorption edge , contoured at 5 , with the same wild - type protein model ( 1ots ) shown for both maps , with subunit a in red and subunit b in blue . crystallographic statistics crystals of clc - ec1-fab complex were formed in 3-l sitting drops , in the presence of 100250 mm nacl or nabr . all crystals were of spacegroup c2 with cell dimensions similar to those of the wild - type complex ( 1ots ) . crystals grown in br , a functionally faithful cl substitute ( accardi et al . , 2004 ) , are particularly advantageous for studying the anion - binding properties of clc - ec1 , since this halide can be visualized by anomalous diffraction , as has been done for wild - type protein and several mutants ( dutzler et al . , 2003 ; lobet and dutzler , 2005 ) . when crystallized from nabr solutions , wild - type protein shows two such sites , both prominently occupied , and br density shows up identically in the e203q mutant ( fig . thus , the crystal structures indicate that this mutant with profoundly altered function adopts an unperturbed conformation with normal anion - binding characteristics . consequently , we first examined the ion transport behavior of the mutant e203q , which resembles an always - protonated glutamate side chain . the results meet expectations : removing the carboxylate group at e203 completely abolishes h coupling to cl transport , as can be seen from several functional indicators . fig . 2 a illustrates e203q currents across lipid bilayers separating symmetrical cl solutions , with or without a ph gradient . with wild - type protein , imposition of a 4-unit ph gradient across the bilayer produces a large shift in reversal potential ( 62 mv , fig . 2 b ) , a direct manifestation of h transport ( accardi and miller , 2004 ) ; in contrast , e203q shows no ph - induced shift whatsoever ( fig . if this loss in h transport reflects specific abolition of h coupling rather than merely protein damage , a gain in cl selectivity should appear in the mutant , since the altered clc would catalyze permeation of only a single ionic species , cl . 2 c , where reversal potentials are plotted against cl gradient at symmetrical ph . for both wild - type and e203q , reversal potential varies logarithmically with cl gradient , and the mutation raises the subnernstian slope observed for wild type ( accardi and miller , 2004 ) close to the ideally cl - selective value ( 53 mv / decade ) . this result directly demonstrates that the mutant protein is competent in cl transport but has lost its ability to move h. loss of h coupling in e203q . ( a ) currents mediated by e203q in symmetrical 300 mm cl , with or without a 4-unit ph gradient . a 3-s voltage pulse ( 100 to + 100 mv in 10-mv steps ) was applied from a holding potential of 0 mv , followed by a 1-s tail pulse to 100 mv . ( b ) i - v curves , normalized to the current in the same bilayer at 100 mv in symmetrical ph 3.0 and 300 mm cl . open circles , wild type , ph 3.0/ph 7 ; black circles , e203 ph 3/ph 3 ; red triangles , e203 ph 3.0/ph 7 ; green squares , e203 ph 7/ph 3 ; yellow triangles , e203 ph 7.0/ph 7 . gradients are indicated by convention cis / trans sides of planar bilayer , with the trans side defined as zero voltage . ( c ) reversal potential , vrev , as a function of [ cl ] for wild type ( black circles ) and e203q ( red triangles ) ; dashed line is nernst potential predicted for ideal cl selectivity . an additional indication of the loss of h coupling can be seen in a direct proton - pumping assay . here , liposomes reconstituted with clc protein and loaded with high cl are exposed to low external cl ; in a coupled exchanger , cl flowing outward down its imposed gradient drives h uphill into the liposomes , as is readily seen with wild - type protein ( fig . 3 ) . in the same assay , however , e203q produces no observable proton flux , while still fully competent to mediate cl uptake ( fig . it is notable that cl influx time courses at ph 4.5 are similar for mutant and wild - type protein , a result implying that the maximal cl turnover rate is not greatly altered by the mutation . these experiments demonstrate that e203q conducts cl but has completely lost coupling of anion movement to h. this behavior is similar to that displayed upon mutation of the extracellular h transfer residue e148 ( accardi et al . , 2004 ) . we also note that substitution by aspartate here does not undermine the transport mechanism , since e203d supports h coupling ( fig . 4 ) as well as cl - driven proton pumping ( not depicted ) ; this result is somewhat surprising in light of the conservation of glutamate at this position in the exchange transporter subclass ( fig fluxes of cl and h. liposomes reconstituted with clc - ec1 ( 15 g / mg lipid ) were assayed for either cl uptake or cl - driven h pumping . top panels , cl uptake at ph 7.0 ( circles ) and ph 4.5 ( triangles ) for wt ( a ) , e203q ( b ) , and e148a / e203q ( c ) . lower traces show h flux assays , upward deflection indicating increase in ph of the liposome suspension . additions of 1 m valinomycin or fccp are denoted by * and + , respectively . reversal potentials of indicated mutants in presence of ( a ) a cl gradient ( 300 mm/45 mm ) at symmetrical ph 3.0 or ( b ) a ph gradient ( 3/7 ) at symmetrical 300 mm cl . dotted lines represent wild - type values under the same conditions . data for wild type and e148a taken from accardi et al . colored bars indicate mutants with h transport inhibited completely ( red ) or partially ( blue ) . since neutralization of either e148 or e203 abolishes h transport , these two residues appear to have identical functions . however , this is not strictly true . while mutation of e148 leads to complete loss of the ph dependence of cl transport rates ( accardi et al . , 2004 ) , neutralization of e203 merely weakens it , as shown by both fluxes and currents ( fig for example , while wild type and e203q have similar influx time courses at ph 4.5 , the mutant influx is well over 10-fold faster than wild type at ph 7 . thus , if e148 is neutralized , cl flux rates become insensitive to the protonation state of e203 ; in contrast , if e203 is neutralized , cl flux rates still respond to ph , presumably through protonation of e148 , which appears to be an absolute requirement for cl transport . to verify that this residual ph dependence arises from e148 in the e203q background , we examined the double mutant e148a / e203q , and found that it displays ph - independent cl uptake and no active h transport ( figs . 3 and 4 ) , as expected . to assess whether the loss of h transport upon mutation is specific to e203 , we performed similar experiments at the seven other carboxylates indicated in fig . 1 a. for each mutant ( except for e113q , which failed to express protein ) , proton coupling was gauged electrically , using reversal potential shifts induced by ph or cl gradients . h coupling is similar to wild type except with e202q and d278n , which display distinctly lower coupling efficiency , as indicated by the smaller shifts in reversal potential induced by a ph gradient and the greater shift in a cl gradient . the former , which is strictly conserved , has been proposed to electrostatically influence the two cl ions bound to wild - type clc - ec1 ( faraldo - gomez and roux , 2004 ) , while the latter forms an intersubunit salt bridge with e203 . however , r28l shows fully wild - type transport behavior , while k131 m still mediates robust proton transport , though with slightly altered characteristics . thus , e203 is unique among all residues tested in leading to complete loss of proton transport . we crystallized e203q and determined its structure at 3.3 resolution by molecular replacement ( fig . 5 ; table i ) . the mutant crystallized isomorphically to wild type with a virtually identical structure ( backbone rmsd = 0.4 ) except close to the site of mutation . in wild - type protein , e203 forms a salt bridge with r28 from the other subunit of the homodimer and an h bond with e113 in the same subunit . in the mutant structure , the q203 side chain occupies the same position as e203 in the wild type , but r28 lacks side chain density and so is most likely disordered . this result raises the intriguing possibility that crosstalk between the two subunits is involved in the proton - coupling mechanism ; however , the wild - type behavior shown by r28l ( fig . 4 ) eliminates this cross - subunit salt bridge as a necessary element of the transport cycle . crystal structure and halide binding . ( a ) wild type ; ( b ) e203q . top panel , 2fo - fc electron density maps ( blue ) contoured at 1.2 , with protein model shown in stick representation . wild - type map was calculated to 3.3 resolution from deposited structure factors ( accession no . bottom panel , anomalous difference density maps ( green ) of crystals grown in nabr , determined from data collected at the br absorption edge , contoured at 5 , with the same wild - type protein model ( 1ots ) shown for both maps , with subunit a in red and subunit b in blue . crystallographic statistics crystals of clc - ec1-fab complex were formed in 3-l sitting drops , in the presence of 100250 mm nacl or nabr . all crystals were of spacegroup c2 with cell dimensions similar to those of the wild - type complex ( 1ots ) . crystals grown in br , a functionally faithful cl substitute ( accardi et al . , 2004 ) , are particularly advantageous for studying the anion - binding properties of clc - ec1 , since this halide can be visualized by anomalous diffraction , as has been done for wild - type protein and several mutants ( dutzler et al . , 2003 ; lobet and dutzler , 2005 ) . when crystallized from nabr solutions , wild - type protein shows two such sites , both prominently occupied , and br density shows up identically in the e203q mutant ( fig . 5 ) . thus , the crystal structures indicate that this mutant with profoundly altered function adopts an unperturbed conformation with normal anion - binding characteristics . all exchange transport mechanisms involve protein conformational changes linked to sided binding and release of substrates ( maloney , 1994 ) . for clc cl / h exchangers , x - ray structures of clc - ec1 tell us where the cl ions bind ( fig . , the structures also suggest a plausible mechanism for concerted movement of cl and h linked to a small conformational change in one part of the transport cycle : simple rotation of the e148 side chain that alters access of both h and cl to the protein interior from the extracellular solution ( dutzler et al . , 2003 ; accardi and miller , 2004 ) . for these reasons , e148 is well established as serving a dual role at the extracellular face of the protein : handing off protons to , and allowing concomitant entry of cl ions from , the external solution . ( this coupled reaction is of course reversible and must operate in both directions . ) since e148 faces the extracellular solution , we supposed that an analogous proton transfer site might exist near the intracellular surface of the protein . indeed , in a conventional exchange mechanism , a single protonatable locus alternates its exposure to the two sides of the membrane through a cycle of conformational changes , as proposed in detail for the lactose permease ( kaback , 2005 ) . but the structure of clc - ec1 , in contrast to that of lactose permease , does not suggest any obvious conformational changes that would alternate exposure of e148 . for this reason , we conjectured that protons may somehow move through the protein during transport . such a picture envisions a proton pathway connecting distant residues on each side of the membrane , each mediating proton transfer between the protein and its corresponding aqueous solution , as in the well - characterized proton transfer residues in the bacterial reaction center , for example ( delroth and brzezinski , 2004 ) . our results do not nail down this conclusion rigorously , but they do suggest it strongly via several lines of argument . first , neutralization of e203 completely and specifically abolishes all proton transport , as expected if the carboxyl group were required for shuttling protons to and from the protein interior , a prerequisite for participation in the coupled - transport cycle . second , e203 is partially buried near the intracellular solution , as is required for this proposed function . third , e203 is unique among all groups tested in being absolutely necessary for proton coupling ; while mutations at e202 and at d278 lower coupling , mutation at e203 alone fully ablates it . fourth , the only other residue that behaves this way is the externally facing e148 , for which a proton transfer function is strongly supported by previous work . finally , the loss of h transport in e203q can not be attributed to mutagenic disruption , since the structure of this mutant is similar to wild type and specific cl transport is maintained . these considerations support the idea that e203 directly catalyzes proton transfer between protein and intracellular solution as a required step in the coupled transport cycle . it argues that the protein contains a pathway along which protons move between e148 on the extracellular side and e203 on the intracellular . proton movement along this pathway must be somehow coupled to occupancy of the cl binding sites , which themselves imply a pathway for anions . discovering the still - unknown rules of this coupling is required for understanding the transport mechanism ( jencks , 1980 ) . we do not yet know how protonation of e203 influences the cl sites , but it is intriguing that computational studies ( faraldo - gomez and roux , 2004 ) suggest a strong electrostatic influence of the protonation state of e203 ( as well as of other nearby residues ) on the affinity of cl binding to the protein . a second inference is that this ion transport pathway is bifurcated , as illustrated in fig . the structure of e148q , which mimics the external open form with e148 protonated , shows an additional cl ion occupying the location vacated by the q148 side chain ( dutzler et al . , 2003 ) . thus , on the external side of the protein , the cl and h pathways overlap ; but they diverge on the intracellular side , since e203 is distant from the internal cl site . a future challenge will be to explore the terra incognita between the two aqueous transfer sites , where h must bind as it traverses the membrane , where the central cl ion is coordinated by hydroxyls of s107 and y445 , and where the two pathways appear to separate . protonation of e203 is a necessary step only for h transport , while protonation of e148 is required for both cl and h movements . clc dimer , with the following side chains highlighted : e148 , e203 ( red ) ; s107 , y445 ( blue ) . the region between e148 and e203 , which the h must traverse , is indicated by the gray box , with side chains of s107 and y445 indicated in blue . as a final point , we should note an odd feature of the cl / h exchange mechanism : that abolition of h transport does not impede cl permeation . extensively studied exchange transporters , mfs exchangers ( ruan et al . 2001 ) , cl / hco3 exchangers ( knauf et al . , 2002 ) , na / ca exchangers ( dong et al . , 2002 ; kang and hilgemann , 2004 ) , mitochondrial nucleotide exchangers ( pebay - peyroula et al . , 2003 ) , display obligatory exchange , whereby removal of either substrate completely inhibits net movement of the other . mutation of either proton transfer residue , e148 or e203 , abolishes h transport but leaves cl transport largely unaffected . our assays are not sufficiently quantitative to tell us precisely how much the unitary rate of cl flux is affected in these mutants , but the overall transport behavior suggests that any effect on cl turnover can not be very large . for this reason , we consider that the mechanism of clc transporters will turn out to be novel , unconventional , and eerily reminiscent of ion channel permeation , and fundamentally unlike the alternating - site schemes that have so productively guided our understanding of long - studied and more familiar classes of transport proteins .

clc - ec1 is a prokaryotic clc - type cl/h+ exchange transporter . little is known about the mechanism of h+ coupling to cl. a critical glutamate residue , e148 , was previously shown to be required for cl/h+ exchange by mediating proton transfer between the protein and the extracellular solution . to test whether an analogous h+ acceptor exists near the intracellular side of the protein , we performed a mutagenesis scan of inward - facing carboxyl - bearing residues and identified e203 as the unique residue whose neutralization abolishes h+ coupling to cl transport . glutamate at this position is strictly conserved in all known clcs of the transporter subclass , while valine is always found here in clc channels . the x - ray crystal structure of the e203q mutant is similar to that of the wild - type protein . cl transport rate in e203q is inhibited at neutral ph , and the double mutant , e148a / e203q , shows maximal cl transport , independent of ph , as does the single mutant e148a . the results argue that substrate exchange by clc - ec1 involves two separate but partially overlapping permeation pathways , one for cl and one for h+ . these pathways are congruent from the protein 's extracellular surface to e148 , and they diverge beyond this point toward the intracellular side . this picture demands a transport mechanism fundamentally different from familiar alternating - access schemes .

INTRODUCTION MATERIALS AND METHODS RESULTS E203 Mediates H pH Dependence of E203Q E203 Is Unique among Internally Facing Carboxylates Structure and Halide Binding of E203Q DISCUSSION

the former catalyze transmembrane movement of small inorganic anions via passive electrodiffusion through an aqueous pore , while the latter mediate stoichiometric exchange of cl for h. of the nine clcs in the human genome , four ( clc-1 , -2 , -ka , and -kb ) are plasma membrane ion channels , and three ( clc-3 , -4 , and -5 ) are exchangers residing in intracellular membranes that bound acidified compartments ; the remaining two homologues , clc-6 and -7 , have not been characterized functionally , but are likely to be exchange transporters as well since they are found in acid - transporting intracellular membranes ( kida et al . , 2001 ; the structure of clc - ec1 , a clc homologue from escherichia coli , has been determined at high resolution by x - ray crystallography ( dutzler et al . the x - ray structure has proven useful in guiding electrophysiological studies of eukaryotic clc channels , for which no direct structural information exists , but its greatest potential utility lies in gaining understanding of the exchange transport mechanism . we know very little about the molecular details by which this protein brings about transmembrane cl movement coupled to h transport in the opposite direction , beyond an initial estimate of the exchange stoichiometry , 2 cl/1 h ( accardi and miller , 2004 ) . one key mechanistic insight is that an extracellular - facing glutamate residue , e148 , specifically mediates the transfer of h between external solution and protein interior . moreover , the strong stimulation of cl flux rates by low ph , a characteristic feature of the wild - type protein , is totally absent in e148 mutants , for which cl flux takes on maximal rates across the entire ph range of 37 ( iyer et al . , thus , in the absence of a carboxyl group at this position , protons are no longer coupled to the transport of cl nor do they control the anion 's flux rate . , 2003 ) , mimicking the opening of wild - type channels at low extracellular ph ( chen and chen , 2001 ) . these phenomena are readily comprehensible from the crystal structure of clc - ec1 ( dutzler et al . in wild - type protein , the e148 side chain lies close to a bound cl ion and blocks the anion 's access to the extracellular solution . in the e148q mutant , the side chain rotates away from the bound cl and thereby opens an extracellular aqueous pathway . for this reason , e148 is thought to act as a proton - linked gate in clc channels ; in the transporter subclass , such a movement provides an obvious means for linking h and cl binding at some point in the transport cycle . the existence of a specific extracellular proton transfer group suggests that a similar site might be present on the intracellular side of this protein . in this work we search for such a residue by mutating inward - facing glutamate and aspartate side chains in clc - ec1 , identifying e203 as a likely inward - facing proton transfer group clc - ec1 and variants were expressed in e. coli , purified , and crystallized after complexation with a fab fragment of antibody 10ec3/g4 ( national cell culture center ) , as previously described ( dutzler et al . diffraction images were indexed and integrated in the hkl program , and electron density and anomalous difference maps were calculated in the ccp4 suite , after molecular replacement with phaser and refinement with refmac5 , using the wild - type clc - ec1-fab model ( accession no . clc - ec1 ion transport functions were assayed by cl and h fluxes in liposomes and voltage - clamped currents in planar lipid bilayers as previously described ( accardi et al . we set out to search for an intracellular proton transfer residue by mutagenesis of clc - ec1 , seeking a dissociable side chain whose neutralization would specifically abolish h transport coupled to cl movement . , 2002 ) , and so low - pka carboxylate residues are likely to be involved in the h transport mechanism . most of the glutamates and aspartates of clc - ec1 are found in two loci , intracellular and extracellular ( fig . the positions of all carboxyl - bearing residues in a single subunit of the homodimeric protein are shown in fig . the alignment reveals a glutamate residue , e203 in clc - ec1 , that is uniformly present in the cl / h exchangers but absent in the cl channels , where valine takes its place . ( a ) ribbon representation of the clc - ec1 dimer , view from within the membrane , with extracellular side up . genes listed are of human origin except for clc-0 ( torpedo marmorata ) , clc - ec1 ( e. coli ) , clc - st1 ( salmonella typhimurium ) , and gef1 ( saccharomyces cerevisiae ) . consequently , we first examined the ion transport behavior of the mutant e203q , which resembles an always - protonated glutamate side chain . the results meet expectations : removing the carboxylate group at e203 completely abolishes h coupling to cl transport , as can be seen from several functional indicators . with wild - type protein , imposition of a 4-unit ph gradient across the bilayer produces a large shift in reversal potential ( 62 mv , fig . for both wild - type and e203q , reversal potential varies logarithmically with cl gradient , and the mutation raises the subnernstian slope observed for wild type ( accardi and miller , 2004 ) close to the ideally cl - selective value ( 53 mv / decade ) . this result directly demonstrates that the mutant protein is competent in cl transport but has lost its ability to move h. loss of h coupling in e203q . here , liposomes reconstituted with clc protein and loaded with high cl are exposed to low external cl ; in a coupled exchanger , cl flowing outward down its imposed gradient drives h uphill into the liposomes , as is readily seen with wild - type protein ( fig . it is notable that cl influx time courses at ph 4.5 are similar for mutant and wild - type protein , a result implying that the maximal cl turnover rate is not greatly altered by the mutation . these experiments demonstrate that e203q conducts cl but has completely lost coupling of anion movement to h. this behavior is similar to that displayed upon mutation of the extracellular h transfer residue e148 ( accardi et al . 4 ) as well as cl - driven proton pumping ( not depicted ) ; this result is somewhat surprising in light of the conservation of glutamate at this position in the exchange transporter subclass ( fig fluxes of cl and h. liposomes reconstituted with clc - ec1 ( 15 g / mg lipid ) were assayed for either cl uptake or cl - driven h pumping . top panels , cl uptake at ph 7.0 ( circles ) and ph 4.5 ( triangles ) for wt ( a ) , e203q ( b ) , and e148a / e203q ( c ) . while mutation of e148 leads to complete loss of the ph dependence of cl transport rates ( accardi et al . thus , if e148 is neutralized , cl flux rates become insensitive to the protonation state of e203 ; in contrast , if e203 is neutralized , cl flux rates still respond to ph , presumably through protonation of e148 , which appears to be an absolute requirement for cl transport . to verify that this residual ph dependence arises from e148 in the e203q background , we examined the double mutant e148a / e203q , and found that it displays ph - independent cl uptake and no active h transport ( figs . to assess whether the loss of h transport upon mutation is specific to e203 , we performed similar experiments at the seven other carboxylates indicated in fig . h coupling is similar to wild type except with e202q and d278n , which display distinctly lower coupling efficiency , as indicated by the smaller shifts in reversal potential induced by a ph gradient and the greater shift in a cl gradient . the former , which is strictly conserved , has been proposed to electrostatically influence the two cl ions bound to wild - type clc - ec1 ( faraldo - gomez and roux , 2004 ) , while the latter forms an intersubunit salt bridge with e203 . however , r28l shows fully wild - type transport behavior , while k131 m still mediates robust proton transport , though with slightly altered characteristics . in wild - type protein , e203 forms a salt bridge with r28 from the other subunit of the homodimer and an h bond with e113 in the same subunit . this result raises the intriguing possibility that crosstalk between the two subunits is involved in the proton - coupling mechanism ; however , the wild - type behavior shown by r28l ( fig . bottom panel , anomalous difference density maps ( green ) of crystals grown in nabr , determined from data collected at the br absorption edge , contoured at 5 , with the same wild - type protein model ( 1ots ) shown for both maps , with subunit a in red and subunit b in blue . all crystals were of spacegroup c2 with cell dimensions similar to those of the wild - type complex ( 1ots ) . , 2004 ) , are particularly advantageous for studying the anion - binding properties of clc - ec1 , since this halide can be visualized by anomalous diffraction , as has been done for wild - type protein and several mutants ( dutzler et al . when crystallized from nabr solutions , wild - type protein shows two such sites , both prominently occupied , and br density shows up identically in the e203q mutant ( fig . consequently , we first examined the ion transport behavior of the mutant e203q , which resembles an always - protonated glutamate side chain . the results meet expectations : removing the carboxylate group at e203 completely abolishes h coupling to cl transport , as can be seen from several functional indicators . with wild - type protein , imposition of a 4-unit ph gradient across the bilayer produces a large shift in reversal potential ( 62 mv , fig . for both wild - type and e203q , reversal potential varies logarithmically with cl gradient , and the mutation raises the subnernstian slope observed for wild type ( accardi and miller , 2004 ) close to the ideally cl - selective value ( 53 mv / decade ) . this result directly demonstrates that the mutant protein is competent in cl transport but has lost its ability to move h. loss of h coupling in e203q . here , liposomes reconstituted with clc protein and loaded with high cl are exposed to low external cl ; in a coupled exchanger , cl flowing outward down its imposed gradient drives h uphill into the liposomes , as is readily seen with wild - type protein ( fig . it is notable that cl influx time courses at ph 4.5 are similar for mutant and wild - type protein , a result implying that the maximal cl turnover rate is not greatly altered by the mutation . these experiments demonstrate that e203q conducts cl but has completely lost coupling of anion movement to h. this behavior is similar to that displayed upon mutation of the extracellular h transfer residue e148 ( accardi et al . 4 ) as well as cl - driven proton pumping ( not depicted ) ; this result is somewhat surprising in light of the conservation of glutamate at this position in the exchange transporter subclass ( fig fluxes of cl and h. liposomes reconstituted with clc - ec1 ( 15 g / mg lipid ) were assayed for either cl uptake or cl - driven h pumping . top panels , cl uptake at ph 7.0 ( circles ) and ph 4.5 ( triangles ) for wt ( a ) , e203q ( b ) , and e148a / e203q ( c ) . dotted lines represent wild - type values under the same conditions . while mutation of e148 leads to complete loss of the ph dependence of cl transport rates ( accardi et al . , 2004 ) , neutralization of e203 merely weakens it , as shown by both fluxes and currents ( fig for example , while wild type and e203q have similar influx time courses at ph 4.5 , the mutant influx is well over 10-fold faster than wild type at ph 7 . thus , if e148 is neutralized , cl flux rates become insensitive to the protonation state of e203 ; in contrast , if e203 is neutralized , cl flux rates still respond to ph , presumably through protonation of e148 , which appears to be an absolute requirement for cl transport . to verify that this residual ph dependence arises from e148 in the e203q background , we examined the double mutant e148a / e203q , and found that it displays ph - independent cl uptake and no active h transport ( figs . to assess whether the loss of h transport upon mutation is specific to e203 , we performed similar experiments at the seven other carboxylates indicated in fig . h coupling is similar to wild type except with e202q and d278n , which display distinctly lower coupling efficiency , as indicated by the smaller shifts in reversal potential induced by a ph gradient and the greater shift in a cl gradient . the former , which is strictly conserved , has been proposed to electrostatically influence the two cl ions bound to wild - type clc - ec1 ( faraldo - gomez and roux , 2004 ) , while the latter forms an intersubunit salt bridge with e203 . however , r28l shows fully wild - type transport behavior , while k131 m still mediates robust proton transport , though with slightly altered characteristics . in wild - type protein , e203 forms a salt bridge with r28 from the other subunit of the homodimer and an h bond with e113 in the same subunit . this result raises the intriguing possibility that crosstalk between the two subunits is involved in the proton - coupling mechanism ; however , the wild - type behavior shown by r28l ( fig . wild - type map was calculated to 3.3 resolution from deposited structure factors ( accession no . bottom panel , anomalous difference density maps ( green ) of crystals grown in nabr , determined from data collected at the br absorption edge , contoured at 5 , with the same wild - type protein model ( 1ots ) shown for both maps , with subunit a in red and subunit b in blue . all crystals were of spacegroup c2 with cell dimensions similar to those of the wild - type complex ( 1ots ) . , 2004 ) , are particularly advantageous for studying the anion - binding properties of clc - ec1 , since this halide can be visualized by anomalous diffraction , as has been done for wild - type protein and several mutants ( dutzler et al . when crystallized from nabr solutions , wild - type protein shows two such sites , both prominently occupied , and br density shows up identically in the e203q mutant ( fig . for clc cl / h exchangers , x - ray structures of clc - ec1 tell us where the cl ions bind ( fig . , the structures also suggest a plausible mechanism for concerted movement of cl and h linked to a small conformational change in one part of the transport cycle : simple rotation of the e148 side chain that alters access of both h and cl to the protein interior from the extracellular solution ( dutzler et al . for these reasons , e148 is well established as serving a dual role at the extracellular face of the protein : handing off protons to , and allowing concomitant entry of cl ions from , the external solution . since e148 faces the extracellular solution , we supposed that an analogous proton transfer site might exist near the intracellular surface of the protein . indeed , in a conventional exchange mechanism , a single protonatable locus alternates its exposure to the two sides of the membrane through a cycle of conformational changes , as proposed in detail for the lactose permease ( kaback , 2005 ) . but the structure of clc - ec1 , in contrast to that of lactose permease , does not suggest any obvious conformational changes that would alternate exposure of e148 . for this reason , we conjectured that protons may somehow move through the protein during transport . such a picture envisions a proton pathway connecting distant residues on each side of the membrane , each mediating proton transfer between the protein and its corresponding aqueous solution , as in the well - characterized proton transfer residues in the bacterial reaction center , for example ( delroth and brzezinski , 2004 ) . first , neutralization of e203 completely and specifically abolishes all proton transport , as expected if the carboxyl group were required for shuttling protons to and from the protein interior , a prerequisite for participation in the coupled - transport cycle . second , e203 is partially buried near the intracellular solution , as is required for this proposed function . fourth , the only other residue that behaves this way is the externally facing e148 , for which a proton transfer function is strongly supported by previous work . finally , the loss of h transport in e203q can not be attributed to mutagenic disruption , since the structure of this mutant is similar to wild type and specific cl transport is maintained . these considerations support the idea that e203 directly catalyzes proton transfer between protein and intracellular solution as a required step in the coupled transport cycle . it argues that the protein contains a pathway along which protons move between e148 on the extracellular side and e203 on the intracellular . discovering the still - unknown rules of this coupling is required for understanding the transport mechanism ( jencks , 1980 ) . we do not yet know how protonation of e203 influences the cl sites , but it is intriguing that computational studies ( faraldo - gomez and roux , 2004 ) suggest a strong electrostatic influence of the protonation state of e203 ( as well as of other nearby residues ) on the affinity of cl binding to the protein . the structure of e148q , which mimics the external open form with e148 protonated , shows an additional cl ion occupying the location vacated by the q148 side chain ( dutzler et al . thus , on the external side of the protein , the cl and h pathways overlap ; but they diverge on the intracellular side , since e203 is distant from the internal cl site . protonation of e203 is a necessary step only for h transport , while protonation of e148 is required for both cl and h movements . as a final point , we should note an odd feature of the cl / h exchange mechanism : that abolition of h transport does not impede cl permeation . mutation of either proton transfer residue , e148 or e203 , abolishes h transport but leaves cl transport largely unaffected . for this reason , we consider that the mechanism of clc transporters will turn out to be novel , unconventional , and eerily reminiscent of ion channel permeation , and fundamentally unlike the alternating - site schemes that have so productively guided our understanding of long - studied and more familiar classes of transport proteins .

the number of people affected by idiopathic environmental intolerances ( iei ) , now also more appropriately called environmental sensitivity - related illnesses ( sri ) with difficult diagnosis , is constantly growing worldwide . iei include the most frequent multiple chemical sensitivity ( mcs ) , chronic fatigue syndrome ( cfs ) , fibromyalgia ( fm ) , electromagnetic hypersensitivity ( ehs ) , amalgam disease , and others . the clinical iei features include multiorgan manifestations in respiratory , nervous , cardiological , endocrine , cutaneous , and gastrointestinal systems , often without classic allergologic and/or immunologic markers [ 13 ] . in many cases , disease onset appears to be preceded by a short - term stress , most commonly a chemical overexposure in the case of mcs , infections in cfs , and physical trauma in fm , and then followed by a chronic condition that typically lasts for years or decades ( for recent review see [ 2 , 4 , 5 ] ) . the inherited or acquired impaired metabolism of xenobiotics has been postulated as a molecular basis for iei pathogenesis . it has recently been demonstrated that some genetic variants of drug metabolizing and detoxifying enzymes , such as cytochrome p450 reductase ( cyp ) , glutathione - s - transferase ( gst ) , n - acetyl - transferase ( nat ) , and superoxide dismutase ( sod2 ) , may be considered genetic determinants of iei risk [ 2 , 69 ] . these genetic polymorphisms seem to be connected to the loss of efficiency of detoxification systems , disturbances of free radical / antioxidant homeostasis , and increased production of inflammatory cytokines [ 1 , 10 , 11 ] . the overlapping symptoms among various iei have prompted several investigators to hypothesize that they may share a common pathogenetic feature , and the most suitable one proposed so far is the increased nitric oxide / peroxynitrite levels . in fact , short - term stressors are able to increase systemic nitric oxide production [ 12 , 13 ] , which in the reaction with superoxide forms the potent oxidant peroxynitrite . peroxynitrite , in turn , can act through six different positive feedback loop mechanisms to increase the levels of nitric oxide and its other precursor , superoxide anion , to form more peroxynitrite in a vicious cycle [ 14 , 15 ] . interestingly , it has recently been reported that gene variants of nos2 are associated with alteration of no levels in inflammatory bowel disorders , asthma , atopy , and migraine [ 1620 ] , all of which are comorbidities shared by iei . furthermore , circulating no levels may also be affected by some variants of nos3 gene . this clinical case - control study aimed at investigating the association , if any , of the promoter pentanucleotide microsatellite 2.5 kb ( ccttt)n and the ser608leu polymorphisms in nos2 gene and the 786t > c polymorphism in nos3 promoter with different iei , such as mcs , suspected mcs ( smcs ) , fm , and cfs . we also attempted to find out a correlation between the indicated polymorphisms and serum levels of nitrite / nitrate . three hundred and sixty - seven italian patients , who presented with symptoms compatible with iei , were enrolled in this study at department of medical pathophysiology , university of rome la sapienza , policlinico umberto i , and at istituto dermopatico dell'immacolata ( idi irccs ) in rome , italy ( ethical committee board approval , istituto dermopatico dell'immacolata , idi irccs , rome , italy , n.121/ce/2008 ) . diagnosis was set based on the medically assessed results of the modified qeesi ( quick environmental exposure and sensitivity inventory ) scoring . qeesi is a validated self - administered questionnaire developed as a screening tool for patients with iei . it is based on five different scales of assessment : symptoms severity , chemical triggers , other triggers , life impact , and finally a masking index to ongoing exposures . a modified qeesi score of 10 common environmental exposures and 10 major symptoms enabled the differential diagnosis of mcs and smcs ( suspected mcs ) : full diagnosis ( 20 score 30 ) or strongly suspected diagnosis , that is , subjects fulfilling diagnostic criteria only partially ( 10 score 20 ) or subjects excluded from enrollment ( 0 score 10 ) . one hundred and seventy of the recruited subjects were consecutive patients with mcs ( 49m/121f ; 49 11 years ) , 108 were consecutive patients with smcs ( 90f/18 m ; 49 12 years ) , who partly corresponded to the above reported diagnostic criteria , and 89 were consecutive patients ( 67f/22 m ; 47 10 years ) presenting with either fibromyalgia or chronic fatigue syndrome ( fm / cfs ) . one hundred and ninety - six healthy italian subjects ( m = 59 , f = 137 ; 45.5 9 years ) were selected as controls among healthy staff members of participant institutions , istituto dermopatico dell'immacolata ( idi irccs ) in rome and university of messina , according to the established criteria as follows : ( i ) an absence of any clinically diagnosed disease , in particular allergic or immunologic disturbances , ( ii ) no drug or nutraceutical supplement since at least six weeks , at the time of blood sampling , and ( iii ) whole blood total production of reactive oxygen and nitrogen species ( ros / rns ) below 650 cps / l , as determined by luminol - dependent chemiluminescent response to phorbol 12-myristate 13-acetate ( subject recruitment under study protocol approved by istituto dermopatico dell'immacolata idi irccs , rome , italy ethical committee , n.52/ce/2010 ) . nonsmokers in the patients ' group were 81.4% , smokers 11.3% , and patients with undetermined smoking habits 7.3% . the study was carried out in accordance with the declaration of helsinki ( 1964 ) , and the study protocol was approved by the local ethics committee . genomic dna was isolated from peripheral leukocytes by puregene dna purification system kit ( gentra qiagen , milan , italy ) . genotyping for the single nucleotide polymorphisms ( snps ) nos2a ser608leu ( c2087 t , rs2297518 ) and nos3 786t > c ( rs2070744 ) was performed by real - time pcr allelic discrimination , using taqman - based genotyping assays ( applied biosystems ; assay i d : c_11889257_10 ; c_15903863_10 ) available from life technologies ( monza , italy ) in a 96-well plate on a 7900ht fast real - time pcr system ( applied biosystems , foster city , ca , usa ) . the reactions were carried out in a final volume of 20 l containing 1x taqman genotyping master mix , 1x taqman - based specific assay , and 10 ng genomic dna , using thermal cycling conditions suggested by manufacturer 's protocols . genotyping for the pentanucleotide microsatellite 2.5 kb ( ccttt)n in nos2a promoter was carried out by dna direct sequencing . pcr reactions were carried out in a final volume of 50 l , containing 100 ng of genomic dna , 1x pcr buffer , 1.7 mm mgcl2 , 0.2 mm dntp , 0.2 m primers , and 1 u eurotaq dna polymerase ( euroclone , milan , italy ) , in one personal thermocycler ( celbio , milan , italy ) , using the following thermal cycling conditions : initial denaturation at 95c for 10 , then 40 cycles of 95c for 20 , 56c for 20 , 72c for 20 , and a final extension step at 72c for 7. pcr primer sequences were the same used by holla and coworkers . pcr products were purified and the sequencing reaction was performed with bigdye terminator v1.1 cycle sequencing kit ( applied biosystems , applera corp . , the reaction was carried out in a final volume of 20 l , containing 30 ng of purified pcr product , 1.6 pmol of forward primer , 2.5x ready reaction mix , 5x sequencing buffer , and dnase / rnase free water , in a thermocycler hybaid thermosprint ( celbio , milan , italy ) . the thermal cycling conditions were 96c for 3 , then 28 cycles of 10 at 96c , 10 at 50c , 4 at 60c , and finally 5 at 4c . the sequencing products were purified and analysed with abi prism 310 genetic analyzer ( applied biosystems , applera corp . , milan , italy ) . to compare nos2a and nos3 allele and genotype frequencies in patients and controls , as well as deviations of genotype distributions from hardy - weinberg equilibrium , statistical analysis was performed using the graphpad prism 4 software ( san diego , ca , usa ) . the linkage disequilibrium extent between the two nos2 polymorphisms was estimated by haploview v4.2 , by collecting the ( ccttt)n alleles in two categories according to the number of repeats ; that is , the alleles with < 12 repeats were designated as s ( short ) and alleles with 12 repeats as l ( long ) alleles . statistical analysis of nitrite / nitrate variations in patients and controls was performed using statistica 7.0 program ( statsoft inc . , nitrite / nitrate plasma concentration values were presented as median , lower , and upper quartiles , minimum and maximum . nitrite / nitrate plasma levels in either patients or controls having different nos2a and nos3 genotypes were analysed by one - way anova . the differences in nitrite / nitrate plasma levels among the subgroups of patients and controls were analysed by two - way anova . if necessary genotype and allele frequencies of nos2a and nos3 snps examined in iei patients ( mcs , smcs , and fm / cfs ) and controls are shown in table 1 . the genotype frequencies did not deviate from the expected value by hardy - weinberg equilibrium . genotyping for the snp c2087 t ( ser608leu ) in nos2a showed that allele frequencies in patients were not statistically significant different in comparison with controls . the wild - type allele c was the most frequent in all groups and almost entirely represented by the homozygous genotype . the cc2087 genotype frequency was higher in fm / cfs group than in all the other groups , and it tended to a statistically significant difference only in comparison with smcs group ( p = 0.08 ) that showed the lowest cc frequency . the frequency of the homozygous genotype tt2087 was higher in patients than in controls , but these differences were not statistically significant . the heterozygous genotype was more represented in controls than in patients , except for smcs patients . the fm / cfs group showed the lowest ct genotype frequency ( table 1 ) . the analysis of the distribution of nos3 gene variant 786t > c showed that allele and genotype frequencies were not significantly different between the groups . genotyping results showed that the nos2a 2.5 kb ( ccttt)n repeat variant was present with nine different alleles ( ranging 816 repeats , 176216 bp ) both in patients and controls , with an unimodal distribution having a peak at the ( ccttt)12 allele that was the most frequent in all examined groups ( table 2 ) . the ( ccttt)7 and ( ccttt)17 alleles were not found in our study cohort . the estimation of linkage disequilibrium for nos2a c2087 t and 2.5 kb ( ccttt)n alleles showed that nos2a c2087 t is not in linkage disequilibrium with the microsatellite s / l allelic classification ( d = 0.112 , lod = 0.24 , r = 0.003 ) . the ( ccttt)8 allele was more frequent in controls than in patients , showing a prevalence three - fourfold higher in controls than in mcs and smcs patients . this difference was highly significant in comparison with mcs patients ( p < 0.01 ) and significant in comparison with smcs patients ( p < 0.05 ) ; this allele was not observed among fm / cfs patients ( table 2 ) . notably , the presence of the ( ccttt)8 allele was associated with a decreased risk of iei , since it reduced by threefold the risk for mcs ( or = 0.3 ; 95% c.i . = 0.120.77 ) , by sixfold the risk for smcs ( or = 0.16 ; 95% c.i . = 0.0211.24 ) , and by tenfold the risk for fm / cfs ( or = 0.09 ; 95% c.i . the ( ccttt)9 allele and the ( ccttt)12 allele had a lower and higher frequency , respectively , in fm / cfs patients than in all other groups that showed similar frequencies . the ( ccttt)10 allele had similar frequencies in all groups ( table 2 ) . the ( ccttt)11 allele frequency was around threefold higher in fm / cfs patients than in controls , and this difference was statistically significant ( p < 0.01 ) ( table 2 ) . moreover , a difference tending to statistical significance was found when comparing fm / cfs patients with mcs ( p = 0.06 ) . this nos2a variant was associated with a significantly increased risk for fm / cfs ( or = 3.557 , 95% c.i . 1.5758.033 ) . the ( ccttt)13 allele was more represented in mcs patients than in other groups and had a frequency almost double than in smcs patients , but these differences were not statistically significant . the ( ccttt)16 allele was more represented in smcs patients than in other groups , with a frequency that was eight- and twofold higher than in mcs patients and controls , respectively , but this difference was statistically significant ( p < 0.01 ) only in comparison with mcs patients , while tended to be significant when compared with controls ( p < 0.06 ) . the lowest frequency was observed in mcs patients , with a significant difference in comparison with controls ( p < 0.05 ) . the ( ccttt)n genotype frequencies did not deviate from the expected value by hardy - weinberg equilibrium . the ( ccttt)8/13 genotype was only found in controls , with a frequency of 4.5% . the ( ccttt)12/12 genotype was the most frequent in all examined group except in fm / cfs ( 16.9% mcs , 12.5% smcs , 12% fm / cfs , and 16.9% controls ) . in this latter group , the most represented genotype was the ( ccttt)10/12 genotype that showed a significantly higher frequency when compared with mcs patients ( 20 versus 2.5% ; p = 0.0044 ) and a borderline significant difference when compared with controls ( 20 versus 6.7% ; p = 0.061 ) , while frequencies in smcs patients and controls were similar ( 6.3% versus 6.7% , p > 0.05 ) . the ( ccttt)10/10 genotype , having a frequency around 7% in mcs , smcs , and controls , was not found in fm / cfs patients . the ( ccttt)10/11 in mcs patients had a frequency double than that in controls ( 10.2 versus 4.5% , not significant ) , while in smcs patients and fm / cfs it accounted for 6.3% and 8.0% , respectively . the ( ccttt)11/11 genotype had the highest frequency in fm / cfs patients ( 8% versus 3% in all other groups ) , but this difference did not reach statistically significant values . the ( ccttt)11/12 genotype had the highest frequency in smcs patients ( 12.5% versus 3.4% mcs versus 8% fm / cfs versus 3.4% controls ) , but this difference was not statistically significant . the ( ccttt)11/13 genotype was more represented in fm / cfs patients than in other groups , with a frequency significantly different when compared with smcs ( 16 versus 0% ; p = 0.032 ) and controls ( 16 versus 1.1% ; p = 0.008 ) and borderline significant when compared with mcs ( 16 versus 4.2% ; p = 0.0501 ) . moreover , this genotype was associated with an increased risk for fm / cfs ( or = 16.8 ; 95% c.i . = 1.78157.9 ) . the ( ccttt)13/13 genotype had the highest frequency in mcs patients ( 6.8% versus 3.1% smcs versus 0% fm / cfs versus 4.5% controls ) , but this difference was not statistically significant . all other allele combinations did not reach a frequency higher than 5% in all groups . in an attempt to better understand whether the presence of increasing 2.5 kb ( ccttt ) repeat numbers of nos2a promoter may be a genetic feature useful to discriminate different types of iei , we grouped together different genotypes by classifying alleles with repeat number < 12 as short ( s ) and alleles with repeat number 12 as long ( l ) . the between - groups comparison of s and l allele frequencies showed that there were no significant differences between different groups of patients and also between patients and controls . the heterozygous genotype ( ccttt)short/(ccttt)long ( sl ) showed the highest frequencies in fm / cfs and smcs patients that were significantly different in comparison with both controls ( p < 0.001 , p = 0.02 , resp . ) and mcs patients ( p < 0.001 , p = 0.005 , resp . ) ; additionally , smcs patients having the ( ccttt)sl genotype were found to be significantly different from fm / cfs ( p = 0.0016 ) . notably , this genotype increased by about fivefold and twofold , respectively , the risk for fm / cfs ( or = 4.8 ; c.i . finally , the heterozygous genotype ( ccttt)s/(ccttt)l may be useful for diagnosis of either smcs or fm / cfs states and discrimination between smcs and either mcs or fm / cfs . the ( ccttt)long/(ccttt)long ( ll ) genotype had the lowest frequency in fm / cfs patients and statistically significant differences were found in comparison with all other groups ( mcs : p < 0.0001 ; smcs : p = 0.04 ; controls : p = 0.0002 ) . therefore , the ( ccttt)ll genotype may be useful to discriminate fm / cfs patients from both mcs and smcs patients . the ( ccttt)short/(ccttt)short ( ss ) genotype showed the lowest frequency in fm / cfs patients that were significantly different in comparison with controls ( p = 0.012 ) and mcs patients ( p = 0.015 ) . moreover , this genotype was protective against fm / cfs since it reduced by 2.5-fold the disease risk ( or = 0.4334 ; c.i . = 0.22690.8280 ) . analysis of nitrite / nitrate plasma concentrations showed that iei patients recruited for this study exhibited similar levels of these proinflammatory markers ( 26.2 13.3 mol / l mcs ; 24.7 12.2 smcs ; 28.4 15.2 fm / cfs ) . notably , nitrite / nitrate concentrations in patients were around or more than twofold higher than in controls ( 27.1 13.8 versus 15.3 5.2 mol / l ) , and this difference was statistically significant ( p = 0.037 ) . we analyzed the variability of nitrite / nitrate concentrations in patients and controls having different nos2a and nos3 genotypes . patients with different nos2 c2087 t genotypes showed different nitrite / nitrate levels , even if these differences did not reach statistical significance . in particular , tt homozygous mutated patients showed higher nitrite / nitrate concentrations than patients with other genotypes , while controls with different genotypes exhibited similar mean nitrite / nitrate plasma levels . tt homozygous patients had higher nitrite / nitrate levels compared with controls with the same genotype , while wild - type patients showed lower nitrite / nitrate levels than controls . however , no significant differences were observed when comparing patients and controls with different genotypes ( figure 1(a ) ) . we tried to better investigate the between - groups differences , if present , by dividing iei patients on the basis of their diagnosis . again , no significant differences were found between different groups of patients and controls ( figure 1(b ) ) . when nitrite / nitrate levels were analyzed in patients and controls with different nos3 786t > c genotypes , / nitrate levels were significantly higher in tt wild - type patients than in either tc heterozygous or cc mutated patients ( p = 0.012 , p = 0.0007 , resp . ) . moreover , when comparing controls and patients a statistically significant difference was observed between tc heterozygous subjects , since controls showed higher nitrite / nitrate levels than patients with the same genotype ( figure 2(a ) ) . when patients were divided in subgroups on the basis of different diagnosis , significant differences were found between either mcs or smcs patients with tc genotype and controls having the same genotype ( p = 0.04 , p = 0.03 , resp . ) . moreover , smcs and fm / cfs patients bearing the cc genotype had significantly lower nitrite / nitrate plasma levels than either controls having the same genotype ( p = 0.002 , p = 0.03 , resp . ) or tc genotype ( p = 0.0005 , p = 0.014 , resp . ) ( figure 2(b ) ) . finally , mcs patients having the tt genotype had nitrite / nitrate levels significantly higher than either smcs or fm / cfs patients with tc ( p = 0.008 , p = 0.04 , resp . ) and cc genotype ( p = 0.0001 , p = 0.004 , resp . ) and mcs patients having either tc or cc genotype ( p = 0.004 , p = 0.03 , resp . ) unfortunately , we could not analyze in detail the influence of the nos2a 2.5 kb ( ccttt)n pentanucleotide microsatellite on the variation of nitrite / nitrate plasma levels due to the dramatically high fragmentation of our study cohort caused by the high number of combinations of different nos2a 2.5 kb ( ccttt ) alleles . however , we tried to detect differences among patient subgroups by grouping ( ccttt ) repeats as short ( s ) when < 12 or long ( l ) when 12 . by this way , we obtained three types of allele combinations , namely , ( ccttt)s/(ccttt)s , ( ccttt)s/(ccttt)l , and ( ccttt)l/(ccttt)l . then , we compared patients , grouped together ( figure 3(a ) ) or separated according to their diagnosis ( figure 3(b ) ) , with controls having the same allele combinations . the results showed that nitrite / nitrate plasma levels were significantly higher in all patients and controls having the ( ccttt)s/(ccttt)s allele combination compared with patients and controls having the ( ccttt)l/(ccttt)l allele combination ( p = 0.0002 , p = 0.004 , resp . ) moreover , while examining the subgroups of patients significant differences were found between mcs patients having the ( ccttt)s/(ccttt)s allele combination and those with ( ccttt)l/(ccttt)l allele combination ( p = 0.008 ) ( figure 3(b ) ) . however , two - way anova , making a between - groups comparison , showed that nitrite / nitrate plasma levels were neither significantly different in subgroups of patients having different ( ccttt)s and ( ccttt)l allele combinations nor significantly different in comparison with controls having the same allele combinations ( figure 3(b ) ) . environment - associated pathologies have been attracting growing attention in the recent years due to increasingly daily exposure to various hazardous stimuli , such as chemicals , drugs , metals , electromagnetic or nuclear radiations , iatrogenic factors ( including synthetic implants ) , specific foods , and microbial and environmental allergens . ethical , medical , and social / occupational impact of iei is worsened by steadily growing population of hypersensitive people , not yet regulated diagnostic protocols , and abuse of not evidence - based treatments . the most important studies on hypersensitive populations , especially mcs patients , have been concentrated on the search of genetic determinants of abnormal sensitivity . based on the primary hypothesis of an impairment in the metabolism of xenobiotics , a wide variety of polymorphisms of genes encoding for phases i and ii of detoxification enzymes were studied , such as cyps , gst , comt , ugt , nat , and pon , with conflicting results [ 6 , 10 , 2426 ] . our group recently demonstrated that gene variants of gst and cyp isoforms , namely , gstp1 , gstm1 , gstt1 , cyp2c92 , cyp2c93 , cyp2c192 , cyp2d64 , and cyp2d641 , could represent genetic determinants for iei and hence may be used as markers for differential diagnosis of various iei [ 7 , 10 ] . the patients recruited for this study were diagnosed clinically by means of standard validated questionnaires ( qeesi ) . previous analyses of specific panels of genetic and metabolic markers identified as pathognomonic for the diseases [ 6 , 7 , 911 ] had confirmed the peculiar alterations of antioxidant enzyme activities ( catalase , glutathione - s - transferase , and glutathione peroxidase ) , low cellular and plasmatic concentrations of antioxidant molecules ( reduced glutathione , plasma total antioxidant activity ) , increased oxidative stress indices ( 4-hydroxy-2-nonenal , whole blood luminol - dependent chemiluminescence ) , increased plasma levels of inflammatory markers ( cytokines ) , and altered concentrations of erythrocyte membrane fatty acid saturated , monounsaturated , and polyunsaturated and fatty acid omega-6 and omega-3 [ 9 , 10 ] . moreover , genetic analyses had shown that patient subgroups were bearing one or more mutated variants of detoxifying enzymes , namely , gstp1 , gstm1 , gstt1 , cyp2c92 , cyp2c93 , cyp2c192 , cyp2d64 , and cyp2d641 , proven to be useful for diagnostic assessment of iei patients [ 6 , 7 , 10 , 11 ] . a number of years ago , pall and satterlee postulated the existence of a vicious nitric oxide / peroxynitrite cycle as the basis for iei , starting from several observations : ( 1 ) several organic solvents , which induce chemical sensitization ( formaldehyde , benzene , carbon tetrachloride , and certain organochlorine pesticides ) , trigger increases in nitric oxide levels ; ( 2 ) organophosphate and carbamate insecticides are suggested to induce mcs by inactivating acetylcholinesterase and thus activating muscarinic response , which is known to produce increases in nitric oxide ; ( 3 ) elevated cytokines , an integral part of the proposed feedback mechanism of the elevated nitric oxide / peroxynitrite theory , are known to be induced by organic solvents through nos2 activation ; ( 4 ) antioxidant therapy has been reported to improve iei symptoms , as expected if the levels of oxidant peroxynitrite are elevated ; ( 5 ) the symptoms exacerbated after chemical exposure may be explained by several known proinflammatory properties of nitric oxide , peroxynitrite , and inflammatory cytokines , each of which have a role in the proposed mechanism ; and ( 6 ) iei are often treated through intramuscular injections of vitamin b12 , that , in the form of hydroxocobalamin , is a potent nitric oxide scavenger in vitro and in vivo . lately , it has been reported that nitrite / nitrate levels , responsible for lipid peroxidation and cytokine increase , are significantly increased in iei patients . interestingly , circulating no and consequently nitrite / nitrate levels may be altered by the presence of nos polymorphisms [ 21 , 27 , 28 ] . moreover , some nos variants , particularly the nos2 ser608leu as well as the pentanucleotide microsatellite 2.5 kb ( ccttt)n and the nos3 786t > c , were associated with either increased nitrite / nitrate levels , total serum immunoglobulin e , and blood eosinophil levels or cytokines in various allergic / inflammatory / autoimmune disorders , including atopy , asthma , migraine , inflammatory bowel disease , or rheumatoid arthritis [ 1620 , 2933 ] . given that allergic / inflammatory symptoms are common in iei patients and that our patients were all exhibiting remarkably elevated nitrite / nitrate plasma levels compared to controls ( 27.1 13.8 versus 15.3 5.2 m , p < 0.05 ) , we chose to investigate the distribution of the polymorphic variants nos2 2.5 kb ( ccttt)n and ser608leu and nos3 786t > c in iei patients . we did not find significant difference in the distribution of the polymorphic variants nos2a c2087 t ( ser608leu ) and nos3 786t > c when patient groups were compared with controls ( table 1 ) . allele and genotype frequencies in healthy subjects were similar to those reported in other european populations . moreover , while investigating the effects of these nos2a and nos3 variants on the variability of nitrite / nitrate plasma levels , we could not observe any effect of nos2a c2087 t . unfortunately , no information is available on the influence of nos c2087 t on variation of these inflammatory markers . so , this is the first report documenting no apparent effect of the nos2a ser608leu on the variation of nitrite / nitrate plasma levels . interestingly , we found that patients with the nos3 786tt wild - type genotype had significantly increased nitrite / nitrate levels in comparison with patients having other genotypes . this latter observation agrees with those previously reported in patients with coronary spastic angina and healthy subjects after exercise [ 21 , 28 ] and is in contrast with others in patients with asthma . we also tried to detect differences in nitrite / nitrate plasma levels between subgroups of patients having different genotypes . what we found is that the significant difference initially observed between plasma levels of nitrite / nitrate in tt patients and those of either tc or cc patients ( figure 2(a ) ) was largely attributable to mcs patients bearing the tt genotype that had nitrite / nitrate concentrations significantly higher than mcs , smcs , and fm / cfs patients bearing either tc of cc genotype . notably , we detected a relationship between the 2.5 kb ( ccttt)n microsatellite in the promoter region of nos2a and iei ( table 2 ) . the nos2a 2.5 kb ( ccttt)n microsatellite has been so far associated with asthma , atopy , rheumatoid arthritis , and inflammatory bowel disease [ 17 , 18 , 2931 ] . it represents an attractive disease - causing candidate for iei , being a highly polymorphic marker ( 11 different alleles , ranging 717 repeats , and 171221 bp ) with a suggested effect in nos2 transcription . indeed , it has been reported that variations in the number of ( ccttt ) repeats are functionally important in the regulation of nos2 transcription , leading to increase in no production . highly significant differences have been reported in the ( ccttt ) allele frequencies between ethnically diverse populations . the observed frequencies of this marker in our cohort of healthy subjects fell within the caucasian pattern , with the peak at the 12-repeat allele [ 29 , 31 , 35 , 36 ] . when we compared the distribution of the ( ccctt ) repeat within the control subjects with that of iei patients , the 8-repeat allele showed a significantly decreased frequency in the groups of patients compared with controls ( table 2 ) . the odds ratio calculation showed that the presence of this allele reduced the risk for the different types of iei examined in this study , suggesting that this nos2a variant may display a protective effect against these disorders . on the contrary , the 11-repeat allele , showing a significantly increased frequency in the group of fm / cfs patients compared with controls , may be considered a genetic determinant for fm / cfs . moreover , the ( ccttt)16 allele , having a significantly different frequency between mcs and smcs , may be used to discriminate these two types of iei . moreover , taking into account different repeat cut offs ( 9 , 11 , 13 , and 16 repeats ) , we found that a different number of nos2a ( ccttt ) repeats could be used to discriminate between the different pathological conditions . interestingly , when we grouped the ( ccttt)n alleles in two categories according to the number of repeats , that is , the alleles with < 12 repeats were designated as s ( short ) and alleles with 12 repeats as l ( long ) alleles , as previously described , we found that the ( ccttt)s/(ccttt)l allele combination had the highest frequencies in fm / cfs and smcs ( table 3 ) . therefore , this nos2a allele combination may be useful for diagnosis of these two types of iei . moreover , the observed distribution of ( ccttt)s/(ccttt)l and ( ccttt)l/(ccttt)l allele combination ( table 3 ) confirms that smcs patients had overlapping features with fm / cfs and mcs and may be considered as an intermediate pathological condition between the two above cited . after grouping of different ( ccttt ) allele combinations , we were able to investigate the relationship between this polymorphic variant and nitrite / nitrate plasma levels in patients . thus , we found that a short number of ( ccttt ) repeats allele was associated with higher concentrations of nitrites / nitrates . our results demonstrated for the first time that the nos2a promoter pentanucleotide microsatellite 2.5 kb ( ccttt)n is associated with fm / cfs and may be feasible for the diagnostic assessment of this type of iei . moreover , the screening for the presence of some nos2a 2.5 kb ( ccttt ) variants , that is , the 8- and 16-repeat alleles , may be useful , respectively , to exclude the diagnosis of iei and discriminate between mcs and smcs .

oxidative stress and inflammation play a pathogenetic role in idiopathic environmental intolerances ( iei ) , namely , multiple chemical sensitivity ( mcs ) , fibromyalgia ( fm ) , and chronic fatigue syndrome ( cfs ) . given the reported association of nitric oxide synthase ( nos ) gene polymorphisms with inflammatory disorders , we aimed to investigate the distribution of nos2a 2.5 kb ( ccttt)n as well as ser608leu and nos3 786t > c variants and their correlation with nitrite / nitrate levels , in a study cohort including 170 mcs , 108 suspected mcs ( smcs ) , 89 fm / cfs , and 196 healthy subjects . patients and controls had similar distributions of nos2a ser608leu and nos3 786t > c polymorphisms . interestingly , the nos3 786tt genotype was associated with increased nitrite / nitrate levels only in iei patients . we also found that the nos2a 2.5 kb ( ccttt)11 allele represents a genetic determinant for fm / cfs , and the ( ccttt)16 allele discriminates mcs from smcs patients . instead , the ( ccttt)8 allele reduces by three- , six- , and tenfold , respectively , the risk for mcs , smcs , and fm / cfs . moreover , a short number of ( ccttt ) repeats is associated with higher concentrations of nitrites / nitrates . here , we first demonstrate that nos3 786t > c variant affects nitrite / nitrate levels in iei patients and that screening for nos2a 2.5 kb ( ccttt)n polymorphism may be useful for differential diagnosis of various iei .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

the number of people affected by idiopathic environmental intolerances ( iei ) , now also more appropriately called environmental sensitivity - related illnesses ( sri ) with difficult diagnosis , is constantly growing worldwide . iei include the most frequent multiple chemical sensitivity ( mcs ) , chronic fatigue syndrome ( cfs ) , fibromyalgia ( fm ) , electromagnetic hypersensitivity ( ehs ) , amalgam disease , and others . peroxynitrite , in turn , can act through six different positive feedback loop mechanisms to increase the levels of nitric oxide and its other precursor , superoxide anion , to form more peroxynitrite in a vicious cycle [ 14 , 15 ] . interestingly , it has recently been reported that gene variants of nos2 are associated with alteration of no levels in inflammatory bowel disorders , asthma , atopy , and migraine [ 1620 ] , all of which are comorbidities shared by iei . this clinical case - control study aimed at investigating the association , if any , of the promoter pentanucleotide microsatellite 2.5 kb ( ccttt)n and the ser608leu polymorphisms in nos2 gene and the 786t > c polymorphism in nos3 promoter with different iei , such as mcs , suspected mcs ( smcs ) , fm , and cfs . one hundred and seventy of the recruited subjects were consecutive patients with mcs ( 49m/121f ; 49 11 years ) , 108 were consecutive patients with smcs ( 90f/18 m ; 49 12 years ) , who partly corresponded to the above reported diagnostic criteria , and 89 were consecutive patients ( 67f/22 m ; 47 10 years ) presenting with either fibromyalgia or chronic fatigue syndrome ( fm / cfs ) . genotyping for the single nucleotide polymorphisms ( snps ) nos2a ser608leu ( c2087 t , rs2297518 ) and nos3 786t > c ( rs2070744 ) was performed by real - time pcr allelic discrimination , using taqman - based genotyping assays ( applied biosystems ; assay i d : c_11889257_10 ; c_15903863_10 ) available from life technologies ( monza , italy ) in a 96-well plate on a 7900ht fast real - time pcr system ( applied biosystems , foster city , ca , usa ) . , the reaction was carried out in a final volume of 20 l , containing 30 ng of purified pcr product , 1.6 pmol of forward primer , 2.5x ready reaction mix , 5x sequencing buffer , and dnase / rnase free water , in a thermocycler hybaid thermosprint ( celbio , milan , italy ) . to compare nos2a and nos3 allele and genotype frequencies in patients and controls , as well as deviations of genotype distributions from hardy - weinberg equilibrium , statistical analysis was performed using the graphpad prism 4 software ( san diego , ca , usa ) . if necessary genotype and allele frequencies of nos2a and nos3 snps examined in iei patients ( mcs , smcs , and fm / cfs ) and controls are shown in table 1 . the analysis of the distribution of nos3 gene variant 786t > c showed that allele and genotype frequencies were not significantly different between the groups . genotyping results showed that the nos2a 2.5 kb ( ccttt)n repeat variant was present with nine different alleles ( ranging 816 repeats , 176216 bp ) both in patients and controls , with an unimodal distribution having a peak at the ( ccttt)12 allele that was the most frequent in all examined groups ( table 2 ) . the estimation of linkage disequilibrium for nos2a c2087 t and 2.5 kb ( ccttt)n alleles showed that nos2a c2087 t is not in linkage disequilibrium with the microsatellite s / l allelic classification ( d = 0.112 , lod = 0.24 , r = 0.003 ) . the ( ccttt)8 allele was more frequent in controls than in patients , showing a prevalence three - fourfold higher in controls than in mcs and smcs patients . notably , the presence of the ( ccttt)8 allele was associated with a decreased risk of iei , since it reduced by threefold the risk for mcs ( or = 0.3 ; 95% c.i . = 0.0211.24 ) , and by tenfold the risk for fm / cfs ( or = 0.09 ; 95% c.i . the ( ccttt)9 allele and the ( ccttt)12 allele had a lower and higher frequency , respectively , in fm / cfs patients than in all other groups that showed similar frequencies . the ( ccttt)11 allele frequency was around threefold higher in fm / cfs patients than in controls , and this difference was statistically significant ( p < 0.01 ) ( table 2 ) . moreover , a difference tending to statistical significance was found when comparing fm / cfs patients with mcs ( p = 0.06 ) . this nos2a variant was associated with a significantly increased risk for fm / cfs ( or = 3.557 , 95% c.i . the ( ccttt)16 allele was more represented in smcs patients than in other groups , with a frequency that was eight- and twofold higher than in mcs patients and controls , respectively , but this difference was statistically significant ( p < 0.01 ) only in comparison with mcs patients , while tended to be significant when compared with controls ( p < 0.06 ) . the ( ccttt)12/12 genotype was the most frequent in all examined group except in fm / cfs ( 16.9% mcs , 12.5% smcs , 12% fm / cfs , and 16.9% controls ) . in this latter group , the most represented genotype was the ( ccttt)10/12 genotype that showed a significantly higher frequency when compared with mcs patients ( 20 versus 2.5% ; p = 0.0044 ) and a borderline significant difference when compared with controls ( 20 versus 6.7% ; p = 0.061 ) , while frequencies in smcs patients and controls were similar ( 6.3% versus 6.7% , p > 0.05 ) . the ( ccttt)10/10 genotype , having a frequency around 7% in mcs , smcs , and controls , was not found in fm / cfs patients . the ( ccttt)10/11 in mcs patients had a frequency double than that in controls ( 10.2 versus 4.5% , not significant ) , while in smcs patients and fm / cfs it accounted for 6.3% and 8.0% , respectively . the ( ccttt)11/12 genotype had the highest frequency in smcs patients ( 12.5% versus 3.4% mcs versus 8% fm / cfs versus 3.4% controls ) , but this difference was not statistically significant . the ( ccttt)11/13 genotype was more represented in fm / cfs patients than in other groups , with a frequency significantly different when compared with smcs ( 16 versus 0% ; p = 0.032 ) and controls ( 16 versus 1.1% ; p = 0.008 ) and borderline significant when compared with mcs ( 16 versus 4.2% ; p = 0.0501 ) . moreover , this genotype was associated with an increased risk for fm / cfs ( or = 16.8 ; 95% c.i . the ( ccttt)13/13 genotype had the highest frequency in mcs patients ( 6.8% versus 3.1% smcs versus 0% fm / cfs versus 4.5% controls ) , but this difference was not statistically significant . in an attempt to better understand whether the presence of increasing 2.5 kb ( ccttt ) repeat numbers of nos2a promoter may be a genetic feature useful to discriminate different types of iei , we grouped together different genotypes by classifying alleles with repeat number < 12 as short ( s ) and alleles with repeat number 12 as long ( l ) . ; additionally , smcs patients having the ( ccttt)sl genotype were found to be significantly different from fm / cfs ( p = 0.0016 ) . notably , this genotype increased by about fivefold and twofold , respectively , the risk for fm / cfs ( or = 4.8 ; c.i . finally , the heterozygous genotype ( ccttt)s/(ccttt)l may be useful for diagnosis of either smcs or fm / cfs states and discrimination between smcs and either mcs or fm / cfs . the ( ccttt)long/(ccttt)long ( ll ) genotype had the lowest frequency in fm / cfs patients and statistically significant differences were found in comparison with all other groups ( mcs : p < 0.0001 ; smcs : p = 0.04 ; controls : p = 0.0002 ) . therefore , the ( ccttt)ll genotype may be useful to discriminate fm / cfs patients from both mcs and smcs patients . analysis of nitrite / nitrate plasma concentrations showed that iei patients recruited for this study exhibited similar levels of these proinflammatory markers ( 26.2 13.3 mol / l mcs ; 24.7 12.2 smcs ; 28.4 15.2 fm / cfs ) . we analyzed the variability of nitrite / nitrate concentrations in patients and controls having different nos2a and nos3 genotypes . patients with different nos2 c2087 t genotypes showed different nitrite / nitrate levels , even if these differences did not reach statistical significance . when nitrite / nitrate levels were analyzed in patients and controls with different nos3 786t > c genotypes , / nitrate levels were significantly higher in tt wild - type patients than in either tc heterozygous or cc mutated patients ( p = 0.012 , p = 0.0007 , resp . ) moreover , smcs and fm / cfs patients bearing the cc genotype had significantly lower nitrite / nitrate plasma levels than either controls having the same genotype ( p = 0.002 , p = 0.03 , resp . ) finally , mcs patients having the tt genotype had nitrite / nitrate levels significantly higher than either smcs or fm / cfs patients with tc ( p = 0.008 , p = 0.04 , resp . ) unfortunately , we could not analyze in detail the influence of the nos2a 2.5 kb ( ccttt)n pentanucleotide microsatellite on the variation of nitrite / nitrate plasma levels due to the dramatically high fragmentation of our study cohort caused by the high number of combinations of different nos2a 2.5 kb ( ccttt ) alleles . the results showed that nitrite / nitrate plasma levels were significantly higher in all patients and controls having the ( ccttt)s/(ccttt)s allele combination compared with patients and controls having the ( ccttt)l/(ccttt)l allele combination ( p = 0.0002 , p = 0.004 , resp . ) our group recently demonstrated that gene variants of gst and cyp isoforms , namely , gstp1 , gstm1 , gstt1 , cyp2c92 , cyp2c93 , cyp2c192 , cyp2d64 , and cyp2d641 , could represent genetic determinants for iei and hence may be used as markers for differential diagnosis of various iei [ 7 , 10 ] . moreover , genetic analyses had shown that patient subgroups were bearing one or more mutated variants of detoxifying enzymes , namely , gstp1 , gstm1 , gstt1 , cyp2c92 , cyp2c93 , cyp2c192 , cyp2d64 , and cyp2d641 , proven to be useful for diagnostic assessment of iei patients [ 6 , 7 , 10 , 11 ] . a number of years ago , pall and satterlee postulated the existence of a vicious nitric oxide / peroxynitrite cycle as the basis for iei , starting from several observations : ( 1 ) several organic solvents , which induce chemical sensitization ( formaldehyde , benzene , carbon tetrachloride , and certain organochlorine pesticides ) , trigger increases in nitric oxide levels ; ( 2 ) organophosphate and carbamate insecticides are suggested to induce mcs by inactivating acetylcholinesterase and thus activating muscarinic response , which is known to produce increases in nitric oxide ; ( 3 ) elevated cytokines , an integral part of the proposed feedback mechanism of the elevated nitric oxide / peroxynitrite theory , are known to be induced by organic solvents through nos2 activation ; ( 4 ) antioxidant therapy has been reported to improve iei symptoms , as expected if the levels of oxidant peroxynitrite are elevated ; ( 5 ) the symptoms exacerbated after chemical exposure may be explained by several known proinflammatory properties of nitric oxide , peroxynitrite , and inflammatory cytokines , each of which have a role in the proposed mechanism ; and ( 6 ) iei are often treated through intramuscular injections of vitamin b12 , that , in the form of hydroxocobalamin , is a potent nitric oxide scavenger in vitro and in vivo . lately , it has been reported that nitrite / nitrate levels , responsible for lipid peroxidation and cytokine increase , are significantly increased in iei patients . interestingly , circulating no and consequently nitrite / nitrate levels may be altered by the presence of nos polymorphisms [ 21 , 27 , 28 ] . moreover , some nos variants , particularly the nos2 ser608leu as well as the pentanucleotide microsatellite 2.5 kb ( ccttt)n and the nos3 786t > c , were associated with either increased nitrite / nitrate levels , total serum immunoglobulin e , and blood eosinophil levels or cytokines in various allergic / inflammatory / autoimmune disorders , including atopy , asthma , migraine , inflammatory bowel disease , or rheumatoid arthritis [ 1620 , 2933 ] . given that allergic / inflammatory symptoms are common in iei patients and that our patients were all exhibiting remarkably elevated nitrite / nitrate plasma levels compared to controls ( 27.1 13.8 versus 15.3 5.2 m , p < 0.05 ) , we chose to investigate the distribution of the polymorphic variants nos2 2.5 kb ( ccttt)n and ser608leu and nos3 786t > c in iei patients . we did not find significant difference in the distribution of the polymorphic variants nos2a c2087 t ( ser608leu ) and nos3 786t > c when patient groups were compared with controls ( table 1 ) . moreover , while investigating the effects of these nos2a and nos3 variants on the variability of nitrite / nitrate plasma levels , we could not observe any effect of nos2a c2087 t . so , this is the first report documenting no apparent effect of the nos2a ser608leu on the variation of nitrite / nitrate plasma levels . interestingly , we found that patients with the nos3 786tt wild - type genotype had significantly increased nitrite / nitrate levels in comparison with patients having other genotypes . what we found is that the significant difference initially observed between plasma levels of nitrite / nitrate in tt patients and those of either tc or cc patients ( figure 2(a ) ) was largely attributable to mcs patients bearing the tt genotype that had nitrite / nitrate concentrations significantly higher than mcs , smcs , and fm / cfs patients bearing either tc of cc genotype . notably , we detected a relationship between the 2.5 kb ( ccttt)n microsatellite in the promoter region of nos2a and iei ( table 2 ) . the nos2a 2.5 kb ( ccttt)n microsatellite has been so far associated with asthma , atopy , rheumatoid arthritis , and inflammatory bowel disease [ 17 , 18 , 2931 ] . indeed , it has been reported that variations in the number of ( ccttt ) repeats are functionally important in the regulation of nos2 transcription , leading to increase in no production . when we compared the distribution of the ( ccctt ) repeat within the control subjects with that of iei patients , the 8-repeat allele showed a significantly decreased frequency in the groups of patients compared with controls ( table 2 ) . on the contrary , the 11-repeat allele , showing a significantly increased frequency in the group of fm / cfs patients compared with controls , may be considered a genetic determinant for fm / cfs . moreover , the ( ccttt)16 allele , having a significantly different frequency between mcs and smcs , may be used to discriminate these two types of iei . moreover , taking into account different repeat cut offs ( 9 , 11 , 13 , and 16 repeats ) , we found that a different number of nos2a ( ccttt ) repeats could be used to discriminate between the different pathological conditions . interestingly , when we grouped the ( ccttt)n alleles in two categories according to the number of repeats , that is , the alleles with < 12 repeats were designated as s ( short ) and alleles with 12 repeats as l ( long ) alleles , as previously described , we found that the ( ccttt)s/(ccttt)l allele combination had the highest frequencies in fm / cfs and smcs ( table 3 ) . moreover , the observed distribution of ( ccttt)s/(ccttt)l and ( ccttt)l/(ccttt)l allele combination ( table 3 ) confirms that smcs patients had overlapping features with fm / cfs and mcs and may be considered as an intermediate pathological condition between the two above cited . after grouping of different ( ccttt ) allele combinations , we were able to investigate the relationship between this polymorphic variant and nitrite / nitrate plasma levels in patients . thus , we found that a short number of ( ccttt ) repeats allele was associated with higher concentrations of nitrites / nitrates . our results demonstrated for the first time that the nos2a promoter pentanucleotide microsatellite 2.5 kb ( ccttt)n is associated with fm / cfs and may be feasible for the diagnostic assessment of this type of iei . moreover , the screening for the presence of some nos2a 2.5 kb ( ccttt ) variants , that is , the 8- and 16-repeat alleles , may be useful , respectively , to exclude the diagnosis of iei and discriminate between mcs and smcs .

bullying is broadly defined as a desire to hurt and the execution of a harmful action ; it is characterised by repetition and either a physical or a psychological power imbalance ( farrington , 1993 ; rigby , 2002 ; smith & brain , 2000 ; smith & sharp , 1994 ) . it may come in the form of verbal abuse , physical aggression or relational victimisation . the first two forms of bullying have sometimes been called direct bullying as they include directly aggressive behaviour . relational victimisation is the manipulation of peer relationships in order to exclude someone ( wolke , woods , bloomfield , & karstadt , 2000 ) . are bullied at some point whilst at school and between 5% and 10% are regularly bullied ( newman , holden , & delville , 2005 ) . research has shown that boys are more likely to be involved in bullying , either as bullies or as victims , than girls ( forero , mclellan , rissel , & bauman , 1999 ; kaltiala - heino , rimpela , rantanen , & rimpela , 2000 ; nansel et al . , 2001 ; rigby , 2000 ; seals & young , 2003 ) . rigby ( 2000 ) found that boys and girls were similarly likely to be bullied through name calling , teasing and deliberate exclusion . boys are more likely to receive threats of harm and be physically bullied ( baldry , 2004 ; owens , shute , & slee , 2000 ; rigby , 2000 ; roland & idsoe , 2000 ) . there is evidence that prevalence of bullying declines with age ( newman et al . , 2005 ; cross - sectional research on the effects of bullying on psychological health has consistently found that those that are bullied exhibit poorer emotional adjustment in early to late adolescence , indicating an association but no direct causality ( alikasifoglu , erginoz , ercan , uysal , & albayrak - kaymak , 2007 ; baldry , 2004 ; brunstein klomak , marrocco , kleinman , schonfeld , & gould , 2007 ; espelage & holt , 2001 ; fekkes , pijpers , & verloove - vanhorick , 2004 ; forero et al . , 1999 ; greco , freeman , & dufton , 2007 ; kaltiala - heino , rimpela , marttunen , rimpela , & rantanen , 1999 ; kaltiala - heino et al . , 2000 ; kumpulainen , rasanen , & puura , 2001 ; saluja et al . , 2004 ; seals & young , 2003 ; smith , talamelli , cowie , naylor , & chauhan , 2004 ; van der wal , de wit , & hirasing , 2003 ) . few studies have prospectively examined the relationship between bullying and the psychological health of the victim . one study found that bullied australian secondary school students in year 8 ( age 13 ) had a higher risk of depression and anxiety over the following school year ( bond , carlin , thomas , rubin , & patton , 2001 ) . the association between bullying and poor psychological health may work in both directions ; fekkes et al . found that victims of bullying had significantly higher chances of psychosomatic and psychosocial problems compared with children who were not bullied ; conversely , children with depressive and anxiety symptoms had a higher chance of being bullied ( fekkes , pijpers , fredriks , vogels , & verloove - vanhorick , 2006 ) . a limited amount of research has been carried out on the relationship between bullying and educational achievement in adolescence , although there have been some studies on the potential effects of bullying ( nansel et al . , 2001 ) . one us study found a link between bullying at elementary school and low achievement ( glew , fan , katon , rivara , & kernic , 2005 ) . a longitudinal study in uk primary schools found that being a victim of relational bullying was the second most important predictor of academic achievement for year 2 ( age 67 years ) ; no effect was found for direct bullying ( woods & wolke , 2004 ) . the study was limited by the short time lag between the measure for bullying and that for academic achievement . other studies have not looked specifically at bullying but have found links between peer rejection and academic achievement ( ladd , 1990 ; wentzel & caldwell , 1997 ) . a prospective study found that , amongst the youngest cohort , those that were most rejected by their peers performed worse on tests . however , they found no direct relationship between peer rejection and later academic achievement ( derosier , kupersmidt , & patterson , 1994 ) . responses to peer rejection have also been found to be important ; aggressive - rejected but not submissive - rejected adolescents had low academic achievement ( wentzel & asher , 1995 ) . although a large number of studies have looked at bullying prevention strategies , few studies have examined processes that may already be operating to mitigate the negative effects of bullying ( baldry & farrington , 2005 ) . however , it has been argued that in adolescence , individuals spend more unsupervised time with their peers and friends are increasingly relied upon for support ( marini , dane , bosacki , & ylc - cura , 2006 ) . adjustment to secondary school may therefore have important links with the ability of young people to initiate and maintain positive peer relationships ( wentzel & caldwell , 1997 ) . there are a number of mechanisms whereby high levels of social support might translate into more positive outcomes for students who are bullied . house ( 1981 ) identified 4 main types of social support : emotional , instrumental , informational and appraisal support . places particular emphasis on the emotionally or instrumentally sustaining quality of social relationships in relation to social support ( house , landis , & umberson , 1988 ) . ( 2005 ) conceptualise bullying as a chronic stressor and social support as one of the coping resources on which bullied students can draw . instrumental support may also be important ; as well as operating to enable individuals to cope with bullying whilst it is occurring , high levels of social support may stop bullying at an earlier stage . bullying is a group phenomenon ; bystanders can have an important impact on the trajectory that the bullying takes by either assisting the bully , not getting involved or by intervening . in most cases bystanders will remain uninvolved which enables the bully to continue ( fekkes , pijpers , & verloove - vanhorick , 2005 ) . it has been shown , however , that if bystanders do try to stop the bullying , it usually ceases ( hawkins , pepler , & craig , 2001 ) . there is also a possibility of reverse causation ; students with low levels of social support may be more likely to be bullied ; their isolation may draw bullies to them ( newman et al . , 2005 ) . one way in which instrumental support might work is through students with more friends receiving more academically - related assistance . in this way social support might have a direct impact on achievement ( wentzel & caldwell , 1997 ) . alternatively , the impact of social support may be indirect ; students with high levels of emotional social support may be less likely to suffer from psychological distress which in turn predicts higher academic achievement ( wentzel , 1994 ; wentzel , weinberger , ford , & feldman , 1990 ) . vygotsky s augmentation of cognitive development theory , which gave prominence to social interaction in influencing language and thought development , also places emphasis on interaction with peers as an important factor in facilitating intellectual progress ( vygotsky , 1978 ) . rigby ( 2000 ) found that social support and bullying contributed independently to poor mental health but that perceived availability of social support did not impact on victimised adolescents more than pupils that were not victimised . another study found evidence for a buffering effect of social support against anxiety and depressive symptoms for victims of bullying ( davidson & demaray , 2007 ) . holt and espelage ( 2007 ) found the lowest levels of anxiety and depression amongst bullies , victims and bully - victims with moderate peer social support ( holt & espelage , 2007 ) . ( 2005 ) looked at isolation as a moderator in reactions to bullying and found that being bullied does the most damage ( measured by stress on reaching college ) to those with high levels of perceived isolation . because bullying has a negative influence on children s health and educational outcomes it is important that healthcare workers and teachers have a good understanding of bullying and its potential consequences . this paper studies the association between bullying and both educational achievement and the exhibition of depressive symptoms in british secondary schools . based on the literature , the key hypotheses are as follows:1.victims of bullying will have a greater propensity to exhibit depressive symptoms2.victims of bullying will be less likely to reach national achievement benchmarks3.social support from friends will be more effective as a protective factor than social support from the family4.bullied students with low levels of perceived social support will exhibit poorer outcomes than bullied students with higher levels of perceived social support . victims of bullying will have a greater propensity to exhibit depressive symptoms victims of bullying will be less likely to reach national achievement benchmarks social support from friends will be more effective as a protective factor than social support from the family bullied students with low levels of perceived social support will exhibit poorer outcomes than bullied students with higher levels of perceived social support . the data come from the research with east london adolescents : community health survey ( relachs ) , a school based epidemiological study of a representative sample of 2790 adolescents from year 7 ( 1112 years ) and year 9 ( 1314 years ) attending 28 comprehensive schools in hackney , newham , and tower hamlets in 2001 ( stansfeld et al . , 2003 ) . of those pupils that did not participate , the majority were not available during due to school absence , illness or other school activities . the item used to measure whether an adolescent had been subjected to bullying was as follows : how often have you been bullied in school this term ? . i havent been bullied in school this term , once or twice , sometimes , about once a week , several times a week . a further category of never bullied was added based on another item : have you ever been bullied at school . the variable was then recoded into a binary variable whereby being a victim constituted experiencing bullying sometimes , about once a week or being bullied once or twice in a term was combined with the category for never being bullied because bullying is defined as a repeated action ( baldry & farrington , 2004 ; olweus , 1993 ; smith & brain , 2000 ) . educational achievement was measured two years after the baseline survey . for the younger age group , the benchmark used for educational achievement at age 1314 was the attainment of level 5 or above in english , mathematics and science in the key stage 3 examinations . these national tests are intended to indicate if a student is working at the target level for their age . for the older group , the benchmark was the attainment of 5 or more general certificate of secondary education examinations ( gcses ) at grades a*c ( taken at age 1516 ) . these benchmarks are used by the department of education and skills as an indicator of adequate performance ( department for education and skills , 2006 ) . data on educational achievement at key stage 3 and gcse was obtained from local education authorities . depressive symptoms were measured using the short moods and feelings questionnaire ( smfq ) two years after the baseline survey ( angold et al . , 1987 ) . statements about the emotions and behaviour of the respondent over the past 2 weeks were rated . there are 13 items in this scale : true , sometimes true , or not true . the scores for these items were summed to produce an overall magnitude of symptoms , with a score of 8 or above indicating the presence of depressive symptoms . in the original validation against the diagnostic interview schedule for children depressive scale this threshold yielded a positive predictive value to 80 per cent and a negative predictive value of 68 per cent ( angold et al . , 1995 ) . the degree of social support derived from family and friends was measured at baseline using the multidimensional scale of perceived social support ( mspss ) ( zimet , dahlem , zimet , & farley , 1988 ) . this is a 12-item scale which produces scores that measure levels of social support from three sources : family , friends and a significant other . this scale has been found to have a high level of internal and test retest reliability ; the overall reliability coefficient is 0.88 ( zimet et al . , 1988 ) . the scale has been found to have good concurrent , construct and discriminant validity ( zimet et al . , 1988 ) . social support scores were split into tertiles ( high , moderate and low support ) . ethnicity was categorised as follows using a revised version of the 2001 census : white uk , bangladeshi , pakistani , asian indian , black african , black caribbean , other . pupils are eligible for free school meals if their parents receive income support , income based jobseekers allowance , support under part vi of the immigration and asylum act 1999 or ( with some conditions ) child tax credit . all 42 schools in the three london boroughs were stratified by borough and school type ( comprehensive , voluntary , other ) . head teachers were informed about the study and asked for permission for their school to participate . within the 28 schools that agreed to take part , four representative , mixed ability classes were selected ( two from year 7 and two from year 9 ) . information sheets explaining the study were given to teachers , parents and pupils a week before the visit to the school . parents were given the opportunity to opt their child out of the study and pupils gave fully informed consent on the day . a team of researchers administered the questionnaire in classrooms in one session of 4050 min . one member of the team led the class , explaining the questionnaire and providing assurance that all answers would be anonymous and confidential . three or four additional researchers assisted , answering queries from participants , ensuring that participants did not confer with each other and checking for missing data on completion of the questionnaires . meetings were held with a community advisory group , consisting of teachers , parents , health and social care professionals , to advise on ethical aspects of the work and on the research process . the study protocol was approved by the east london and the city local research ethics committee . because the primary sampling unit for the study was the school , it was necessary to make adjustments for the clustered survey design in the analyses . failing to adjust for this would result in an overstatement of precision by ignoring the possible lack of independence of observations within the same school . an equal number of classes were selected in each school regardless of school size , so that probability of selection varied by school . data were reweighted to ensure that the data were representative of all adolescents attending comprehensive schools in the three boroughs at the time of the baseline survey . missing data was analysed using crosstabulation and the chi - squared option in stata . haenszel analysis was carried out to identify possible confounding variables . for the achievement models , these were gender , age group , ethnicity and eligibility for free school meals . independent variables were dropped from the model if they did not result in a change in the odds ratio for bullying . for the analysis examining the impact of bullying on depressive symptoms , however , if bullied children with a specific problem at baseline were to be included , it is harder to study the causal relationship between bullying and health outcomes . two sets of analyses were therefore carried out firstly including those depressed at baseline ( reported here ) and then excluding them . the results were in the same direction , although there was greater statistical evidence for the associations when those depressed at baseline were included . 48.6% of the participants were male ; 27.0% were white , 25.1% bangladeshi , 20.9% black , 9.1% indian , 6.7% pakistani and 11.2% of other ethnic origin . the average age of the younger age group was 12.18 ( standard deviation = 0.33 ) and the older age group 14.22 ( standard deviation = 0.33 ) ; 49.5% of the sample was in the younger age group . those who did not reach the academic benchmark were less likely to have data on bullying ( p = 0.003 ) . pupils who were bullied were less likely to have data on achievement ( p = 0.007 ) , as were white pupils ( p < 0.0001 ) and pupils eligible for free school meals ( p = 0.076 ) . those that were bullied ( p < 0.0001 ) , white pupils ( p < 0.0001 ) and pupils eligible for free school meals ( p = 0.001 ) were less likely to have complete data on depressive symptoms . 9.1% of the sample reported being bullied sometimes or more often in the term in which the survey was taken . table 1 shows the crude odds ratios for the association between bullying and the variables of interest . there was very strong evidence that those in the older age group were less likely to have been bullied ( or = 0.56 , 95% ci 0.39 , 0.80 ) . there was some evidence that indian pupils were less likely to have been subject to bullying in the last term ( or = 0.53 , 95% ci 0.29 , 0.96 ) . there was evidence that those with moderate ( or = 0.57 , 95% ci 0.46 , 0.71 ) and high ( or = 0.54 , 95% ci 0.35 , 0.83 ) levels of social support from friends were about half as likely to have been bullied . table 2 shows the crude odds ratios for the association between achievement and the main exposure variables . those that had been bullied in the last term were approximately half as likely to achieve the benchmark ( or = 0.46 , 95% ci 0.29 , 0.72 ) . those in the older age group were 1.5 times more likely to reach the benchmark than those in the younger age group ( or = 1.50 , 95% ci 1.12 , 2.03 ) . girls were more likely to reach the benchmark than boys ( or = 1.84 , 95% ci 1.18 , 2.86 ) . indian children were approximately twice as likely to reach the achievement as white pupils ( or = 1.97 , 95% ci 1.37 , 2.84 ) . there was very strong evidence for lower performance by those pupils eligible for free school meals ( or = 0.55 , 95% ci 0.43 , 0.71 ) . there was some evidence that high levels of social support from family were associated with lower levels of achievement ( or = 0.74 , 95% ci 0.59 , 0.93 ) . adjusting for age group , gender , ethnicity and eligibility for free school meals reduced the crude odds ratio for the sample to 0.46 ( 95% ci 0.28 , 0.76 ) , a very modest reduction . there was little evidence for social support from friends as a confounder of the relationship between bullying and achievement ; the odds ratio fell to 0.45 when this was added to the model ( 95% ci 0.27 , 0.75 ) . support from family also had little effect ( or = 0.46 , 95% ci 0.28 , 0.76 ) . mantel haenszel analysis provided some evidence for an interaction between bullying and social support from friends ( test for homogeneity of odds ratios p - value = 0.013 ) and a very weak suggestion of an interaction between bullying and social support from family ( test for homogeneity of odds ratios p - value = 0.130 ) . after adjustment for a bullying friends social support interaction , bullying was not associated with educational achievement for students with high levels of support from friends . for students who did not have high levels of support from friends , bullying was associated with lower odds of achieving the academic benchmark . bullied students with moderate ( or = 0.27 , 95% ci 0.110.65 ) or low ( or = 0.38 , 95% ci 0.190.76 ) levels of support had approximately a third of the odds of achieving the academic benchmark , compared to students who were not bullied . introducing the bullying family social support interaction resulted in an interesting finding ; for students with moderate levels of support from family , bullying did not appear to be associated with lower odds of achieving the academic benchmark ( or = 1.00 , 95% ci 0.492.06 ) . however , there was evidence that bullied students with either high ( or = 0.20 , 95% ci 0.070.51 ) or low ( or = 0.40 , 95% ci 0.210.76 ) levels of social support from family had lower odds of achieving the benchmark compared to those students who were not bullied . table 4 shows the crude odds ratios for the association between depressive symptoms and the main exposure variables . those bullied in the last term were approximately one and a half times more likely to have depressive symptoms ( or = 1.42 , 95% ci 1.05 , 1.94 ) . there was weak evidence that those in the older age group were more likely to be depressed ( or = 1.25 , 95% ci 0.99 , 1.59 ) . they were more than twice as likely to show depressive symptoms as boys ( or = 2.16 , 95% ci 1.74 , 2.68 ) . pupils with high levels of family social support were less than half as likely to have depressive symptoms ( or = 0.44 , 95% ci 0.31 , 0.61 ) . moderate family social support was also associated with lower odds of depressive symptoms ( or = 0.60 , 95% ci 0.49 , 0.72 ) . there was evidence from the mantel haenszel analysis for a difference in odds ratios by gender ( test for homogeneity of odds ratios p - value = 0.019 ) . adjusting for age group , gender and ethnicity with a gender bullying interaction led to an odds ratio for bullying for boys of 2.34 ( 95% ci 1.41 , 3.87 ) and an odds ratio of 1.11 ( 95% ci 0.67 , 1.84 ) for girls . bullying thus appeared to have an effect on the mental health of boys , but not that of girls . adjusting for social support from friends led to a reduction in the odds ratio for boys to 2.17 ( 95% ci 1.31 , 3.61 ) although bullying still had a significant impact ( p = 0.004 ) . adjusting for social support from family did not result in the impact of bullying on depression being attenuated ( or = 2.29 , 95% ci 1.34 , 3.93 ) . adding depressive symptoms at baseline to the model resulted in a reduction in the odds ratio for bullying to 1.60 ( 95% ci 0.92 , 2.77 ) . weak evidence for an association between being bullied and depressive symptoms for boys at follow - up remained ( p = 0.091 ) . boys and girls were equally likely to be victims of bullying . there were few differences in the percentage of students who were bullied across ethnic groups . there was evidence that bullying decreased with age and that those with high levels of social support from their friends were less likely to be bullied . there was evidence that a high level of support from friends was able to protect bullied adolescents from poor achievement at school . a moderate ( but not high ) level of support from the family was also protective . other research has observed a decline in bullying prevalence with age , as is found here ( newman et al . , 2005 ; the importance of social support from friends ( as opposed to family ) in reducing an adolescent s chance of being bullied is of interest . this supports previous literature which has argued that adolescents spend increasing amounts of unsupervised time with their peers rather than their family ( marini et al . , 2006 ; it has been argued that their support may be more important than family support as it is in adolescence that independence from parents is developed ( sebald , 1992 ; youniss & smollar , 1985 ) . being bullied had a strong impact on an adolescent s chances of achieving the national academic benchmark for their age . one possible mechanism for the finding is that those that are bullied have been found to be more likely to play truant or be absent from school for other reasons ( smith et al . , 2004 ) . it does not support accounts that suggest that bullied pupils are able to turn to and succeed at school work in order to escape from the problems that they are experiencing ( sharp , 1995 ) . family social support protects against poor outcomes only when it is provided at a moderate level . low or high support is associated with decreased odds of reaching the academic benchmark for pupils who are bullied . it may be that social support operates to produce a variety of outcomes . whilst some argue that parental support is important for the development of resilience in adolescents , over - protective parenting may have the opposite effect and make young people less assertive and independent ( bowen , smith , & binney , 1994 ; olweus , 1993 ; swihart & cotter , 1997 ) . high levels of social support from friends , on the other hand , do appear to be able to protect against the negative impact of bullying . it may be that a high level of social support from friends has a particular role in limiting the extent of any bullying that does occur . friends may intervene rather than standing by ; it has been shown that if bystanders become involved in preventing their friends being bullied , they are usually successful ( hawkins et al . , 2001 ) . when a gender bullying interaction was introduced into the analysis , it was found that this was only the case for boys . a key question raised by the analysis , then , is why boys are affected by the stress of bullying and girls are not . a finnish study found that girls more often reported using the more constructive tactic of stress - recognition ( for example crying or screaming in addition to seeking support ) as a strategy than boys ( olafsen & viemero , 2000 ) . another study in finland found that a higher proportion of girls than boys used the effective strategy of nonchalance in response to bullying , although both boys and girls frequently used less helpful responses . in the case of girls strategies that seek compromise may also be effective ( feldman & gowan , 1998 ) . it has been found that girls are more likely to run for help or comfort the victim or demonstrate conflict resolution strategies ( feldman & gowan , 1998 ; osterman et al . , 1997 ; salmivalli , lagerspetz , bjorkqvist , osterman , & kaukiainen , 1996 ) . the main strengths of the study lie in its use of a representative sample of young people and the availability of standardised and previously validated measures of psychological distress . the large minority ethnic component of the sample enabled analysis by ethnic grouping ; this has often been a problem in analysis of educational differentials by ethnicity ( demack , drew , & grimsley , 2000 ; drew & gray , 1990 ) . the data is also unusual in that it provides comprehensive measures of psychological distress alongside information on educational achievement ; few british datasets provide this . however , the term bullying is more familiar in english than in a number of other languages and has been in usage for a long time ( smith , cowie , olafsson , & liefooghe , 2002 ) . ( 2002 ) found that fourteen year olds in their sample were able to clearly separate physical aggression and physical bullying using cartoon depictions . one is the unmeasured results of an adolescent s realisation that they are being bullied . it may be that adolescents who recognise the bullying that they are being subjected to are better equipped than those that do not as they are more prepared to respond in an assertive manner . it is possible , then , that the analysis reported here underestimates the negative effects of bullying as it does not take account of those adolescents that are bullied but are unable or unwilling to recognise the nature of the treatment that they are experiencing . however , perceived support has been found to be protective , even when perceptions are not accurate ( lakey & cassady , 1990 ) . there was evidence that those who did not reach the academic benchmark and who had depressive symptoms were less likely to have data on bullying . this may have led to an over- or underestimation of the association between bullying and the key outcomes . sensitivity analysis was carried out whereby it was assumed in the first set of analyses that all pupils with missing data on bullying had experienced being bulled and in the second set of analyses that they had not . although support from family and friends can protect adolescents against poor academic outcomes , the analysis here has demonstrated that support from friends and family alone can not mitigate against the strong negative effect that bullying has on mental health amongst secondary school pupils in east london . it has been shown in other studies that being bullied can have a longer term impact and contribute to both poor mental health , trouble with personal relationships and unemployment risk in adulthood ( gladstone , parker , & malhi , 2006 ; hugh - jones & smith , 1999 ; varhama & bjorkqvist , 2005 ) . given its substantial impact , it seems vital that strategies to tackle the problem are developed to prevent the development of the most serious consequences such as suicide attempts ( rigby & slee , 1999 ) . early and wide - ranging intervention may avoid the need for specialist psychiatric consultation , to which bullied children are disproportionately referred ( kumpulainen et al .

this paper investigates the extent to which social support can have a buffering effect against the potentially adverse consequences of bullying on school achievement and mental health . it uses a representative multiethnic sample of adolescents attending east london secondary schools in three boroughs . bullied adolescents were less likely to achieve the appropriate academic achievement benchmark for their age group and bullied boys ( but not girls ) were more likely to exhibit depressive symptoms compared to those not bullied . high levels of social support from family were important in promoting good mental health . there was evidence that high levels of support from friends and moderate ( but not high ) family support was able to protect bullied adolescents from poor academic achievement . support from friends and family was not sufficient to protect adolescents against mental health difficulties that they might face as a result of being bullied . more active intervention from schools is recommended .

Introduction Methods Results Discussion Conclusions and future research

bullying is broadly defined as a desire to hurt and the execution of a harmful action ; it is characterised by repetition and either a physical or a psychological power imbalance ( farrington , 1993 ; rigby , 2002 ; smith & brain , 2000 ; smith & sharp , 1994 ) . the first two forms of bullying have sometimes been called direct bullying as they include directly aggressive behaviour . research has shown that boys are more likely to be involved in bullying , either as bullies or as victims , than girls ( forero , mclellan , rissel , & bauman , 1999 ; kaltiala - heino , rimpela , rantanen , & rimpela , 2000 ; nansel et al . rigby ( 2000 ) found that boys and girls were similarly likely to be bullied through name calling , teasing and deliberate exclusion . boys are more likely to receive threats of harm and be physically bullied ( baldry , 2004 ; owens , shute , & slee , 2000 ; rigby , 2000 ; roland & idsoe , 2000 ) . there is evidence that prevalence of bullying declines with age ( newman et al . , 2005 ; cross - sectional research on the effects of bullying on psychological health has consistently found that those that are bullied exhibit poorer emotional adjustment in early to late adolescence , indicating an association but no direct causality ( alikasifoglu , erginoz , ercan , uysal , & albayrak - kaymak , 2007 ; baldry , 2004 ; brunstein klomak , marrocco , kleinman , schonfeld , & gould , 2007 ; espelage & holt , 2001 ; fekkes , pijpers , & verloove - vanhorick , 2004 ; forero et al . found that victims of bullying had significantly higher chances of psychosomatic and psychosocial problems compared with children who were not bullied ; conversely , children with depressive and anxiety symptoms had a higher chance of being bullied ( fekkes , pijpers , fredriks , vogels , & verloove - vanhorick , 2006 ) . a longitudinal study in uk primary schools found that being a victim of relational bullying was the second most important predictor of academic achievement for year 2 ( age 67 years ) ; no effect was found for direct bullying ( woods & wolke , 2004 ) . the study was limited by the short time lag between the measure for bullying and that for academic achievement . however , they found no direct relationship between peer rejection and later academic achievement ( derosier , kupersmidt , & patterson , 1994 ) . responses to peer rejection have also been found to be important ; aggressive - rejected but not submissive - rejected adolescents had low academic achievement ( wentzel & asher , 1995 ) . although a large number of studies have looked at bullying prevention strategies , few studies have examined processes that may already be operating to mitigate the negative effects of bullying ( baldry & farrington , 2005 ) . there are a number of mechanisms whereby high levels of social support might translate into more positive outcomes for students who are bullied . house ( 1981 ) identified 4 main types of social support : emotional , instrumental , informational and appraisal support . places particular emphasis on the emotionally or instrumentally sustaining quality of social relationships in relation to social support ( house , landis , & umberson , 1988 ) . ( 2005 ) conceptualise bullying as a chronic stressor and social support as one of the coping resources on which bullied students can draw . instrumental support may also be important ; as well as operating to enable individuals to cope with bullying whilst it is occurring , high levels of social support may stop bullying at an earlier stage . bullying is a group phenomenon ; bystanders can have an important impact on the trajectory that the bullying takes by either assisting the bully , not getting involved or by intervening . there is also a possibility of reverse causation ; students with low levels of social support may be more likely to be bullied ; their isolation may draw bullies to them ( newman et al . in this way social support might have a direct impact on achievement ( wentzel & caldwell , 1997 ) . alternatively , the impact of social support may be indirect ; students with high levels of emotional social support may be less likely to suffer from psychological distress which in turn predicts higher academic achievement ( wentzel , 1994 ; wentzel , weinberger , ford , & feldman , 1990 ) . rigby ( 2000 ) found that social support and bullying contributed independently to poor mental health but that perceived availability of social support did not impact on victimised adolescents more than pupils that were not victimised . another study found evidence for a buffering effect of social support against anxiety and depressive symptoms for victims of bullying ( davidson & demaray , 2007 ) . holt and espelage ( 2007 ) found the lowest levels of anxiety and depression amongst bullies , victims and bully - victims with moderate peer social support ( holt & espelage , 2007 ) . ( 2005 ) looked at isolation as a moderator in reactions to bullying and found that being bullied does the most damage ( measured by stress on reaching college ) to those with high levels of perceived isolation . because bullying has a negative influence on children s health and educational outcomes it is important that healthcare workers and teachers have a good understanding of bullying and its potential consequences . this paper studies the association between bullying and both educational achievement and the exhibition of depressive symptoms in british secondary schools . based on the literature , the key hypotheses are as follows:1.victims of bullying will have a greater propensity to exhibit depressive symptoms2.victims of bullying will be less likely to reach national achievement benchmarks3.social support from friends will be more effective as a protective factor than social support from the family4.bullied students with low levels of perceived social support will exhibit poorer outcomes than bullied students with higher levels of perceived social support . victims of bullying will have a greater propensity to exhibit depressive symptoms victims of bullying will be less likely to reach national achievement benchmarks social support from friends will be more effective as a protective factor than social support from the family bullied students with low levels of perceived social support will exhibit poorer outcomes than bullied students with higher levels of perceived social support . the data come from the research with east london adolescents : community health survey ( relachs ) , a school based epidemiological study of a representative sample of 2790 adolescents from year 7 ( 1112 years ) and year 9 ( 1314 years ) attending 28 comprehensive schools in hackney , newham , and tower hamlets in 2001 ( stansfeld et al . the variable was then recoded into a binary variable whereby being a victim constituted experiencing bullying sometimes , about once a week or being bullied once or twice in a term was combined with the category for never being bullied because bullying is defined as a repeated action ( baldry & farrington , 2004 ; olweus , 1993 ; smith & brain , 2000 ) . for the younger age group , the benchmark used for educational achievement at age 1314 was the attainment of level 5 or above in english , mathematics and science in the key stage 3 examinations . these national tests are intended to indicate if a student is working at the target level for their age . depressive symptoms were measured using the short moods and feelings questionnaire ( smfq ) two years after the baseline survey ( angold et al . the scores for these items were summed to produce an overall magnitude of symptoms , with a score of 8 or above indicating the presence of depressive symptoms . in the original validation against the diagnostic interview schedule for children depressive scale this threshold yielded a positive predictive value to 80 per cent and a negative predictive value of 68 per cent ( angold et al . the degree of social support derived from family and friends was measured at baseline using the multidimensional scale of perceived social support ( mspss ) ( zimet , dahlem , zimet , & farley , 1988 ) . this is a 12-item scale which produces scores that measure levels of social support from three sources : family , friends and a significant other . this scale has been found to have a high level of internal and test retest reliability ; the overall reliability coefficient is 0.88 ( zimet et al . social support scores were split into tertiles ( high , moderate and low support ) . all 42 schools in the three london boroughs were stratified by borough and school type ( comprehensive , voluntary , other ) . the study protocol was approved by the east london and the city local research ethics committee . data were reweighted to ensure that the data were representative of all adolescents attending comprehensive schools in the three boroughs at the time of the baseline survey . for the achievement models , these were gender , age group , ethnicity and eligibility for free school meals . for the analysis examining the impact of bullying on depressive symptoms , however , if bullied children with a specific problem at baseline were to be included , it is harder to study the causal relationship between bullying and health outcomes . the results were in the same direction , although there was greater statistical evidence for the associations when those depressed at baseline were included . the average age of the younger age group was 12.18 ( standard deviation = 0.33 ) and the older age group 14.22 ( standard deviation = 0.33 ) ; 49.5% of the sample was in the younger age group . those who did not reach the academic benchmark were less likely to have data on bullying ( p = 0.003 ) . pupils who were bullied were less likely to have data on achievement ( p = 0.007 ) , as were white pupils ( p < 0.0001 ) and pupils eligible for free school meals ( p = 0.076 ) . those that were bullied ( p < 0.0001 ) , white pupils ( p < 0.0001 ) and pupils eligible for free school meals ( p = 0.001 ) were less likely to have complete data on depressive symptoms . there was very strong evidence that those in the older age group were less likely to have been bullied ( or = 0.56 , 95% ci 0.39 , 0.80 ) . there was some evidence that indian pupils were less likely to have been subject to bullying in the last term ( or = 0.53 , 95% ci 0.29 , 0.96 ) . there was evidence that those with moderate ( or = 0.57 , 95% ci 0.46 , 0.71 ) and high ( or = 0.54 , 95% ci 0.35 , 0.83 ) levels of social support from friends were about half as likely to have been bullied . table 2 shows the crude odds ratios for the association between achievement and the main exposure variables . those that had been bullied in the last term were approximately half as likely to achieve the benchmark ( or = 0.46 , 95% ci 0.29 , 0.72 ) . those in the older age group were 1.5 times more likely to reach the benchmark than those in the younger age group ( or = 1.50 , 95% ci 1.12 , 2.03 ) . girls were more likely to reach the benchmark than boys ( or = 1.84 , 95% ci 1.18 , 2.86 ) . indian children were approximately twice as likely to reach the achievement as white pupils ( or = 1.97 , 95% ci 1.37 , 2.84 ) . there was very strong evidence for lower performance by those pupils eligible for free school meals ( or = 0.55 , 95% ci 0.43 , 0.71 ) . there was some evidence that high levels of social support from family were associated with lower levels of achievement ( or = 0.74 , 95% ci 0.59 , 0.93 ) . adjusting for age group , gender , ethnicity and eligibility for free school meals reduced the crude odds ratio for the sample to 0.46 ( 95% ci 0.28 , 0.76 ) , a very modest reduction . there was little evidence for social support from friends as a confounder of the relationship between bullying and achievement ; the odds ratio fell to 0.45 when this was added to the model ( 95% ci 0.27 , 0.75 ) . support from family also had little effect ( or = 0.46 , 95% ci 0.28 , 0.76 ) . mantel haenszel analysis provided some evidence for an interaction between bullying and social support from friends ( test for homogeneity of odds ratios p - value = 0.013 ) and a very weak suggestion of an interaction between bullying and social support from family ( test for homogeneity of odds ratios p - value = 0.130 ) . after adjustment for a bullying friends social support interaction , bullying was not associated with educational achievement for students with high levels of support from friends . for students who did not have high levels of support from friends , bullying was associated with lower odds of achieving the academic benchmark . bullied students with moderate ( or = 0.27 , 95% ci 0.110.65 ) or low ( or = 0.38 , 95% ci 0.190.76 ) levels of support had approximately a third of the odds of achieving the academic benchmark , compared to students who were not bullied . introducing the bullying family social support interaction resulted in an interesting finding ; for students with moderate levels of support from family , bullying did not appear to be associated with lower odds of achieving the academic benchmark ( or = 1.00 , 95% ci 0.492.06 ) . however , there was evidence that bullied students with either high ( or = 0.20 , 95% ci 0.070.51 ) or low ( or = 0.40 , 95% ci 0.210.76 ) levels of social support from family had lower odds of achieving the benchmark compared to those students who were not bullied . those bullied in the last term were approximately one and a half times more likely to have depressive symptoms ( or = 1.42 , 95% ci 1.05 , 1.94 ) . there was weak evidence that those in the older age group were more likely to be depressed ( or = 1.25 , 95% ci 0.99 , 1.59 ) . they were more than twice as likely to show depressive symptoms as boys ( or = 2.16 , 95% ci 1.74 , 2.68 ) . pupils with high levels of family social support were less than half as likely to have depressive symptoms ( or = 0.44 , 95% ci 0.31 , 0.61 ) . moderate family social support was also associated with lower odds of depressive symptoms ( or = 0.60 , 95% ci 0.49 , 0.72 ) . there was evidence from the mantel haenszel analysis for a difference in odds ratios by gender ( test for homogeneity of odds ratios p - value = 0.019 ) . bullying thus appeared to have an effect on the mental health of boys , but not that of girls . adjusting for social support from friends led to a reduction in the odds ratio for boys to 2.17 ( 95% ci 1.31 , 3.61 ) although bullying still had a significant impact ( p = 0.004 ) . adjusting for social support from family did not result in the impact of bullying on depression being attenuated ( or = 2.29 , 95% ci 1.34 , 3.93 ) . adding depressive symptoms at baseline to the model resulted in a reduction in the odds ratio for bullying to 1.60 ( 95% ci 0.92 , 2.77 ) . weak evidence for an association between being bullied and depressive symptoms for boys at follow - up remained ( p = 0.091 ) . boys and girls were equally likely to be victims of bullying . there was evidence that bullying decreased with age and that those with high levels of social support from their friends were less likely to be bullied . there was evidence that a high level of support from friends was able to protect bullied adolescents from poor achievement at school . a moderate ( but not high ) level of support from the family was also protective . , 2005 ; the importance of social support from friends ( as opposed to family ) in reducing an adolescent s chance of being bullied is of interest . being bullied had a strong impact on an adolescent s chances of achieving the national academic benchmark for their age . one possible mechanism for the finding is that those that are bullied have been found to be more likely to play truant or be absent from school for other reasons ( smith et al . it does not support accounts that suggest that bullied pupils are able to turn to and succeed at school work in order to escape from the problems that they are experiencing ( sharp , 1995 ) . low or high support is associated with decreased odds of reaching the academic benchmark for pupils who are bullied . it may be that social support operates to produce a variety of outcomes . high levels of social support from friends , on the other hand , do appear to be able to protect against the negative impact of bullying . it may be that a high level of social support from friends has a particular role in limiting the extent of any bullying that does occur . friends may intervene rather than standing by ; it has been shown that if bystanders become involved in preventing their friends being bullied , they are usually successful ( hawkins et al . a key question raised by the analysis , then , is why boys are affected by the stress of bullying and girls are not . a finnish study found that girls more often reported using the more constructive tactic of stress - recognition ( for example crying or screaming in addition to seeking support ) as a strategy than boys ( olafsen & viemero , 2000 ) . it has been found that girls are more likely to run for help or comfort the victim or demonstrate conflict resolution strategies ( feldman & gowan , 1998 ; osterman et al . the main strengths of the study lie in its use of a representative sample of young people and the availability of standardised and previously validated measures of psychological distress . one is the unmeasured results of an adolescent s realisation that they are being bullied . it may be that adolescents who recognise the bullying that they are being subjected to are better equipped than those that do not as they are more prepared to respond in an assertive manner . it is possible , then , that the analysis reported here underestimates the negative effects of bullying as it does not take account of those adolescents that are bullied but are unable or unwilling to recognise the nature of the treatment that they are experiencing . there was evidence that those who did not reach the academic benchmark and who had depressive symptoms were less likely to have data on bullying . although support from family and friends can protect adolescents against poor academic outcomes , the analysis here has demonstrated that support from friends and family alone can not mitigate against the strong negative effect that bullying has on mental health amongst secondary school pupils in east london . it has been shown in other studies that being bullied can have a longer term impact and contribute to both poor mental health , trouble with personal relationships and unemployment risk in adulthood ( gladstone , parker , & malhi , 2006 ; hugh - jones & smith , 1999 ; varhama & bjorkqvist , 2005 ) . early and wide - ranging intervention may avoid the need for specialist psychiatric consultation , to which bullied children are disproportionately referred ( kumpulainen et al .

we performed a retrospective study of individuals with domestically acquired c. jejuni infection in order to assess factors associated with related hospitalisations in sweden between november 2011 and october 2012 . a total of 8,585 notifications of cases with laboratory - confirmed campylobacter infection were received at the public health agency of sweden during the study period , and 3,434 from those were classified as having been acquired in sweden . clinical microbiological laboratories in sweden were asked to submit all isolates obtained from domestically acquired campylobacter cases to the national veterinary institute ( sva ) in uppsala for mlst typing during the study period ; a total of 1,914 samples were received from 25 laboratories . from these samples , 1,139 were selected for typing by stratified random sampling to generate an equal sampling fraction through the year and the region . a total of 1,139 c. jejuni stool isolates obtained from domestically acquired cases between november 2011 and october 2012 were included in our study . these isolates had already been typed at the sva according to the previously published mlst protocol ( 7 ) . data collected included swedish personal identification number , age , sex , and name of the laboratory where primary diagnostic testing was performed as well as the date of sampling and mlst type . the personal identification numbers from all 1,139 c. jejuni cases were used to match the microbiological data to the registry of hospitalisation from the swedish board of health and welfare . clinical data obtained included all hospitalisation events for these patients from january 2011 to december 2012 , including admission and discharge dates , primary diagnosis , and other diagnosis encoded with the icd-10 system . hospitalisation events occurring within 2 weeks from the c. jejuni isolation were considered potentially related to the campylobacter infection ; these diagnostic codes were checked individually and if unlikely to be related to campylobacter infection ( i.e. road traffic accident or burns ) , hospitalisation was considered as non - related . in addition , patients without matching hospitalisation records or hospitalisation outside the window period were considered not having been hospitalised due to campylobacter infection . campylobacter infection was defined as domestic if the individual had not travelled abroad in the 2-week period before the laboratory diagnosis . co - morbidity was defined as having a chronic underlying health condition , including chronic heart , lung , kidney , and liver disease , or being immunocompromised ( any form of neoplasm or acquired immunodeficiency ) based on provided icd-10 codes . typed isolates were excluded from this study if it was not possible to match the sample identifier to a record from registry of hospitalisations from the swedish board of health and welfare . the factors associated with two separate outcome measures , hospitalisation ( yes / no ) and length of hospitalisation ( in days ) , were investigated . the factors included c. jejuni st , county of residence , age , sex , and co - morbidity . variables which were present in less than 33 cases ( i.e. detection frequency < 3% ) were omitted from the analysis ; p - values under 0.05 were considered statistically significant.hospitalisation . univariable analyses to investigate associations between the risk factors and hospitalisation were performed using the 22 chi - square test . risk ratios ( rrs ) with 95% confidence intervals [ ci ] were calculated . to adjust the rrs for potential confounders such as age , sex , and co - morbidity , a multivariable analysis using a logistic regression model was performed . risk factors with p<0.20 in the univariable analysis were included in a multivariable model built through stepwise forward selection using the likelihood ratio test.length of hospitalisation . univariable analysis to assess the association between the risk factors and length of hospitalisation ( i.e. time to discharge ) was performed using a cox proportional hazard model . risk factors with p<0.20 in the univariable analysis were included in a multivariable model built through stepwise forward selection using the likelihood ratio test.estimation of hospitalisation linked to campylobacter infections in sweden . based on the reported numbers of campylobacter infections and the hospitalisation rate obtained in this study , we have estimated the total number of hospitalisations linked to domestic campylobacter infections in sweden during the 1-year study period . univariable analyses to investigate associations between the risk factors and hospitalisation were performed using the 22 chi - square test . to adjust the rrs for potential confounders such as age , sex , and co - morbidity , a multivariable analysis using a logistic regression model was performed . risk factors with p<0.20 in the univariable analysis were included in a multivariable model built through stepwise forward selection using the likelihood ratio test . univariable analysis to assess the association between the risk factors and length of hospitalisation ( i.e. time to discharge ) was performed using a cox proportional hazard model . risk factors with p<0.20 in the univariable analysis were included in a multivariable model built through stepwise forward selection using the likelihood ratio test . estimation of hospitalisation linked to campylobacter infections in sweden . based on the reported numbers of campylobacter infections and the hospitalisation rate obtained in this study , we have estimated the total number of hospitalisations linked to domestic campylobacter infections in sweden during the 1-year study period . the confidentiality of study subjects was protected via anonymisation of the data at the sva . we performed a retrospective study of individuals with domestically acquired c. jejuni infection in order to assess factors associated with related hospitalisations in sweden between november 2011 and october 2012 . a total of 8,585 notifications of cases with laboratory - confirmed campylobacter infection were received at the public health agency of sweden during the study period , and 3,434 from those were classified as having been acquired in sweden . clinical microbiological laboratories in sweden were asked to submit all isolates obtained from domestically acquired campylobacter cases to the national veterinary institute ( sva ) in uppsala for mlst typing during the study period ; a total of 1,914 samples were received from 25 laboratories . from these samples , 1,139 were selected for typing by stratified random sampling to generate an equal sampling fraction through the year and the region . a total of 1,139 c. jejuni stool isolates obtained from domestically acquired cases between november 2011 and october 2012 were included in our study . these isolates had already been typed at the sva according to the previously published mlst protocol ( 7 ) . data collected included swedish personal identification number , age , sex , and name of the laboratory where primary diagnostic testing was performed as well as the date of sampling and mlst type . the personal identification numbers from all 1,139 c. jejuni cases were used to match the microbiological data to the registry of hospitalisation from the swedish board of health and welfare . clinical data obtained included all hospitalisation events for these patients from january 2011 to december 2012 , including admission and discharge dates , primary diagnosis , and other diagnosis encoded with the icd-10 system . hospitalisation events occurring within 2 weeks from the c. jejuni isolation were considered potentially related to the campylobacter infection ; these diagnostic codes were checked individually and if unlikely to be related to campylobacter infection ( i.e. road traffic accident or burns ) , hospitalisation was considered as non - related . in addition , patients without matching hospitalisation records or hospitalisation outside the window period were considered not having been hospitalised due to campylobacter infection . campylobacter infection was defined as domestic if the individual had not travelled abroad in the 2-week period before the laboratory diagnosis . co - morbidity was defined as having a chronic underlying health condition , including chronic heart , lung , kidney , and liver disease , or being immunocompromised ( any form of neoplasm or acquired immunodeficiency ) based on provided icd-10 codes . typed isolates were excluded from this study if it was not possible to match the sample identifier to a record from registry of hospitalisations from the swedish board of health and welfare . the factors associated with two separate outcome measures , hospitalisation ( yes / no ) and length of hospitalisation ( in days ) , were investigated . the factors included c. jejuni st , county of residence , age , sex , and co - morbidity . variables which were present in less than 33 cases ( i.e. detection frequency < 3% ) were omitted from the analysis ; p - values under 0.05 were considered statistically significant.hospitalisation . univariable analyses to investigate associations between the risk factors and hospitalisation were performed using the 22 chi - square test . risk ratios ( rrs ) with 95% confidence intervals [ ci ] were calculated . to adjust the rrs for potential confounders such as age , sex , and co - morbidity , a multivariable analysis using a logistic regression model was performed . risk factors with p<0.20 in the univariable analysis were included in a multivariable model built through stepwise forward selection using the likelihood ratio test.length of hospitalisation . univariable analysis to assess the association between the risk factors and length of hospitalisation ( i.e. time to discharge ) was performed using a cox proportional hazard model . risk factors with p<0.20 in the univariable analysis were included in a multivariable model built through stepwise forward selection using the likelihood ratio test.estimation of hospitalisation linked to campylobacter infections in sweden . based on the reported numbers of campylobacter infections and the hospitalisation rate obtained in this study , we have estimated the total number of hospitalisations linked to domestic campylobacter infections in sweden during the 1-year study period . univariable analyses to investigate associations between the risk factors and hospitalisation were performed using the 22 chi - square test . risk ratios ( rrs ) with 95% confidence intervals [ ci ] were calculated . to adjust the rrs for potential confounders such as age , sex , and co - morbidity , a multivariable analysis using a logistic regression model was performed . risk factors with p<0.20 in the univariable analysis were included in a multivariable model built through stepwise forward selection using the likelihood ratio test . univariable analysis to assess the association between the risk factors and length of hospitalisation ( i.e. time to discharge ) was performed using a cox proportional hazard model . risk factors with p<0.20 in the univariable analysis were included in a multivariable model built through stepwise forward selection using the likelihood ratio test . estimation of hospitalisation linked to campylobacter infections in sweden . based on the reported numbers of campylobacter infections and the hospitalisation rate obtained in this study , we have estimated the total number of hospitalisations linked to domestic campylobacter infections in sweden during the 1-year study period . the confidentiality of study subjects was protected via anonymisation of the data at the sva . a total of 1,139 c. jejuni isolates were included in this study , but 64 of these were excluded as it was not possible to match their sample identifiers to the data from the swedish board of health and welfare . the remaining 1,075 c. jejuni isolates consisted of 119 distinct c. jejuni sts and 26 ccs . the most frequent sts identified and included in statistical analyses were st-21 ( 139 isolates ; 12.9% [ belonging to the cc-21 ] ) , st-50 ( 116 ; 10.8% [ cc-21 ] ) , st-19 ( 104 ; 9.7% [ cc-21 ] ) , st-45 ( 105 ; 9.8% [ cc-45 ] ) , st-677 ( 90 ; 8.4% [ cc-677 ] ) , st-48 ( 88 ; 8.2% [ cc-48 ] ) , and st-257 ( 41 ; 3.8% [ cc-257 ] ) ; all the remaining sts were identified in less than 33 cases and were clustered into their own group named other ( 392 isolates ; 36.5% ) ( table 1 ) . although sts were further grouped into ccs , analyses have been performed based on sts as genetic diversity is less within sts than within the ccs ( and results based on the cc were in line with the results based on sts ; data not shown ) . overall , 76 sts were represented by only one or two isolates in the collection . samples were obtained from all 21 swedish counties ; those counties which had less than 33 campylobacter cases in total ( i.e. frequency < 3% regardless of st ) were further clustered into their own group named the sample was geographically and temporally representative of all domestic cases reported in sweden during the study period ( data not shown ) . the frequency of detection and hospital admission , length of hospital admission and mean age of individuals infected with c. jejuni mlst sequence types ( st ) in sweden , between november 2011 and october 2012 sequence types which were identified in les s than 33 isolates have been combined into this a total of 289 individuals with c. jejuni infection were hospitalised ( 26.9% ; table 1 ) : the smallest proportion of hospitalisation was noted for those infected with c. jejuni st-19 ( 19.4% ) and highest for those infected with st-257 ( 40% ) . most individuals ( 98% ; 292/298 ) were hospitalised within 7 days ( from 2 days before to 5 days after ) from microbiological diagnosis of c. jejuni ; the remaining six were hospitalised either 4 days ( three individuals ) or 5 days ( three individuals ) before the diagnosis . most individuals hospitalised with c. jejuni infection were admitted either with the diagnosis of enteritis or colitis ( n=265 ) or with symptoms of diarrhoea and abdominal pain ( n=6 ) , and the reason for admission was not given for five individuals . however , campylobacter - specific icd-10 diagnostic code was used for 148 hospitalised individuals ( a405 ; 51% ) . a total of seven individuals with colitis were known to have either ulcerative colitis ( n=4 ) , diverticular disease ( n=1 ) , or crohn 's disease ( n=2 ) . isolates from gastroenteritis were represented in all sts , whereas most isolates obtained from individuals with inflammatory bowel disease were found in st-45 ( 4/6 ) , gbs in st-22 ( 1/1 ) , and sepsis in st-677 ( 1/1 ) . mean age of individuals infected with c. jejuni was 42.5 years , whereas individuals hospitalised with c. jejuni infections were generally older with a mean age of 50 years ( table 1 ) . the individuals infected and hospitalised with c. jejuni st-677 were the oldest ( mean age of 50.5 and 57.9 years , respectively ) . furthermore , individuals infected with c. jejuni st-21 ( 37.3 years ) and individuals hospitalised with c. jejuni st-50 ( mean age of 42.7 years ) were the youngest . infection with c. jejuni st-257 ( rr : 1.57 ; 95% ci : 1.082.30 ) , an age of 60 years or older ( rr : 2.13 ; 95% ci : 1.762.57 ) , and underlying co - morbidity ( rr : 4.49 ; 95% ci : 2.916.92 ) were factors significantly associated with hospitalisation among patients who had c. jejuni infection in sweden based on univariable analysis ( table 2 ) . most of the cases with reported co - morbidities , including being immunocompromised and those with chronic underlying conditions admitted to hospital , also had a laboratory - confirmed c. jejuni infection ( 83% ; 80/97 ) , whereas a smaller proportion of individuals without these co - morbidities were hospitalised with c. jejuni infection ( 22% ; 209/978 ) . furthermore , age under 20 years ( rr : 0.69 ; 95% ci : 0.500.95 ) or between 40 and 60 years ( rr : 0.69 ; 95% ci : 0.550.87 ) as well as being diagnosed in halland and skne counties ( rr : 0.45 ; 95% ci : 0.210.97 and rr : 0.67 ; 95% ci : 0.460.97 , respectively ) were identified as statistically significant protective factors for hospitalisation coinciding with c. jejuni infection . no significant differences in proportion of individuals with co - morbidities or in age distribution of individuals were observed between counties . potential covariates and their association with hospitalisation evaluated in univariable analysis using a 22 chi - square test in laboratory - confirmed domestic cases of c. jejuni in sweden , november 2011october 2012 variables with more than two categories ( st , county , and age group ) were dichotomised by category , using all the remaining observations were as the reference group ( e.g. st-21 vs all other sts ) . results for variables shown to be statistically significantly associated with hospitalisation ( p>0.05 ) are shown in bold face . nd = not determined for variables with prevalence < 3% . after correction for potential confounders , co - morbidity ( or : 13.89 ; 95% ci : 7.8824.46 ) , an age of 60 years or older ( or : 1.98 ; 95% ci : 1.223.25 ) , and c. jejuni st-257 ( or : 2.35 ; 95% ci : 1.154.81 ) were found to be independently associated with hospitalisation ( table 3 ) . in addition , vstra gtaland ( or : 0.59 ; 95% ci : 0.360.96 ) , halland ( or : 0.30 ; 95% ci : 0.210.97 ) , and skne ( or : 0.43 ; 95% ci : 0.460.97 ) counties were identified as statistically significant protecting factors for hospitalisation with c. jejuni infection in multivariable analysis . result of multivariable logistic regression analysis of variables associated with hospitalisation of laboratory - confirmed domestic cases of c. jejuni in sweden , november 2011october 2012 the results for variables shown to be statistically significantly associated with hospitalisation ( p>0.05 ) are shown in bold face . none of the factors investigated were significantly associated with duration of hospitalisation based on univariable analysis ( table 4 ) or multivariable analysis ( data not shown ) . duration of hospitalisation was not statistically different between cases infected with st-677 or any other c. jejuni sts . it varied from 2.51 days ( st-50 ) to 4.21 days ( st-48 ) , with the mean hospital stay of 3.20 days . duration of hospitalisation was not statistically significant between different age groups , but those older than 60 years were hospitalised longer than those under 60 years of age ( 4.07 days vs. 2.53 days [ < 20 years ] , 2.14 days [ 2039 years ] , and 3.12 days [ 4059 years ] ) . furthermore , those infected with c. jejuni who had underlying co - morbidities were also hospitalised for longer than those without ( 4.16 days vs. 2.84 days ) . variables investigated for their association with length of hospitalisation evaluated in univariable analysis using cox regression in laboratory - confirmed domestic cases of c. jejuni in sweden , november 2011october 2012 as the mean length of hospitalisation was 3.2 days , and 289 individuals with domestically acquired c. jejuni infections were hospitalised , it can be calculated that a total of 925 hospital bed days were used ( 95% ci : 879971 ) . as only a proportion of domestic c. jejuni infections were included in our study ( 1,075 from 3,434 ) , the true numbers would be bigger ( estimated around 2,954 bed days ) . furthermore , one case of gbs among our study population would translate into three cases among all hospitalised individuals due to domestic c. jejuni infections in sweden during the study period . infection with c. jejuni st-257 ( rr : 1.57 ; 95% ci : 1.082.30 ) , an age of 60 years or older ( rr : 2.13 ; 95% ci : 1.762.57 ) , and underlying co - morbidity ( rr : 4.49 ; 95% ci : 2.916.92 ) were factors significantly associated with hospitalisation among patients who had c. jejuni infection in sweden based on univariable analysis ( table 2 ) . most of the cases with reported co - morbidities , including being immunocompromised and those with chronic underlying conditions admitted to hospital , also had a laboratory - confirmed c. jejuni infection ( 83% ; 80/97 ) , whereas a smaller proportion of individuals without these co - morbidities were hospitalised with c. jejuni infection ( 22% ; 209/978 ) . furthermore , age under 20 years ( rr : 0.69 ; 95% ci : 0.500.95 ) or between 40 and 60 years ( rr : 0.69 ; 95% ci : 0.550.87 ) as well as being diagnosed in halland and skne counties ( rr : 0.45 ; 95% ci : 0.210.97 and rr : 0.67 ; 95% ci : 0.460.97 , respectively ) were identified as statistically significant protective factors for hospitalisation coinciding with c. jejuni infection . no significant differences in proportion of individuals with co - morbidities or in age distribution of individuals were observed between counties . potential covariates and their association with hospitalisation evaluated in univariable analysis using a 22 chi - square test in laboratory - confirmed domestic cases of c. jejuni in sweden , november 2011october 2012 variables with more than two categories ( st , county , and age group ) were dichotomised by category , using all the remaining observations were as the reference group ( e.g. st-21 vs all other sts ) . results for variables shown to be statistically significantly associated with hospitalisation ( p>0.05 ) are shown in bold face . nd = not determined for variables with prevalence < 3% . after correction for potential confounders , co - morbidity ( or : 13.89 ; 95% ci : 7.8824.46 ) , an age of 60 years or older ( or : 1.98 ; 95% ci : 1.223.25 ) , and c. jejuni st-257 ( or : 2.35 ; 95% ci : 1.154.81 ) in addition , vstra gtaland ( or : 0.59 ; 95% ci : 0.360.96 ) , halland ( or : 0.30 ; 95% ci : 0.210.97 ) , and skne ( or : 0.43 ; 95% ci : 0.460.97 ) counties were identified as statistically significant protecting factors for hospitalisation with c. jejuni infection in multivariable analysis . result of multivariable logistic regression analysis of variables associated with hospitalisation of laboratory - confirmed domestic cases of c. jejuni in sweden , november 2011october 2012 the results for variables shown to be statistically significantly associated with hospitalisation ( p>0.05 ) are shown in bold face . none of the factors investigated were significantly associated with duration of hospitalisation based on univariable analysis ( table 4 ) or multivariable analysis ( data not shown ) . duration of hospitalisation was not statistically different between cases infected with st-677 or any other c. jejuni sts . it varied from 2.51 days ( st-50 ) to 4.21 days ( st-48 ) , with the mean hospital stay of 3.20 days . duration of hospitalisation was not statistically significant between different age groups , but those older than 60 years were hospitalised longer than those under 60 years of age ( 4.07 days vs. 2.53 days [ < 20 years ] , 2.14 days [ 2039 years ] , and 3.12 days [ 4059 years ] ) . furthermore , those infected with c. jejuni who had underlying co - morbidities were also hospitalised for longer than those without ( 4.16 days vs. 2.84 days ) . variables investigated for their association with length of hospitalisation evaluated in univariable analysis using cox regression in laboratory - confirmed domestic cases of c. jejuni in sweden , november 2011october 2012 as the mean length of hospitalisation was 3.2 days , and 289 individuals with domestically acquired c. jejuni infections were hospitalised , it can be calculated that a total of 925 hospital bed days were used ( 95% ci : 879971 ) . as only a proportion of domestic c. jejuni infections were included in our study ( 1,075 from 3,434 ) , the true numbers would be bigger ( estimated around 2,954 bed days ) . furthermore , one case of gbs among our study population would translate into three cases among all hospitalised individuals due to domestic c. jejuni infections in sweden during the study period . this is the first study to determine the risk factors associated with the hospitalisation and the length of hospitalisation of individuals with domestically acquired c. jejuni infections in sweden . our study included 1,075 c. jejuni infected individuals , representing approximately 30% of all reported domestic c. jejuni cases in sweden during the 1-year study period . a high diversity of mlsts among human c. jejuni isolates with a total of 119 distinct sts were observed , from which the seven frequent sts ( st-21 , st-50 , st-19 , st-45 , st-677 , st-48 , and st-257 ) covered 64% of all isolates in accordance with previously published studies from the united kingdom , finland , denmark , and canada ( 914 , 24 , 26 ) . these sts were recently shown to have been responsible for a large proportion of domestic c. jejuni infections in sweden already in 2000 , and have also been isolated from swedish broilers ( 23 ) . it has been suggested that the poultry is likely the most important source of domestic c. jejuni infections ( 19 ) . although campylobacter infections are usually considered mild and self - limiting , they can also lead to more severe infections requiring hospitalisation ( 35 , 31 ) . up to 26.9% of individuals included in this study were admitted to hospital with the domestically acquired c. jejuni infection . we have matched the data on campylobacter infections obtained from the public health agency of sweden and from the registry of hospitalisations from the swedish board of health and welfare . based on our data , only 50% of hospitalisations were registered with campylobacter - specific icd-10 coding ( a04.5 ) , and the remaining were registered with non - specific gastroenteritis and colitis coding . the numbers on the hospitalisations related to campylobacter infections are likely underestimated in studies where only specific icd-10 codes are used without matching ( 3134 ) . only 5% of reported campylobacter cases were hospitalised according to the review of three national databases over a 4-year period in canada ( 32 ) , whereas an annual hospitalisation rate of 12.3% was obtained in a danish registry - based study ( 33 ) and 10% in us surveillance study ( 34 ) . as all the studies have used different methods to obtain the hospitalisation estimates , we can not state if the hospitalisation rates are truly increasing , if they are higher since a smaller proportion of milder infections have been sampled , or simply more accurately estimated than recorded previously . the correlation between co - morbidities and hospitalisation due to domestic c. jejuni infection is not surprising , given that most co - morbidities result in decreased immune function and thus more likely will lead to symptomatic c. jejuni enterocolitis . co - morbidity category also included all known immunocompromised individuals ( i.e. those with known malignancy ) . furthermore , individuals with co - morbidities are more likely to seek health care with gastrointestinal symptoms and are more likely to be tested for campylobacter than those without co - morbidities . there was also a trend towards more severe infections among the individuals with co - morbidities ; they were hospitalised for longer than those without ( 4.16 days vs. 2.84 days ) . although these differences were not statistically significant , they suggest more severe infections in those with co - morbidities . in a previous study , a high hospitalisation rate of 47.6% was described in patients with ulcerative colitis ( 35 ) . however , the main focus of the arora study ( 35 ) was to determine the risk factors for c. jejuni infection and not to investigate the risk factors associated with hospital admissions due to campylobacter infections . furthermore , previous studies have shown that those aged over 60 years are three times more likely to be hospitalised with campylobacter infection than younger individuals ( 34 ) , and that age > 60 years can be a risk factor for a longer hospital stay ( 28 ) . it is most likely that the older individuals have more diagnosed co - morbidities than the younger ones ; the factor not considered in those studies . we clearly demonstrate that co - morbidity is the most significant risk factor for hospitalisation while having c. jejuni infection , not old age . indeed , over 60% of all those hospitalised were younger than 60 years of age . it is not fully understood why the hospitalisation rates were lower in the southwestern parts of sweden ( vstra gtaland , halland , and skne counties ) than elsewhere . none of the swedish microbiology laboratories were using molecular methods for campylobacter detection during the study period . a potential explanation could be differences in study population , such as overrepresentation of the elderly or those with co - morbidities , but no significant differences were observed between counties . the lower hospitalisation rates in these counties could also reflect differences between counties in primary health care policies with regard to testing patients with gastrointestinal illness for campylobacter , where counties with a more liberal testing policy would be identified as protective in this literature due to the relatively larger number of cases detected , and not hospitalised . the finding could also be due to differences in hospitalisation policy ( i.e. stricter criteria for hospital admissions ) . only one study has previously investigated the association between c. jejuni sts and hospitalisation ; based on that it was expected that c. jejuni st-677 would be associated with more frequent hospitalisations and also with increased length of hospitalisation than other campylobacter types ( 13 ) . this hypothesis was also supported with more recent evidence demonstrating that c. jejuni st-677 is specifically associated with invasive infections ( i.e. campylobacteremia ) ( 27 ) . in our study , the association between c. jejuni st-677 and the increased hospitalisations or a longer hospital stay could not be demonstrated . however , it should be noted that the feodoroff study investigated blood culture isolates of c. jejuni collected over a 10-year study period , whereas the number of invasive c. jejuni infections during our 1-year study period would have been minimal ( 36 ) . interestingly , individuals infected with another less common c. jejuni type , st-257 , were shown to be at increased risk of hospital admissions in our study . although st-257 was recently isolated from domestic dogs in sweden ( 37 ) , only 4% of dogs were positive for c. jejuni and thus the importance of dogs as a source of human infections needs to be clarified further . in addition , st-50 was linked to the highest number of campylobacter related hospitalisations in sweden ( 37/289 ) . both of these types , st-257 and st-50 , although the pathogenesis of c. jejuni remains poorly understood , some c. jejuni types have been associated with severe infections . in our study , one individual was diagnosed with gbs and c. jejuni st-22 infection , whereas another individual with systemic invasive infection had c. jejuni st-677 , consistent with the previous literature ( 7 , 26 , 27 ) . furthermore , we identified another c. jejuni type , st-257 , as a risk factor significantly associated with hospitalisation . this st has not been linked to a specific disease outcome and thus the reason(s ) for the observed increased rate of hospitalisation is not currently known . however , c. jejuni st-257 was shown to be the most virulent type measured as larval survival in the insect galleria mellonella model ( 38 ) . it is likely that the genetic differences between c. jejuni types will determine their pathogenicity , as previously demonstrated by combining the data from whole - genome sequencing and phenotypical characterisation of c. jejuni st-677 ( 27 , 39 ) . although most campylobacter infections are self - limiting and do not require treatment , antibiotics are often given to immunocompromised individuals ( 25 ) , and thus it is important to consider possibility of resistance . in that context , it is important to note that a previous study from slovakia demonstrated equally high frequency of antibiotic resistance among isolates obtained from humans , animals , and food . these included universal persistence of ciprofloxacin - resistance among c. jejuni st-50 isolates ( 40 ) ; the second most common c. jejuni type in sweden . high levels of ciprofloxacin resistance were also previously identified not only among c. jejuni st-50 isolates but also among st-257 isolates in belgium ( 41 ) . however , much lower levels of ciprofloxacin - resistance was observed among these types in the united kingdom ( 10 ) . data on antibiotic resistance in human campylobacter isolates is not routinely collected in sweden , but a high level of ciprofloxacin resistance for untyped human campylobacter isolates was reported in 20022011 ( 49% ; ( 42 ) ) . for these reasons , further systematic monitoring of antimicrobial resistance in sweden for human campylobacter should also be considered . the clinical data obtained in this study were based on the diagnostic codes used and reported . as they have been assigned by individual doctors , they might differ according to the doctor and hospital practice . in addition , since only the diagnostic codes were used , we do lack other information on disease severity ( i.e. data on itu admission , need for iv fluids , antibiotic treatment , and resistance ) . it should also be noted that only individuals who have been diagnosed with certain disease entities will have been given the diagnostic codes ( i.e. young individuals with newly diagnosed neoplasm might still be under ongoing investigations and thus definite diagnosis may not have been reported as yet ) . however , these limitations are likely to result in underestimation of associations and can not be minimised within this study design . in addition , only campylobacter cases who sought medical care could be included in this study ( prerequisite for sampling ) and thus milder infections that did not require medical attention were not included in this study . this may result in an under- or over - estimation of the effect of genotype on hospitalisation since cases of mild illness may not be uniformly distributed among genotypes of c. jejuni , as well as overestimation of the population proportion of hospitalised cases . we have demonstrated that over a quarter of all individuals diagnosed with domestic c. jejuni infection are hospitalised in sweden , and that those infected with st-50 or st-257 are slightly more likely to be subject to hospital care than those infected with other sts . although over 50% of all hospitalised individuals were less than 60 years of age and did not have any co - morbidities and were not known to be immunocompromised in this study , the biggest risk factors for hospitalisation identified included co - morbidities and old age . we have shown that individuals with co - morbidities are at a 14-time higher risk of becoming hospitalised with a domestic c. jejuni infection than those without . as these co - morbidities are often seen in older people , the burden of domestic c. jejuni infections is likely to increase even further in the future , considering the ageing population trend . targeted public health messages including strict advice on kitchen hygiene and safe drinking water sources for those with co - morbidities as well as further investigations on antimicrobial resistance in campylobacter in humans in sweden the clinical data obtained in this study were based on the diagnostic codes used and reported . as they have been assigned by individual doctors , they might differ according to the doctor and hospital practice . in addition , since only the diagnostic codes were used , we do lack other information on disease severity ( i.e. data on itu admission , need for iv fluids , antibiotic treatment , and resistance ) . it should also be noted that only individuals who have been diagnosed with certain disease entities will have been given the diagnostic codes ( i.e. young individuals with newly diagnosed neoplasm might still be under ongoing investigations and thus definite diagnosis may not have been reported as yet ) . however , these limitations are likely to result in underestimation of associations and can not be minimised within this study design . in addition , only campylobacter cases who sought medical care could be included in this study ( prerequisite for sampling ) and thus milder infections that did not require medical attention were not included in this study . this may result in an under- or over - estimation of the effect of genotype on hospitalisation since cases of mild illness may not be uniformly distributed among genotypes of c. jejuni , as well as overestimation of the population proportion of hospitalised cases . we have demonstrated that over a quarter of all individuals diagnosed with domestic c. jejuni infection are hospitalised in sweden , and that those infected with st-50 or st-257 are slightly more likely to be subject to hospital care than those infected with other sts . although over 50% of all hospitalised individuals were less than 60 years of age and did not have any co - morbidities and were not known to be immunocompromised in this study , the biggest risk factors for hospitalisation identified included co - morbidities and old age . we have shown that individuals with co - morbidities are at a 14-time higher risk of becoming hospitalised with a domestic c. jejuni infection than those without . as these co - morbidities are often seen in older people , the burden of domestic c. jejuni infections is likely to increase even further in the future , considering the ageing population trend . targeted public health messages including strict advice on kitchen hygiene and safe drinking water sources for those with co - morbidities as well as further investigations on antimicrobial resistance in campylobacter in humans in sweden the authors have not received any funding or benefits from industry or elsewhere to conduct this study .

backgroundcampylobacter jejuni is among the most frequent causes of bacterial gastroenteritis in europe . over 8,000 c. jejuni multilocus sequence typing sequence types ( sts ) have been described ; st-21 and st-45 have been identified as the most frequent types in all human studies so far . in contrast to other sts , st-22 has been associated with the guillain barr syndrome and st-677 was recently linked to severe systemic infections in finland . we investigated risk factors associated with hospitalisation in individuals with c. jejuni infections acquired in sweden.methodsa total of 1,075 individuals with domestically acquired c. jejuni infection diagnosed between november 2011 and october 2012 in sweden were included in this retrospective cohort study . typing data for the isolates as well as clinical data including hospitalisation dates and diagnosis codes for individuals with c. jejuni infection were obtained . factors associated with hospitalisation and length of hospitalisation were investigated by multivariable analysis.resultsa total of 289 individuals were hospitalised due to c. jejuni infection ( 26.8% ) ; those with co - morbidities were over 14 times more likely to become hospitalised than those without ( odds ratio [ or ] : 14.39 , 95% confidence interval [ ci ] : 6.8430.26 ) . those with underlying co - morbidities were also hospitalised longer than those without ( 4.22 days vs. 2.86 days ) , although this was not statistically significant . c. jejuni st-257 ( or : 2.38 ; ci : 1.085.23 ) , but not st-22 or st-677 , was significantly associated with hospitalisation.conclusionst-677 was not associated with increased hospitalisation or a longer hospital stay in our study whilst st-257 was . however , individuals with c. jejuni infections were generally more frequently hospitalised than previously demonstrated ; this requires further consideration including possible targeted interventions .

Material and methods Study design Description of cohort Microbiological data Clinical data Definitions Statistical analysis Ethics Results Risk factors associated with hospitalisation and the length of hospitalisation Estimation of hospitalisation linked to the domestic Discussion Limitations Recommendations Conflict of interest and funding

we performed a retrospective study of individuals with domestically acquired c. jejuni infection in order to assess factors associated with related hospitalisations in sweden between november 2011 and october 2012 . a total of 8,585 notifications of cases with laboratory - confirmed campylobacter infection were received at the public health agency of sweden during the study period , and 3,434 from those were classified as having been acquired in sweden . clinical microbiological laboratories in sweden were asked to submit all isolates obtained from domestically acquired campylobacter cases to the national veterinary institute ( sva ) in uppsala for mlst typing during the study period ; a total of 1,914 samples were received from 25 laboratories . a total of 1,139 c. jejuni stool isolates obtained from domestically acquired cases between november 2011 and october 2012 were included in our study . the factors associated with two separate outcome measures , hospitalisation ( yes / no ) and length of hospitalisation ( in days ) , were investigated . risk factors with p<0.20 in the univariable analysis were included in a multivariable model built through stepwise forward selection using the likelihood ratio test.length of hospitalisation . univariable analysis to assess the association between the risk factors and length of hospitalisation ( i.e. risk factors with p<0.20 in the univariable analysis were included in a multivariable model built through stepwise forward selection using the likelihood ratio test.estimation of hospitalisation linked to campylobacter infections in sweden . we performed a retrospective study of individuals with domestically acquired c. jejuni infection in order to assess factors associated with related hospitalisations in sweden between november 2011 and october 2012 . a total of 8,585 notifications of cases with laboratory - confirmed campylobacter infection were received at the public health agency of sweden during the study period , and 3,434 from those were classified as having been acquired in sweden . a total of 1,139 c. jejuni stool isolates obtained from domestically acquired cases between november 2011 and october 2012 were included in our study . the factors associated with two separate outcome measures , hospitalisation ( yes / no ) and length of hospitalisation ( in days ) , were investigated . risk factors with p<0.20 in the univariable analysis were included in a multivariable model built through stepwise forward selection using the likelihood ratio test.length of hospitalisation . univariable analysis to assess the association between the risk factors and length of hospitalisation ( i.e. risk factors with p<0.20 in the univariable analysis were included in a multivariable model built through stepwise forward selection using the likelihood ratio test.estimation of hospitalisation linked to campylobacter infections in sweden . univariable analysis to assess the association between the risk factors and length of hospitalisation ( i.e. a total of 1,139 c. jejuni isolates were included in this study , but 64 of these were excluded as it was not possible to match their sample identifiers to the data from the swedish board of health and welfare . the most frequent sts identified and included in statistical analyses were st-21 ( 139 isolates ; 12.9% [ belonging to the cc-21 ] ) , st-50 ( 116 ; 10.8% [ cc-21 ] ) , st-19 ( 104 ; 9.7% [ cc-21 ] ) , st-45 ( 105 ; 9.8% [ cc-45 ] ) , st-677 ( 90 ; 8.4% [ cc-677 ] ) , st-48 ( 88 ; 8.2% [ cc-48 ] ) , and st-257 ( 41 ; 3.8% [ cc-257 ] ) ; all the remaining sts were identified in less than 33 cases and were clustered into their own group named other ( 392 isolates ; 36.5% ) ( table 1 ) . the frequency of detection and hospital admission , length of hospital admission and mean age of individuals infected with c. jejuni mlst sequence types ( st ) in sweden , between november 2011 and october 2012 sequence types which were identified in les s than 33 isolates have been combined into this a total of 289 individuals with c. jejuni infection were hospitalised ( 26.9% ; table 1 ) : the smallest proportion of hospitalisation was noted for those infected with c. jejuni st-19 ( 19.4% ) and highest for those infected with st-257 ( 40% ) . most individuals hospitalised with c. jejuni infection were admitted either with the diagnosis of enteritis or colitis ( n=265 ) or with symptoms of diarrhoea and abdominal pain ( n=6 ) , and the reason for admission was not given for five individuals . mean age of individuals infected with c. jejuni was 42.5 years , whereas individuals hospitalised with c. jejuni infections were generally older with a mean age of 50 years ( table 1 ) . infection with c. jejuni st-257 ( rr : 1.57 ; 95% ci : 1.082.30 ) , an age of 60 years or older ( rr : 2.13 ; 95% ci : 1.762.57 ) , and underlying co - morbidity ( rr : 4.49 ; 95% ci : 2.916.92 ) were factors significantly associated with hospitalisation among patients who had c. jejuni infection in sweden based on univariable analysis ( table 2 ) . most of the cases with reported co - morbidities , including being immunocompromised and those with chronic underlying conditions admitted to hospital , also had a laboratory - confirmed c. jejuni infection ( 83% ; 80/97 ) , whereas a smaller proportion of individuals without these co - morbidities were hospitalised with c. jejuni infection ( 22% ; 209/978 ) . furthermore , age under 20 years ( rr : 0.69 ; 95% ci : 0.500.95 ) or between 40 and 60 years ( rr : 0.69 ; 95% ci : 0.550.87 ) as well as being diagnosed in halland and skne counties ( rr : 0.45 ; 95% ci : 0.210.97 and rr : 0.67 ; 95% ci : 0.460.97 , respectively ) were identified as statistically significant protective factors for hospitalisation coinciding with c. jejuni infection . no significant differences in proportion of individuals with co - morbidities or in age distribution of individuals were observed between counties . after correction for potential confounders , co - morbidity ( or : 13.89 ; 95% ci : 7.8824.46 ) , an age of 60 years or older ( or : 1.98 ; 95% ci : 1.223.25 ) , and c. jejuni st-257 ( or : 2.35 ; 95% ci : 1.154.81 ) were found to be independently associated with hospitalisation ( table 3 ) . in addition , vstra gtaland ( or : 0.59 ; 95% ci : 0.360.96 ) , halland ( or : 0.30 ; 95% ci : 0.210.97 ) , and skne ( or : 0.43 ; 95% ci : 0.460.97 ) counties were identified as statistically significant protecting factors for hospitalisation with c. jejuni infection in multivariable analysis . result of multivariable logistic regression analysis of variables associated with hospitalisation of laboratory - confirmed domestic cases of c. jejuni in sweden , november 2011october 2012 the results for variables shown to be statistically significantly associated with hospitalisation ( p>0.05 ) are shown in bold face . duration of hospitalisation was not statistically significant between different age groups , but those older than 60 years were hospitalised longer than those under 60 years of age ( 4.07 days vs. 2.53 days [ < 20 years ] , 2.14 days [ 2039 years ] , and 3.12 days [ 4059 years ] ) . furthermore , those infected with c. jejuni who had underlying co - morbidities were also hospitalised for longer than those without ( 4.16 days vs. 2.84 days ) . variables investigated for their association with length of hospitalisation evaluated in univariable analysis using cox regression in laboratory - confirmed domestic cases of c. jejuni in sweden , november 2011october 2012 as the mean length of hospitalisation was 3.2 days , and 289 individuals with domestically acquired c. jejuni infections were hospitalised , it can be calculated that a total of 925 hospital bed days were used ( 95% ci : 879971 ) . as only a proportion of domestic c. jejuni infections were included in our study ( 1,075 from 3,434 ) , the true numbers would be bigger ( estimated around 2,954 bed days ) . furthermore , one case of gbs among our study population would translate into three cases among all hospitalised individuals due to domestic c. jejuni infections in sweden during the study period . infection with c. jejuni st-257 ( rr : 1.57 ; 95% ci : 1.082.30 ) , an age of 60 years or older ( rr : 2.13 ; 95% ci : 1.762.57 ) , and underlying co - morbidity ( rr : 4.49 ; 95% ci : 2.916.92 ) were factors significantly associated with hospitalisation among patients who had c. jejuni infection in sweden based on univariable analysis ( table 2 ) . most of the cases with reported co - morbidities , including being immunocompromised and those with chronic underlying conditions admitted to hospital , also had a laboratory - confirmed c. jejuni infection ( 83% ; 80/97 ) , whereas a smaller proportion of individuals without these co - morbidities were hospitalised with c. jejuni infection ( 22% ; 209/978 ) . furthermore , age under 20 years ( rr : 0.69 ; 95% ci : 0.500.95 ) or between 40 and 60 years ( rr : 0.69 ; 95% ci : 0.550.87 ) as well as being diagnosed in halland and skne counties ( rr : 0.45 ; 95% ci : 0.210.97 and rr : 0.67 ; 95% ci : 0.460.97 , respectively ) were identified as statistically significant protective factors for hospitalisation coinciding with c. jejuni infection . no significant differences in proportion of individuals with co - morbidities or in age distribution of individuals were observed between counties . after correction for potential confounders , co - morbidity ( or : 13.89 ; 95% ci : 7.8824.46 ) , an age of 60 years or older ( or : 1.98 ; 95% ci : 1.223.25 ) , and c. jejuni st-257 ( or : 2.35 ; 95% ci : 1.154.81 ) in addition , vstra gtaland ( or : 0.59 ; 95% ci : 0.360.96 ) , halland ( or : 0.30 ; 95% ci : 0.210.97 ) , and skne ( or : 0.43 ; 95% ci : 0.460.97 ) counties were identified as statistically significant protecting factors for hospitalisation with c. jejuni infection in multivariable analysis . result of multivariable logistic regression analysis of variables associated with hospitalisation of laboratory - confirmed domestic cases of c. jejuni in sweden , november 2011october 2012 the results for variables shown to be statistically significantly associated with hospitalisation ( p>0.05 ) are shown in bold face . duration of hospitalisation was not statistically significant between different age groups , but those older than 60 years were hospitalised longer than those under 60 years of age ( 4.07 days vs. 2.53 days [ < 20 years ] , 2.14 days [ 2039 years ] , and 3.12 days [ 4059 years ] ) . furthermore , those infected with c. jejuni who had underlying co - morbidities were also hospitalised for longer than those without ( 4.16 days vs. 2.84 days ) . variables investigated for their association with length of hospitalisation evaluated in univariable analysis using cox regression in laboratory - confirmed domestic cases of c. jejuni in sweden , november 2011october 2012 as the mean length of hospitalisation was 3.2 days , and 289 individuals with domestically acquired c. jejuni infections were hospitalised , it can be calculated that a total of 925 hospital bed days were used ( 95% ci : 879971 ) . as only a proportion of domestic c. jejuni infections were included in our study ( 1,075 from 3,434 ) , the true numbers would be bigger ( estimated around 2,954 bed days ) . furthermore , one case of gbs among our study population would translate into three cases among all hospitalised individuals due to domestic c. jejuni infections in sweden during the study period . this is the first study to determine the risk factors associated with the hospitalisation and the length of hospitalisation of individuals with domestically acquired c. jejuni infections in sweden . a high diversity of mlsts among human c. jejuni isolates with a total of 119 distinct sts were observed , from which the seven frequent sts ( st-21 , st-50 , st-19 , st-45 , st-677 , st-48 , and st-257 ) covered 64% of all isolates in accordance with previously published studies from the united kingdom , finland , denmark , and canada ( 914 , 24 , 26 ) . these sts were recently shown to have been responsible for a large proportion of domestic c. jejuni infections in sweden already in 2000 , and have also been isolated from swedish broilers ( 23 ) . up to 26.9% of individuals included in this study were admitted to hospital with the domestically acquired c. jejuni infection . the correlation between co - morbidities and hospitalisation due to domestic c. jejuni infection is not surprising , given that most co - morbidities result in decreased immune function and thus more likely will lead to symptomatic c. jejuni enterocolitis . furthermore , individuals with co - morbidities are more likely to seek health care with gastrointestinal symptoms and are more likely to be tested for campylobacter than those without co - morbidities . there was also a trend towards more severe infections among the individuals with co - morbidities ; they were hospitalised for longer than those without ( 4.16 days vs. 2.84 days ) . although these differences were not statistically significant , they suggest more severe infections in those with co - morbidities . however , the main focus of the arora study ( 35 ) was to determine the risk factors for c. jejuni infection and not to investigate the risk factors associated with hospital admissions due to campylobacter infections . furthermore , previous studies have shown that those aged over 60 years are three times more likely to be hospitalised with campylobacter infection than younger individuals ( 34 ) , and that age > 60 years can be a risk factor for a longer hospital stay ( 28 ) . we clearly demonstrate that co - morbidity is the most significant risk factor for hospitalisation while having c. jejuni infection , not old age . a potential explanation could be differences in study population , such as overrepresentation of the elderly or those with co - morbidities , but no significant differences were observed between counties . only one study has previously investigated the association between c. jejuni sts and hospitalisation ; based on that it was expected that c. jejuni st-677 would be associated with more frequent hospitalisations and also with increased length of hospitalisation than other campylobacter types ( 13 ) . in our study , the association between c. jejuni st-677 and the increased hospitalisations or a longer hospital stay could not be demonstrated . although st-257 was recently isolated from domestic dogs in sweden ( 37 ) , only 4% of dogs were positive for c. jejuni and thus the importance of dogs as a source of human infections needs to be clarified further . both of these types , st-257 and st-50 , although the pathogenesis of c. jejuni remains poorly understood , some c. jejuni types have been associated with severe infections . in our study , one individual was diagnosed with gbs and c. jejuni st-22 infection , whereas another individual with systemic invasive infection had c. jejuni st-677 , consistent with the previous literature ( 7 , 26 , 27 ) . however , c. jejuni st-257 was shown to be the most virulent type measured as larval survival in the insect galleria mellonella model ( 38 ) . we have demonstrated that over a quarter of all individuals diagnosed with domestic c. jejuni infection are hospitalised in sweden , and that those infected with st-50 or st-257 are slightly more likely to be subject to hospital care than those infected with other sts . although over 50% of all hospitalised individuals were less than 60 years of age and did not have any co - morbidities and were not known to be immunocompromised in this study , the biggest risk factors for hospitalisation identified included co - morbidities and old age . we have shown that individuals with co - morbidities are at a 14-time higher risk of becoming hospitalised with a domestic c. jejuni infection than those without . as these co - morbidities are often seen in older people , the burden of domestic c. jejuni infections is likely to increase even further in the future , considering the ageing population trend . targeted public health messages including strict advice on kitchen hygiene and safe drinking water sources for those with co - morbidities as well as further investigations on antimicrobial resistance in campylobacter in humans in sweden the clinical data obtained in this study were based on the diagnostic codes used and reported . we have demonstrated that over a quarter of all individuals diagnosed with domestic c. jejuni infection are hospitalised in sweden , and that those infected with st-50 or st-257 are slightly more likely to be subject to hospital care than those infected with other sts . although over 50% of all hospitalised individuals were less than 60 years of age and did not have any co - morbidities and were not known to be immunocompromised in this study , the biggest risk factors for hospitalisation identified included co - morbidities and old age . we have shown that individuals with co - morbidities are at a 14-time higher risk of becoming hospitalised with a domestic c. jejuni infection than those without . as these co - morbidities are often seen in older people , the burden of domestic c. jejuni infections is likely to increase even further in the future , considering the ageing population trend . targeted public health messages including strict advice on kitchen hygiene and safe drinking water sources for those with co - morbidities as well as further investigations on antimicrobial resistance in campylobacter in humans in sweden the authors have not received any funding or benefits from industry or elsewhere to conduct this study .

malaria remains a significant , debilitating and often lethal disease in many parts of south america , although its incidence , severity and impact is highly regional - dependent [ 1 , 2 ] . in lowland colombia , for example , malaria has been rated by focus groups as one of the most important health problems facing communities . in colombian and ecuadorian communities , the cost of malaria prevention is much less expensive than the sum of direct costs ( e.g. , treatment , travel ) and indirect costs ( e.g. , days of work lost ) . in both countries , national malaria control programs monitor the disease and carry out mosquito control programs in malaria endemic areas [ 5 , 6 ] . due to the success of these programs , statistics in ecuador indicate that overall malaria incidence has declined over the last fifteen years , with occasional regional - scale epidemics at lower elevations ( ca . < 1500 m ) . at higher elevations , malaria was epidemic in the inter - andean valleys of ecuador ( > 1500 m ) prior to the mid-1940s , when large - scale eradication efforts prevented ongoing malaria transmission in these regions , mainly through the reduction and possible elimination of local populations of anopheles pseudopunctipennis . since that time , malaria has only caused illness in highland regions among migrants and travelers returning from low altitudes [ 1 , 7 ] . several reviews of the effects of various forms of global change on the incidence of insect - borne disease have stressed that malaria might move into higher - altitude regions as highland habitats become increasingly suitable for anopheles mosquitoes [ 914 ] . indeed , highland malaria has recently been observed in higher - altitude regions of kenya , rwanda , and uganda [ 1517 ] , as well as in andean bolivia [ 18 , 19 ] . plasmodium vivax has been incriminated as the parasite in highland malaria epidemics in both 1940s ecuador and recently in bolivia [ 8 , 18 ] . additionally , recent widespread highland records of three coastal malaria vectors : anopheles albimanus , an . , it is believed that in higher - altitude regions , residents and health care practitioners may lack experiential knowledge of the disease and therefore do not know how to recognize malaria or protect themselves from mosquito bites . as well , a person 's experience with malaria could potentially raise a greater perception of malaria risk . therefore , highland communities may not rate malaria risk strongly or recognize symptoms in order to seek treatment . residents may also not approach mosquito habitat elimination and personal mosquito bite protection as seriously as residents of malaria - endemic lower altitudes . although there is essentially no data regarding the perceptions of malaria in the south american andes , there have been several studies conducted in lower - altitude regions . the knowledge of a connection between mosquito bites and malaria is regionally variable , for example , 56% of respondents in a rural community in northern coastal ecuador versus 85% of respondents in an urban community in pacific coastal colombia [ 3 , 22 ] . in colombia , malaria has been inaccurately attributed to strenuous activities , ingesting hot foods , poor nutrition , drinking contaminated water , and/or proximity to garbage . individual differences in knowledge and perceptions have also been attributed to gender , with women having limited access to malaria - relevant information and treatment , among other factors . malaria prevention in colombia incorporates both modern techniques ( e.g. , bed nets and anopheline mosquito larval habitat elimination ) as well as traditional techniques more commonly used in rural areas [ 3 , 23 , 25 ] . urban residents in amazonian colombia also have been reported to feel less at risk of malaria than rural residents . malaria diagnosis by national bodies in south america is often based on passive reporting , where misdiagnosis of malaria could lead to a general underreporting of the disease over the landscape . the actual risk and occurrence of malaria in lowlands is complicated by human migration patterns between endemic and nonendemic regions , as well as by proximity to mosquito breeding sites . landowners and land managers , rather than lower - status workers , have the capability to eliminate anopheline larval habitats on their property and to promote and enforce the use of malaria control measures such as bed nets among their families and employees . in this study , a quantitative kap ( knowledge , attitudes and practices ) approach was used to assess the degree of knowledge , concerns and beliefs of land managers and landowners in both lowland and highland regions of ecuador for topics relevant to malaria prevention . the larval habitat management practices used and motivations for eliminating larval habitat were also examined . finally , the study assessed mosquito bite protection that is currently in use in both lowland and highland regions and compared it to protection that would be used by respondents for hypothetical travel to a known malaria - endemic area . to assess the knowledge , beliefs , concerns , land management practices , and forms of bite protection used by ecuadorian land owners and/or managers , researchers conducted a series of 262 structured , questionnaire - based interviews of these individuals throughout all road - accessible regions of ecuador between 2008 and 2010 ( figure 1 ) . most questionnaires were administered during the period from july to december in 2009 and 2010 . although the regions are endemic for malaria , we did not assess respondents in northern esmeraldas , sucumbos , and orellana provinces due to travel safety regulations ( figure 1 ) . we attempted to eliminate clustering of respondents by traveling along as many different roads as possible . however , some clusters of respondents occurred ( e.g. , in pichincha and santo domingo provinces ) due to a higher number of potential respondents present outside their homes at the time of visiting , as well as a greater population and road density in that particular lowland - highland transition area , which may have led to a regional bias in our analysis ( figure 1 ) . to permit statistical inference , we interviewed a pre - established number of land owners and/or managers from each of five generalized land uses : cattle pasture ( whether for meat or dairy ) , fish farms , plantations , human residences , and nature reserves . fish farm and nature reserve managers were given fewer interviews than the other categories since they were encountered less frequently ( table 1 ) . no more than one person was interviewed on the same property for the same land use . although all five land use categories are present in all general regions of the country , there is some clustering of land uses in particular regions which may have caused small levels of spatial bias due to availability ( figure 1 ) . land owners and managers were stratified on a second level by gender since women and men are expected to have different access to information and women may more readily apply knowledge toward prevention and treatment than men ( table 1 ) . although we attempted to interview an equal number of men and women , we were not able to interview many female owners or managers of nature reserves due to the scarcity of women encountered in these positions ( table 1 ) . we further stratified interviews by interviewing an approximately equal number of landowners and/or managers above and below 1500 m ( table 1 ) , which travel medical literature suggests as an appropriate altitudinal division for malaria endemism in ecuador . current statistics from the sistema nacional de eradicacin de malaria ( snem ) indicate that almost all areas below 1500 m have been affected by at least occasional cases of malaria during the last 10 years ( figure 1 ) , which we assume should have provided residents of the below 1500 m category with some experiential knowledge of malaria . entomological data further supports this altitudinal risk division , since densities of anopheles mosquitoes ( malaria vectors ) are more prevalent below 1500 m , although a few larvae have been collected up to an elevation of nearly 2000 m . this altitudinal division was used rather than actual reported malaria rates , since the distribution of malaria varies substantially from year to year , data comes from urban reporting centers rather than rural communities , and we expect rural parts of the country to underreport malaria cases due to poorer access to health centres . therefore , for the purposes of this study we also assume that all respondents below this altitude are potentially at risk for malaria . to locate possible respondents , researchers working in groups of two or three traveled along all roads deemed suitable for travel and potential respondents were approached when visible from the road or other public domain . to introduce randomization , after spotting a potential respondent for a given land use , a coin toss was used to determine if the person would be approached . however , due to the scarcity of fish farm and nature reserve managers / owners , all of these possible respondents were approached when observed . often , the land owner and/or manager was not present and the researchers did not interview any person on the site . to avoid a possible feeling of deference toward the interviewer due to any perceived class differences , for example , , questionnaires were conducted by l. pinault as well as one or two ecuadorian interviewers who spoke the local dialects ( although all field researchers spoke fluent spanish ) and a few contradictory statements were placed into the interview to identify respondents who might not be answering truthfully or might not understand the questions ( e.g. , all affirmative answers ) . questions were asked directly in spanish by both interviewers to respondents , rather than through translation to english . voluntary participation and oral consent were received after researchers explained the study , including rights to withdraw at any stage during the interview process . all research was approved brock university 's ethics in human research committee , file number 07 - 336 . aspects of the interview included : ( 1 ) knowledge of where malaria occurs in ecuador , ( 2 ) knowledge of the ecological relationship among standing water and mosquitoes , and malaria , ( 3 ) belief in climate change , and belief that climate change could potentially cause malaria vectors to move into higher altitudes , ( 4 ) concern about insect - borne disease , the presence of mosquitoes on their property , and the presence of malaria on their property , ( 5 ) land management practices for standing water ( mosquito habitat ) reduction , and ( 6 ) mosquito bite protection used and bite protection that respondents would use to travel to an area known to have malaria . researchers also recorded the geographic coordinates and elevation of the site using a handheld gps unit ( garmin gps e - trex summit ) . respondents answered most questions with the options : yes ( 1 ) or no ( 0 ) . to determine the role of altitudinal category and gender on the frequency of positive answers , a binary logistic regression ( logit ) was used . those who answered do not know or maybe were considered uncertain answers and were eliminated from analysis when they were reported less than 10% of the time , although the percentage of uncertain answers is summarized in the text when provided more frequently . since multiple binary logistic regressions were used in some cases , significance levels were modified using a bonferroni correction , where /n ( n = number of tests ) are used to decrease the probability of false positive results . some questions , including land management practices and bite protection methods , asked respondents to list all possible answers . chi - square tests ( of equal variances among groups of gender and altitude class , unless otherwise indicated ) were used to compare count data from responses between land use and elevation categories . all statistical analyses were conducted in minitab v.15 software ( 2007 ) . at the end of the interview , we asked respondents to list all sources of water on their property , with researchers prompting the respondents with a long list of options that included streams , ponds , and laundry washing areas , as well as any answers beyond the list that may occur to them . all potentially suitable habitats listed by respondents were considered by researchers for comparison to standing water reported from earlier in the interview to assess the relationship between reported potential habitat and actual potential habitat . we also judged the approximate socioeconomic status of respondents based on housing type and signs of disposable income . according to the ecuadorian census , approximately 38% of households lack sufficient income to provide basic necessities of life and 46% of citizens are rated under the nbi income - poverty index based on statistics collected in 2006 , therefore , signs of disposable income such as working televisions indicated above - average wealth . all respondents were land owners , or in the cases of larger properties , land managers ; therefore , respondents in our study were expected to have a higher average economic status and live in rural settings more often than most ecuadorian citizens . only one residential respondent lived in an urban area ( quito ) , while all other respondents lived in rural regions or small communities . although we did not formally evaluate the socioeconomic status of respondents , approximately 56% of our respondents were judged to be of a higher - than - average economic status ( i.e. , living in concrete houses with completed roofing and doors , and some signs of disposable income such as televisions , radios , and cars ) , 23% of our respondents were judged to be of a lower socioeconomic status ( i.e. , living in basic housing sometimes lacking doors and sufficient roofing , with no signs of disposable income ) , and 20% of our respondents were judged to be very wealthy ( i.e. , living in large , finished homes with some degree of landscaping , on expansive properties ) . greater than 90% of our respondents were ethnically identifiable as mestizo ( a mixture of indigenous , african , and spanish ancestry found in all regions of ecuador ) , while fewer than 10 respondents were each of indigenous , black , or white . five people who were approached by interviewers declined to participate in the study , therefore , the interviews did not proceed . eleven percent of respondents answering certainly above 1500 m stated that they could become ill with malaria on their property at present , whereas only 52.6% of respondents below 1500 m provided the same answer ( table 2 ) . however , 96.4% of respondents correctly agreed with the statement that one could get malaria on the coast and/or in the amazon region of ecuador , with the responses not differing among genders or altitudinal categories . agreement with the statement that one could get malaria above 3000 m was affected by both gender and altitude category of the respondent ( table 2 ) : women and low - altitude dwellers were more likely to erroneously agree than men and high - altitude dwellers ( women : above 1500 m : 29.7% , below 1500 m : 55.9% ; men above 1500 m : 18.5% , below 1500 m : 30.6% ) . thirty - three percent of respondents were uncertain if malaria could occur above 3000 m. most respondents answering certainly ( 89.9% ) agreed that eliminating standing water reduces the local population of mosquitoes , thereby acknowledging that they understand where larvae live . a slightly lesser percentage ( 83.8% ) agreed that eliminating standing water reduces the risk of malaria , thereby demonstrating an understanding that mosquito adults arise from larval stages in standing water , and the resulting adults can transmit malaria . there was no effect of gender or altitude category on responses to either statement . most respondents ( 93.3% of those answering certainly , 74.8% total ) believed that climate change was occurring , although 19.4% of were uncertain . fewer ( 75.6% of those answering certainly , 54.9% total ) believed that it may be possible for malaria to move into highland regions with climate change , although 26.1% were uncertain about the possibility of this phenomenon . a degree of concern regarding insect - borne diseases was reported significantly more often at elevations below 1500 m ( 83.8% ) than above 1500 m ( 57.7% ) , but did not differ between genders ( table 2 ) . concern about the presence of mosquitoes on a person 's property was greater for respondents below 1500 m ( 87.2% ) than above 1500 m ( 61.4% ) , although there were insignificant differences among genders after the bonferroni correction ( table 2 ) . concern about the presence of malaria on a person 's property was reported more often below 1500 m ( 82.3% ) than above 1500 m ( 60.0% ) , regardless of gender ( table 2 ) . different numbers of respondents reported the presence of some type of standing water on their property depending on their land use ( = 14.82 ; df = 4 ; p = 0.005 ) . this significant difference was due mainly to cattle farmers that reported more standing water , and fish farmers that reported less standing water than expected ( figure 2 ) . there was no difference in the reporting of standing water presence above and below 1500 m ( = 0.05 ; df = 1 ; p = 0.816 ) . after being asked generally if there was some type of standing water present on the property , all sources of water on the property ( standing water or otherwise ) were enumerated by respondents and later evaluated by the researchers as potentially suitable larval habitat or not . figure 2 compares the presence of potentially suitable habitat ( enumerated by respondents ) to the rate at which the presence of some type of standing water was reported , for each land use . there was a significant association between the land use type and the proportion that reported standing water elimination versus the proportion with standing water reported in the list at the end of the interview ( two - way chi - square contingency table : = 12.9 , df = 4 , p = 0.012 ) , with strongest statistical effects due to the strong initial underreporting of some type of standing water by fish farmers ( figure 2 ) . all other respondents under - reported the actual presence of standing water by about half ( figure 2 ) . it is also possible that respondents are reporting the elimination of some standing water , though not all sources of water . the types of standing water observed on each of the different land uses are summarized in figure 3 . approximately 77.5% of fish farms had at least one fish pond or tank , with an area of stable water surface that could potentially support mosquito larvae ( figure 3(a ) ) . ponds were the most common type of standing water for nature reserves ( figure 3(c ) ) , while irrigation canals or inland ditches were the most common for cattle farms ( figure 3(b ) ) , residences ( figure 3(d ) ) , and plantations ( figure 3(e ) ) . it should be noted that not all standing water types listed would be suitable for anopheles mosquito larvae , although they may be suitable for other species of mosquitoes . the percentage of respondents that presently eliminate their standing water differed significantly among land uses ( = 16.1 ; df = 4 ; p = 0.003 ) , with the strongest effect due to a smaller number of cattle farmers than expected reporting that they eliminate standing water ( figure 4(a ) ) . however , the proportions of respondents that eliminate standing water for each land use did not differ between altitude categories ( above / below 1500 m ) ( = 2.4 ; df = 4 ; p = 0.655 ) ( figure 4(a ) ) . reasons provided for not eliminating standing water were enumerated ( figure 4(b ) ) , and approximately twice the number of reasons were given by respondents below 1500 m than above ( figure 4(b ) ) . the top three reasons provided were logistical difficulty ( 6.2% ) , lacking human assistance ( 3.5% ) , and lacking time to eliminate the water ( 3.5% ) . water being natural was only provided as a reason for not eliminating water by nature reserves . not being able to eliminate standing water due to economic use of the water body was reported for rice farms and fish ponds below 1500 m. four possible motivations that might encourage respondents to eliminate standing water were compared and all four appear to be strong motivators to eliminate water : ( 1 ) the presence of malaria nearby ( 95.3% ) , ( 2 ) financial support to eliminate water ( 93.9% ) , ( 3 ) increased frequency of mosquito bites ( 93.3% ) , and ( 4 ) recommendations by government or medical authorities ( 89.9% ) . there was no statistical difference in agreement with motivational sources between genders or altitude categories . respondents were asked if they currently use any form of mosquito bite protection , and if they would use protection if they visited an area known to have malaria . use of some type of malaria protection differed between altitude categories as well as current use versus use for a known malaria - endemic region , although bite prevention did not differ between genders ( table 3 ) . current use of protection was higher below than above 1500 m ( figure 5 ; table 3 ) . respondents reported that they would use malaria protection more often in an area with known malaria than what they currently use , even if they already live in a possibly malaria - endemic area ( below 1500 m ) ( figure 5 ; table 3 ) . the most commonly used prevention methods above 1500 m are ( 1 ) insecticides for the home , ( 2 ) long sleeves , and ( 3 ) chemical repellent ( figure 6(a ) ) . if those respondents visited an area with known malaria , ( 1 ) bed nets , ( 2 ) long sleeves , and ( 3 ) insecticides for the home would be the most commonly used ( figure 6(b ) ) . the most common types of prevention methods used below 1500 m are ( 1 ) bed nets , ( 2 ) insecticides in the home , ( 3 ) chemical repellent , ( 4 ) long sleeves , ( 5 ) screen or curtain on doors and windows , and ( 6 ) chlorine or chemicals in standing water ( figure 6(c ) ) . the same top six prevention methods are listed by respondents for hypothetical travel to an area with known cases of malaria , although the order in which they are listed is different ( figure 6(d ) ) . the less - often reported prevention methods for respondents living below 1500 m include calling the sistema nacional de eradicacin de malaria ( government agency ) , use of a fan or air conditioning , or a community effort to eliminate mosquito habitat , all of which have the potential to prevent mosquito bites . since the present study focuses on land owners and land managers who are of a higher socioeconomic status than the average ecuadorian citizen , it is also likely that respondents had received more education than average , which we expect to relate to a better understanding of the topics under discussion . therefore , the present study should not be extrapolated to the knowledge , attitudes , and beliefs of all ecuadorians , but rather should provide a baseline of these factors among land owners and/or land managers , that is , the decision makers for larval habitat modification in the overall landscape . although most participants in this study correctly identified general regions where malaria does and does not occur in their country , it is troubling that 52.6% of respondents below 1500 m did not feel that they were at risk of contracting malaria on their property . we did not formally discuss personal experiences with malaria with respondents , which would likely explain some of the variation in this response , and is one of the shortcomings of our study . although a small percentage of these are probably not at immediate risk , several respondents appeared to deny personal risk despite anecdotally providing us with examples of neighbours or nearby towns with recent malaria cases . another explanation might be that respondents feel that malaria is a controllable risk , and they might not be susceptible to infection because they use mosquito bite protection , for example , . the current findings might indicate that lowland residents would benefit from an educational program in their community that provides updated data concerning their immediate and potential risk of malaria . generally , most respondents understood that standing water could increase mosquito populations , and thereby malaria incidence . along the same lines , a large number were also receptive to the possibility of highland regions becoming affected by malaria in the future with climate change . such a widespread open - mindedness would be useful for rapid identification of malaria cases and acceptance of risk should malaria spread to previously - unaffected highland regions due to meteorological or other causes . as we would predict from experience , those at altitudes lower than 1500 m reported being more concerned by the presence of mosquitoes , malaria , and other types of insect - borne disease . substantially , fewer respondents reported the presence of standing water on their property early in the interview than those reported later in the interview . although part of this could be due to denial , or to a lack of awareness of their own property , a more likely reason for many respondents to deny the presence of water could be the too - specific definition of standing water for mosquito habitat . many respondents did not likely consider often - flowing water types such as irrigation canals to be standing water . anecdotally , several respondents talked about the necessity of swamps and marshes for mosquito habitat , whereas the most commonly observed standing water types on properties were ponds , reservoirs , and irrigation canals . therefore , we suggest that the malaria education programs that target standing water elimination need to define potential larval habitat as any water where the surface of the water is not moving or moving slowly , rather than the more typical standing water , or aguas estancadas . since the type of standing water available is dependent on land use , it is also important for future studies to more extensively define suitable versus unsuitable habitat types for anopheles larvae to include those that could be considered to flow , such as irrigation canals and roadside ditches . although ecuadorian land owners / managers could be motivated to eliminate standing water through direct observation or advice from authorities , the largest reason provided for not eliminating the water is difficulty ( usually due to topography)a practical problem that would be expensive to address . a large number of respondents below 1500 m used some type of mosquito protection ( 84.0% ) . bed nets were the most prevalent mosquito bite prevention method in low - altitude regions ( 66.4% ) , and were more commonly used than in a community in northern coastal ecuador ( 23% ) , lowland colombia ( 59% ) , or rural uganda ( 26% ) , possibly due to greater socioeconomic standing among our interviewed landowners / managers than other community members [ 3 , 22 , 31 ] . we did not personally assess whether or not the respondents ' bed nets were in good condition , or if they were treated with insecticide , which is important for bite prevention or might help to reduce resting populations of adult mosquitoes within homes [ 32 , 33 ] . the numbers might have also been inflated due to the common use of bed nets to prevent the annoyance of mosquitoes rather than malaria . insecticides within the home , including sprays , were also often employed in low - altitude areas ( 57.5% ) , similar to communities in the colombian pacific coast ( 57% ) . their common usage is positive , since one study in the highlands of kenya reports that indoor residual house - spraying actually reduces infection rates more than bed nets . interestingly , mosquito spirals were not presently used by ecuadorian respondents , despite 60% usage in the colombian pacific coast . mosquito protection methods that would be employed by ecuadorian land owners / managers during travel to a malaria - endemic area differ slightly from those reported in the travel medical literature . although 8090% of our respondents state that they would use a bed net in a malaria - endemic area of their own country , in one study only 58% of foreign travelers to a malaria - endemic area would do the same . however , a higher proportion of travelers in the same paper would use long sleeves ( 93% ) and chemical repellent ( 90% ) than our respondents ( approximately 5060% ) . travelers from developed countries also rely heavily on chemical prophylaxis for malaria prevention [ 3638 ] , while fewer than 5% of our respondents would use it for travel into a region with known cases of malaria . we had expected more folklore - based and traditional medicine and mosquito prevention methods from our respondents , since it is known that plant - based malaria prevention and treatments are still quite common in parts of ecuador , for example , [ 4 , 22 , 39 ] and in colombia [ 3 , 23 ] , as well as in rural parts of africa [ 31 , 40 , 41 ] . this may have been in part due to the higher average socioeconomic status of our respondents , who may have been more able to purchase nontraditional mosquito bite prevention methods , such as deet - based chemical repellent . some of the plant - based repellents that were mentioned by respondents included citrus fruits ( e.g. , lemon juice ) and menthol . the use of smoke as a mosquito repellent included the traditional burning of termite nests in amazonian regions , as well as burning further studies are required to evaluate the malaria kap of ecuadorian citizens who do not own properties in ecuador , as well as citizens residing elsewhere in the andes , and to elucidate cultural and educational differences among perceptions of malaria risk . the present study focused on individual responses , although community approaches to larval source reduction can also play an important role in habitat elimination and highland malaria prevention . a large proportion of land owners / managers presented a good understanding of the occurrence of malaria on the ecuadorian coast or in amazonia and were able to form a basic causative link between standing water , mosquitoes , and malaria . however , about half of the landowners / managers in potential risk areas ( i.e. , altitudes < 1500 m ) did not believe that it was possible to become ill from malaria on their own property . most respondents expressed a belief in climate change and were open - minded to the possibility of anopheline mosquitoes moving into higher - altitude regions . different generalized land uses provided different types of standing water , which may vary in suitability for anopheline larvae . most respondents reported eliminating standing water on their property . when it was not reported to be eliminated , respondents were most often unable to eliminate water due to logistical difficulties . in many cases , respondents reported eliminating standing water despite later reporting sources of standing water that might provide potentially suitable anopheline habitat . this may have been due to partial , but not thorough , elimination of standing water . most respondents in lowland ( at - risk ) regions use some type of mosquito bite prevention , most commonly bed nets . during hypothetical travel to a known malaria - endemic region , therefore , residents of malaria - endemic regions might be expected to increase their use of prevention if informed that their property lies in a malaria - endemic area . generally , it is hoped that the findings of this study will assist malaria control organizations by providing useful data to improve the efficiency of potential educational programs . while knowledge of malaria 's occurrence patterns is strong in ecuador , even in highland regions , it might be necessary to inform residents of actual malaria risk , so that risk does not continue to be underestimated . although most respondents understand that there is a link between standing water and malaria , the definition of standing water is often too restrictive and does not encompass all potential mosquito habitats on a person 's property . ecuadorian land owners / managers appear to be strongly motivated to eliminate mosquito habitat on their property , and when they do not eliminate standing water , it is usually due to factors beyond their control ( difficulty or limited financial resources ) . most at - risk ecuadorian land owners / managers currently use mosquito bite protection methods , although the use of protection might increase if they were informed that they live in a potentially malarious area .

to control malaria effectively , it is essential to understand the current knowledge , beliefs , concerns , land management practices , and mosquito bite protection methods in use by citizens . this study presents a comparative , quantitative , interview - based study of land owners and/or managers ( n = 262 ) in the ecuadorian lowlands ( presently considered malarious ) ( n = 131 ) and highlands ( potentially malarious in the future ) ( n = 131 ) . although respondents had a strong understanding of where the disease occurs in their own country and of the basic relationship among standing water , mosquitoes , and malaria , about half of respondents in potential risk areas denied the current possibility of malaria infection on their own property . as well , about half of respondents with potential anopheline larval habitat did not report its presence , likely due to a highly specific definition of suitable mosquito habitat . most respondents who are considered at risk of malaria currently use at least one type of mosquito bite prevention , most commonly bed nets .

1. Introduction 2. Materials and Methods 3. Results 4. Personal Knowledge, Belief, and Concern 5. Land Management Perspectives for Standing Water Elimination 6. Mosquito Bite Protection 7. Discussion 8. Conclusions

indeed , highland malaria has recently been observed in higher - altitude regions of kenya , rwanda , and uganda [ 1517 ] , as well as in andean bolivia [ 18 , 19 ] . , it is believed that in higher - altitude regions , residents and health care practitioners may lack experiential knowledge of the disease and therefore do not know how to recognize malaria or protect themselves from mosquito bites . as well , a person 's experience with malaria could potentially raise a greater perception of malaria risk . residents may also not approach mosquito habitat elimination and personal mosquito bite protection as seriously as residents of malaria - endemic lower altitudes . although there is essentially no data regarding the perceptions of malaria in the south american andes , there have been several studies conducted in lower - altitude regions . the knowledge of a connection between mosquito bites and malaria is regionally variable , for example , 56% of respondents in a rural community in northern coastal ecuador versus 85% of respondents in an urban community in pacific coastal colombia [ 3 , 22 ] . , bed nets and anopheline mosquito larval habitat elimination ) as well as traditional techniques more commonly used in rural areas [ 3 , 23 , 25 ] . urban residents in amazonian colombia also have been reported to feel less at risk of malaria than rural residents . malaria diagnosis by national bodies in south america is often based on passive reporting , where misdiagnosis of malaria could lead to a general underreporting of the disease over the landscape . the actual risk and occurrence of malaria in lowlands is complicated by human migration patterns between endemic and nonendemic regions , as well as by proximity to mosquito breeding sites . landowners and land managers , rather than lower - status workers , have the capability to eliminate anopheline larval habitats on their property and to promote and enforce the use of malaria control measures such as bed nets among their families and employees . in this study , a quantitative kap ( knowledge , attitudes and practices ) approach was used to assess the degree of knowledge , concerns and beliefs of land managers and landowners in both lowland and highland regions of ecuador for topics relevant to malaria prevention . the larval habitat management practices used and motivations for eliminating larval habitat were also examined . finally , the study assessed mosquito bite protection that is currently in use in both lowland and highland regions and compared it to protection that would be used by respondents for hypothetical travel to a known malaria - endemic area . to assess the knowledge , beliefs , concerns , land management practices , and forms of bite protection used by ecuadorian land owners and/or managers , researchers conducted a series of 262 structured , questionnaire - based interviews of these individuals throughout all road - accessible regions of ecuador between 2008 and 2010 ( figure 1 ) . although the regions are endemic for malaria , we did not assess respondents in northern esmeraldas , sucumbos , and orellana provinces due to travel safety regulations ( figure 1 ) . , in pichincha and santo domingo provinces ) due to a higher number of potential respondents present outside their homes at the time of visiting , as well as a greater population and road density in that particular lowland - highland transition area , which may have led to a regional bias in our analysis ( figure 1 ) . to permit statistical inference , we interviewed a pre - established number of land owners and/or managers from each of five generalized land uses : cattle pasture ( whether for meat or dairy ) , fish farms , plantations , human residences , and nature reserves . although all five land use categories are present in all general regions of the country , there is some clustering of land uses in particular regions which may have caused small levels of spatial bias due to availability ( figure 1 ) . current statistics from the sistema nacional de eradicacin de malaria ( snem ) indicate that almost all areas below 1500 m have been affected by at least occasional cases of malaria during the last 10 years ( figure 1 ) , which we assume should have provided residents of the below 1500 m category with some experiential knowledge of malaria . this altitudinal division was used rather than actual reported malaria rates , since the distribution of malaria varies substantially from year to year , data comes from urban reporting centers rather than rural communities , and we expect rural parts of the country to underreport malaria cases due to poorer access to health centres . therefore , for the purposes of this study we also assume that all respondents below this altitude are potentially at risk for malaria . to avoid a possible feeling of deference toward the interviewer due to any perceived class differences , for example , , questionnaires were conducted by l. pinault as well as one or two ecuadorian interviewers who spoke the local dialects ( although all field researchers spoke fluent spanish ) and a few contradictory statements were placed into the interview to identify respondents who might not be answering truthfully or might not understand the questions ( e.g. aspects of the interview included : ( 1 ) knowledge of where malaria occurs in ecuador , ( 2 ) knowledge of the ecological relationship among standing water and mosquitoes , and malaria , ( 3 ) belief in climate change , and belief that climate change could potentially cause malaria vectors to move into higher altitudes , ( 4 ) concern about insect - borne disease , the presence of mosquitoes on their property , and the presence of malaria on their property , ( 5 ) land management practices for standing water ( mosquito habitat ) reduction , and ( 6 ) mosquito bite protection used and bite protection that respondents would use to travel to an area known to have malaria . those who answered do not know or maybe were considered uncertain answers and were eliminated from analysis when they were reported less than 10% of the time , although the percentage of uncertain answers is summarized in the text when provided more frequently . since multiple binary logistic regressions were used in some cases , significance levels were modified using a bonferroni correction , where /n ( n = number of tests ) are used to decrease the probability of false positive results . some questions , including land management practices and bite protection methods , asked respondents to list all possible answers . at the end of the interview , we asked respondents to list all sources of water on their property , with researchers prompting the respondents with a long list of options that included streams , ponds , and laundry washing areas , as well as any answers beyond the list that may occur to them . all potentially suitable habitats listed by respondents were considered by researchers for comparison to standing water reported from earlier in the interview to assess the relationship between reported potential habitat and actual potential habitat . all respondents were land owners , or in the cases of larger properties , land managers ; therefore , respondents in our study were expected to have a higher average economic status and live in rural settings more often than most ecuadorian citizens . although we did not formally evaluate the socioeconomic status of respondents , approximately 56% of our respondents were judged to be of a higher - than - average economic status ( i.e. however , 96.4% of respondents correctly agreed with the statement that one could get malaria on the coast and/or in the amazon region of ecuador , with the responses not differing among genders or altitudinal categories . thirty - three percent of respondents were uncertain if malaria could occur above 3000 m. most respondents answering certainly ( 89.9% ) agreed that eliminating standing water reduces the local population of mosquitoes , thereby acknowledging that they understand where larvae live . a slightly lesser percentage ( 83.8% ) agreed that eliminating standing water reduces the risk of malaria , thereby demonstrating an understanding that mosquito adults arise from larval stages in standing water , and the resulting adults can transmit malaria . different numbers of respondents reported the presence of some type of standing water on their property depending on their land use ( = 14.82 ; df = 4 ; p = 0.005 ) . this significant difference was due mainly to cattle farmers that reported more standing water , and fish farmers that reported less standing water than expected ( figure 2 ) . there was no difference in the reporting of standing water presence above and below 1500 m ( = 0.05 ; df = 1 ; p = 0.816 ) . after being asked generally if there was some type of standing water present on the property , all sources of water on the property ( standing water or otherwise ) were enumerated by respondents and later evaluated by the researchers as potentially suitable larval habitat or not . figure 2 compares the presence of potentially suitable habitat ( enumerated by respondents ) to the rate at which the presence of some type of standing water was reported , for each land use . there was a significant association between the land use type and the proportion that reported standing water elimination versus the proportion with standing water reported in the list at the end of the interview ( two - way chi - square contingency table : = 12.9 , df = 4 , p = 0.012 ) , with strongest statistical effects due to the strong initial underreporting of some type of standing water by fish farmers ( figure 2 ) . it is also possible that respondents are reporting the elimination of some standing water , though not all sources of water . approximately 77.5% of fish farms had at least one fish pond or tank , with an area of stable water surface that could potentially support mosquito larvae ( figure 3(a ) ) . ponds were the most common type of standing water for nature reserves ( figure 3(c ) ) , while irrigation canals or inland ditches were the most common for cattle farms ( figure 3(b ) ) , residences ( figure 3(d ) ) , and plantations ( figure 3(e ) ) . the percentage of respondents that presently eliminate their standing water differed significantly among land uses ( = 16.1 ; df = 4 ; p = 0.003 ) , with the strongest effect due to a smaller number of cattle farmers than expected reporting that they eliminate standing water ( figure 4(a ) ) . however , the proportions of respondents that eliminate standing water for each land use did not differ between altitude categories ( above / below 1500 m ) ( = 2.4 ; df = 4 ; p = 0.655 ) ( figure 4(a ) ) . reasons provided for not eliminating standing water were enumerated ( figure 4(b ) ) , and approximately twice the number of reasons were given by respondents below 1500 m than above ( figure 4(b ) ) . not being able to eliminate standing water due to economic use of the water body was reported for rice farms and fish ponds below 1500 m. four possible motivations that might encourage respondents to eliminate standing water were compared and all four appear to be strong motivators to eliminate water : ( 1 ) the presence of malaria nearby ( 95.3% ) , ( 2 ) financial support to eliminate water ( 93.9% ) , ( 3 ) increased frequency of mosquito bites ( 93.3% ) , and ( 4 ) recommendations by government or medical authorities ( 89.9% ) . respondents were asked if they currently use any form of mosquito bite protection , and if they would use protection if they visited an area known to have malaria . use of some type of malaria protection differed between altitude categories as well as current use versus use for a known malaria - endemic region , although bite prevention did not differ between genders ( table 3 ) . respondents reported that they would use malaria protection more often in an area with known malaria than what they currently use , even if they already live in a possibly malaria - endemic area ( below 1500 m ) ( figure 5 ; table 3 ) . if those respondents visited an area with known malaria , ( 1 ) bed nets , ( 2 ) long sleeves , and ( 3 ) insecticides for the home would be the most commonly used ( figure 6(b ) ) . the most common types of prevention methods used below 1500 m are ( 1 ) bed nets , ( 2 ) insecticides in the home , ( 3 ) chemical repellent , ( 4 ) long sleeves , ( 5 ) screen or curtain on doors and windows , and ( 6 ) chlorine or chemicals in standing water ( figure 6(c ) ) . the same top six prevention methods are listed by respondents for hypothetical travel to an area with known cases of malaria , although the order in which they are listed is different ( figure 6(d ) ) . since the present study focuses on land owners and land managers who are of a higher socioeconomic status than the average ecuadorian citizen , it is also likely that respondents had received more education than average , which we expect to relate to a better understanding of the topics under discussion . therefore , the present study should not be extrapolated to the knowledge , attitudes , and beliefs of all ecuadorians , but rather should provide a baseline of these factors among land owners and/or land managers , that is , the decision makers for larval habitat modification in the overall landscape . although most participants in this study correctly identified general regions where malaria does and does not occur in their country , it is troubling that 52.6% of respondents below 1500 m did not feel that they were at risk of contracting malaria on their property . we did not formally discuss personal experiences with malaria with respondents , which would likely explain some of the variation in this response , and is one of the shortcomings of our study . another explanation might be that respondents feel that malaria is a controllable risk , and they might not be susceptible to infection because they use mosquito bite protection , for example , . the current findings might indicate that lowland residents would benefit from an educational program in their community that provides updated data concerning their immediate and potential risk of malaria . generally , most respondents understood that standing water could increase mosquito populations , and thereby malaria incidence . along the same lines , a large number were also receptive to the possibility of highland regions becoming affected by malaria in the future with climate change . such a widespread open - mindedness would be useful for rapid identification of malaria cases and acceptance of risk should malaria spread to previously - unaffected highland regions due to meteorological or other causes . as we would predict from experience , those at altitudes lower than 1500 m reported being more concerned by the presence of mosquitoes , malaria , and other types of insect - borne disease . substantially , fewer respondents reported the presence of standing water on their property early in the interview than those reported later in the interview . although part of this could be due to denial , or to a lack of awareness of their own property , a more likely reason for many respondents to deny the presence of water could be the too - specific definition of standing water for mosquito habitat . many respondents did not likely consider often - flowing water types such as irrigation canals to be standing water . anecdotally , several respondents talked about the necessity of swamps and marshes for mosquito habitat , whereas the most commonly observed standing water types on properties were ponds , reservoirs , and irrigation canals . therefore , we suggest that the malaria education programs that target standing water elimination need to define potential larval habitat as any water where the surface of the water is not moving or moving slowly , rather than the more typical standing water , or aguas estancadas . since the type of standing water available is dependent on land use , it is also important for future studies to more extensively define suitable versus unsuitable habitat types for anopheles larvae to include those that could be considered to flow , such as irrigation canals and roadside ditches . although ecuadorian land owners / managers could be motivated to eliminate standing water through direct observation or advice from authorities , the largest reason provided for not eliminating the water is difficulty ( usually due to topography)a practical problem that would be expensive to address . a large number of respondents below 1500 m used some type of mosquito protection ( 84.0% ) . bed nets were the most prevalent mosquito bite prevention method in low - altitude regions ( 66.4% ) , and were more commonly used than in a community in northern coastal ecuador ( 23% ) , lowland colombia ( 59% ) , or rural uganda ( 26% ) , possibly due to greater socioeconomic standing among our interviewed landowners / managers than other community members [ 3 , 22 , 31 ] . we did not personally assess whether or not the respondents ' bed nets were in good condition , or if they were treated with insecticide , which is important for bite prevention or might help to reduce resting populations of adult mosquitoes within homes [ 32 , 33 ] . their common usage is positive , since one study in the highlands of kenya reports that indoor residual house - spraying actually reduces infection rates more than bed nets . mosquito protection methods that would be employed by ecuadorian land owners / managers during travel to a malaria - endemic area differ slightly from those reported in the travel medical literature . although 8090% of our respondents state that they would use a bed net in a malaria - endemic area of their own country , in one study only 58% of foreign travelers to a malaria - endemic area would do the same . however , a higher proportion of travelers in the same paper would use long sleeves ( 93% ) and chemical repellent ( 90% ) than our respondents ( approximately 5060% ) . we had expected more folklore - based and traditional medicine and mosquito prevention methods from our respondents , since it is known that plant - based malaria prevention and treatments are still quite common in parts of ecuador , for example , [ 4 , 22 , 39 ] and in colombia [ 3 , 23 ] , as well as in rural parts of africa [ 31 , 40 , 41 ] . this may have been in part due to the higher average socioeconomic status of our respondents , who may have been more able to purchase nontraditional mosquito bite prevention methods , such as deet - based chemical repellent . some of the plant - based repellents that were mentioned by respondents included citrus fruits ( e.g. the use of smoke as a mosquito repellent included the traditional burning of termite nests in amazonian regions , as well as burning further studies are required to evaluate the malaria kap of ecuadorian citizens who do not own properties in ecuador , as well as citizens residing elsewhere in the andes , and to elucidate cultural and educational differences among perceptions of malaria risk . a large proportion of land owners / managers presented a good understanding of the occurrence of malaria on the ecuadorian coast or in amazonia and were able to form a basic causative link between standing water , mosquitoes , and malaria . however , about half of the landowners / managers in potential risk areas ( i.e. , altitudes < 1500 m ) did not believe that it was possible to become ill from malaria on their own property . different generalized land uses provided different types of standing water , which may vary in suitability for anopheline larvae . most respondents reported eliminating standing water on their property . this may have been due to partial , but not thorough , elimination of standing water . most respondents in lowland ( at - risk ) regions use some type of mosquito bite prevention , most commonly bed nets . generally , it is hoped that the findings of this study will assist malaria control organizations by providing useful data to improve the efficiency of potential educational programs . while knowledge of malaria 's occurrence patterns is strong in ecuador , even in highland regions , it might be necessary to inform residents of actual malaria risk , so that risk does not continue to be underestimated . although most respondents understand that there is a link between standing water and malaria , the definition of standing water is often too restrictive and does not encompass all potential mosquito habitats on a person 's property . ecuadorian land owners / managers appear to be strongly motivated to eliminate mosquito habitat on their property , and when they do not eliminate standing water , it is usually due to factors beyond their control ( difficulty or limited financial resources ) . most at - risk ecuadorian land owners / managers currently use mosquito bite protection methods , although the use of protection might increase if they were informed that they live in a potentially malarious area .

mouse antichicken iggbiotin ( clone g1 ) and mouse antihuman igg4biotin ( clone hp6025 ) were purchased from southern biotech ( birmingham , al ) . staining buffer , anticd45 v500 ( clone h13 ) , anticd3 af488 ( clone sp342 ) , strepavidin apc ( gmp grade ) , facs lysing solution , cytofix fixation solution were purchased from ( becton dickinson , san jose , ca ) . human igg4 isotype control 2269biotin ( clone 2269 ) , antisema4dbiotin ( clone 2282 ) and the study drug , vx15/2503 , were provided by vaccinex ( rochester , ny ) . samples were acquired on two facscanto ii flow cytometers equipped with 405 nm , 488 nm , and 633 nm lasers from becton dickinson ( san jose , ca ) . each instrument has the same configuration and version of the operating system facsdiva software 6.1.2 from becton dickinson ( san jose , ca ) . the instruments were cross standardized in collaboration with bd biosciences 20 , 21 . briefly , several instruments were characterized using the bd cytometer setup and tracking ( cs&t ) beads and bd facsdiva software . next a virtual predicate instrument was created using the linearity and relative sd of electronic noise ( rsden ) baseline data from all instruments . next application settings were created using 2.5 rsden . after confirming that brightly stained cells would be within the linear range of the instrument , the brightest cs&t bead was used to create the corresponding target channels which were saved in worksheets as application settings . in order to cancel out daytoday variations within individual instruments , quality control data were evaluated using unity real time ( urt ) from biorad laboratories ( hercules , ca ) . samples : peripheral whole blood sample was collected in cytochex bct tubes ( streck labs , omaha , ne ) . clinical specimens were obtained from patients enrolled in the firstinhuman study involving vx15/2503 entitled , safety , tolerability , pharmacokinetics and pharmacodynamics of vx15/2503 , a humanized igg4 antisema4d antibody , in a firstinhuman phase 1 study of patients with advanced solid tumors . clinical specimens were also obtained from the phase 1 ms study in humans involving vx15/2503 entitled , a phase 1 , multicenter , randomized , doubleblind , placebocontrolled , ascending singledose study of the safety , tolerability , and pharmacokinetics of intravenous vx15/2503 in patients with multiple sclerosis . institutional review board approvals for the study protocols , amendments and informed consent documents were obtained prior to study initiation ; study procedures were conducted in accordance with the declaration of helsinki . csema4d saturation assay : two ml of whole blood was washed with pbs and resuspended in one ml of staining buffer . one hundred microliters of washed whole blood were dispensed into each of five polystyrene tubes and incubated with 100 l of mab vx15/2503 ( 20 g / ml ) , or 100 l staining buffer for 30 min at 4c . treated cells were then washed with staining buffer and incubated 30 min at 4c with either 100 l of mouse antichicken igg biotin ( 10 g / ml ) , 100 l of mouse antihuman igg4biotin ( 10 g / ml ) , 100 l of mab 2269 biotin ( 5 g / ml ) , or 100 l of mab 2282 biotin ( 5 g / ml ) . samples were then washed with staining buffer and incubated 30 min at 4c with a cocktail containing 5 l anticd45 v500 , 0.25 l anticd3 af488 and 20 l strepavidin apc . next , samples were incubated for 15 min at room temperature in 3 ml of facs lysing solution , washed with 2 ml pbs and resuspended in 0.5 ml pbs and acquired on a bd facscanto ii . method validation : samples for intraassay imprecision were created by spiking varying amounts of the compound vx15/2503 into peripheral whole blood collected from each of three apparently healthy volunteers . the resulting samples containing low , mid and high levels of vx15/2503 were then assayed in triplicate in a single analytical run . for each sample and each reportable result , the mean , sd and % cv were calculated . interassay imprecision was assessed by analyzing three replicates of immunotrol and immunotrol low ( described below ) in four analytical runs performed by two technicians and distributed over each of two instruments . for each sample , the grand mean ( mean of the daily means ) , sd ( of the daily means ) and % cv ( of all four runs ) were calculated . immunotrol reagents are commercial whole blood products intended to deliver consistent cell populations that can be repeatedly measured to verify reagent performance as well as method performance for staining and analysis . for qc performance monitoring during sample analysis , the qc tubes contain all of the same reagents as the sample tubes , except that immunotrol is used as the sample instead of whole blood . each product ( immunotrol and immunotrol low ) is manufactured to deliver a specific concentration of cells to better ascertain performance across a range of numerical values . for example , a particular lot number of immunotrol reagent may be expected to generate a value of 46% cd4 + cells . a particular lot number of immunotrol low may expect to generate a value of 21% cd4 + cells . as individual patient samples can contain varying sema4d levels , the use of a constant cell concentration can assist in ensuring the assay is performing as expected . for the stability evaluation , peripheral whole blood was collected in ethylenediaminetetraacetic acid ( edta ) and acid citrate dextrose ( acd ) vacutainer tubes ( bectondickinson vacutainer systems , franklin lakes , nj ) as well as cytochex blood collection tube ( bct ) ( streck , inc . samples were spiked ex vivo with 100 ng / ml of vx15/2503 and held at ambient temperature ( 1822c ) or refrigerated ( 4c ) for 24 h , 48 h , 72 h , and 96 h. the percent change from baseline of the percent saturation of sema4d at each time point ( baseline , 24 , 48 , 72 , 96 h ) at a concentration of 100 ng / ml vx15/2503 mab was calculated using the following formula : % change = time point baselinebaseline 100 mouse antichicken iggbiotin ( clone g1 ) and mouse antihuman igg4biotin ( clone hp6025 ) were purchased from southern biotech ( birmingham , al ) . staining buffer , anticd45 v500 ( clone h13 ) , anticd3 af488 ( clone sp342 ) , strepavidin apc ( gmp grade ) , facs lysing solution , cytofix fixation solution were purchased from ( becton dickinson , san jose , ca ) . human igg4 isotype control 2269biotin ( clone 2269 ) , antisema4dbiotin ( clone 2282 ) and the study drug , vx15/2503 , were provided by vaccinex ( rochester , ny ) . samples were acquired on two facscanto ii flow cytometers equipped with 405 nm , 488 nm , and 633 nm lasers from becton dickinson ( san jose , ca ) . each instrument has the same configuration and version of the operating system facsdiva software 6.1.2 from becton dickinson ( san jose , ca ) . the instruments were cross standardized in collaboration with bd biosciences 20 , 21 . briefly , several instruments were characterized using the bd cytometer setup and tracking ( cs&t ) beads and bd facsdiva software . next a virtual predicate instrument was created using the linearity and relative sd of electronic noise ( rsden ) baseline data from all instruments . next application settings were created using 2.5 rsden . after confirming that brightly stained cells would be within the linear range of the instrument , the brightest cs&t bead was used to create the corresponding target channels which were saved in worksheets as application settings . in order to cancel out daytoday variations within individual instruments , quality control data were evaluated using unity real time ( urt ) from biorad laboratories ( hercules , ca ) . samples : peripheral whole blood sample was collected in cytochex bct tubes ( streck labs , omaha , ne ) . clinical specimens were obtained from patients enrolled in the firstinhuman study involving vx15/2503 entitled , safety , tolerability , pharmacokinetics and pharmacodynamics of vx15/2503 , a humanized igg4 antisema4d antibody , in a firstinhuman phase 1 study of patients with advanced solid tumors . clinical specimens were also obtained from the phase 1 ms study in humans involving vx15/2503 entitled , a phase 1 , multicenter , randomized , doubleblind , placebocontrolled , ascending singledose study of the safety , tolerability , and pharmacokinetics of intravenous vx15/2503 in patients with multiple sclerosis . institutional review board approvals for the study protocols , amendments and informed consent documents were obtained prior to study initiation ; study procedures were conducted in accordance with the declaration of helsinki . csema4d saturation assay : two ml of whole blood was washed with pbs and resuspended in one ml of staining buffer . one hundred microliters of washed whole blood were dispensed into each of five polystyrene tubes and incubated with 100 l of mab vx15/2503 ( 20 g / ml ) , or 100 l staining buffer for 30 min at 4c . treated cells were then washed with staining buffer and incubated 30 min at 4c with either 100 l of mouse antichicken igg biotin ( 10 g / ml ) , 100 l of mouse antihuman igg4biotin ( 10 g / ml ) , 100 l of mab 2269 biotin ( 5 g / ml ) , or 100 l of mab 2282 biotin ( 5 g / ml ) . samples were then washed with staining buffer and incubated 30 min at 4c with a cocktail containing 5 l anticd45 v500 , 0.25 l anticd3 af488 and 20 l strepavidin apc . next , samples were incubated for 15 min at room temperature in 3 ml of facs lysing solution , washed with 2 ml pbs and resuspended in 0.5 ml pbs and acquired on a bd facscanto ii . method validation : samples for intraassay imprecision were created by spiking varying amounts of the compound vx15/2503 into peripheral whole blood collected from each of three apparently healthy volunteers . the resulting samples containing low , mid and high levels of vx15/2503 were then assayed in triplicate in a single analytical run . for each sample and each reportable result , the mean , sd and % cv were calculated . interassay imprecision was assessed by analyzing three replicates of immunotrol and immunotrol low ( described below ) in four analytical runs performed by two technicians and distributed over each of two instruments . for each sample , the grand mean ( mean of the daily means ) , sd ( of the daily means ) and % cv ( of all four runs ) were calculated . immunotrol reagents are commercial whole blood products intended to deliver consistent cell populations that can be repeatedly measured to verify reagent performance as well as method performance for staining and analysis . for qc performance monitoring during sample analysis , the qc tubes contain all of the same reagents as the sample tubes , except that immunotrol is used as the sample instead of whole blood . each product ( immunotrol and immunotrol low ) is manufactured to deliver a specific concentration of cells to better ascertain performance across a range of numerical values . for example , a particular lot number of immunotrol reagent may be expected to generate a value of 46% cd4 + cells . a particular lot number of immunotrol low may expect to generate a value of 21% cd4 + cells . as individual patient samples can contain varying sema4d levels , the use of a constant cell concentration can assist in ensuring the assay is performing as expected . for the stability evaluation , peripheral whole blood was collected in ethylenediaminetetraacetic acid ( edta ) and acid citrate dextrose ( acd ) vacutainer tubes ( bectondickinson vacutainer systems , franklin lakes , nj ) as well as cytochex blood collection tube ( bct ) ( streck , inc . samples were spiked ex vivo with 100 ng / ml of vx15/2503 and held at ambient temperature ( 1822c ) or refrigerated ( 4c ) for 24 h , 48 h , 72 h , and 96 h. the percent change from baseline of the percent saturation of sema4d at each time point ( baseline , 24 , 48 , 72 , 96 h ) at a concentration of 100 ng / ml vx15/2503 mab was calculated using the following formula : % change = time point baselinebaseline 100 the csema4d saturation assay uses a peripheral whole blood matrix and a bound receptor design to calculate receptor saturation ( figs . 1a and 1b ) . the bound study drug , vx15/2503 , a human igg4 antisema4d mab , is detected with a fluorescently labeled antiigg4 monoclonal antibody . total available receptors were detected two ways ; the first in the same manner as above except with specimens spiked ex vivo with supersaturating levels of study drug . an advantage of this approach is that the bound drug and available receptors are detected with the same reagents , thus affinity differences and reagent competition do not confound the signal . in addition , a second measurement of total receptor is generated by using a proprietary , noncompeting antisema4d mab ( clone 2282 ) . the csema4d saturation assay uses fewer fluorochromes and more assay tubes to further minimize the effects of unintended antibody and fluorochrome interactions . assay specificity is increased by subtracting the geometric mean fluorescence intensity ( gmfi ) signal from control tubes with secondary reagents alone ( tube 1 ) from tubes containing both primary and secondary reagents ( tubes 2 and 3 ) . t cells were selected as the cell of interest as they express the highest levels of csema4d in human blood ( unpublished results , vaccinex ) . the csema4d saturation assay is a threecolor , fivetube , wash / lyse / wash flow cytometric assay designed to evaluate the level of sema4d and sema4d saturation by vx15/2503 on peripheral t cells . peripheral whole blood was collected in edta , acd , and cytochex bct blood collection tubes . samples were spiked with samples spiked ex vivo with 10 g / ml and 100 ng / ml of vx15/2503 . the three reportable results generated are : 1 the percent saturation of sema4d on peripheral t cells ; 2 the fold over isotype ( foi ) for vx15/2503 ; and 3 foi for mab 2282biotin . the geometric mean fluorescence intensity ( gmfi ) derived from the saapc fl4 for each tube was used to calculate percent saturation and foi using the following equations : percent saturation = | baca 100| vx15/2503 foi = ca mab 2282biotin foi = edwhere : a = fl4 gmfi generated from assay panel tube 1 which represents the background signal . b = fl4 gmfi generated from assay panel tube 2 which represents the unknown sample signal . c = fl4 gmfi generated from assay panel tube 3 which represents the vx15/2503 mab saturated ( maximum ) signal . d = fl4 gmfi generated from assay panel tube 4 which represents the mab 2269 isotype . e = fl4 gmfi generated from assay panel tube 5 which represents the mab 2282 saturated ( maximum ) signal . the assay was capable of detecting differences in receptor saturation in whole blood dosed ex vivo with varying levels of vx15/2503 . assay performance was comparable when the whole blood was collected in a variety of anticoagulants . samples spiked ex vivo with vx15/2503 were stable for up to 3 days in either edta held under refrigerated conditions or in acd or cytochex bct held at ambient temperature ( fig . 2 ) . cytochex bct was selected for further validation and clinical specimens , given that the variability up to day three was slightly less in cytochex bct ( 4.24%cv ) compared to acd ( 5.98%cv ) . the use of refrigerated samples in edta was not implemented due to logistical considerations and the expense of shipping refrigerated specimens . the mean intraassay imprecision for the samples spiked with low , mid , and high levels of vx15/2503 was 5.44%cv ( range of 3.84 to 7.24%cv ) ( table 1 ) . the mean intraassay imprecision values for vx15/2503 foi and for mab 2282biotin foi were 7.35%cv ( range of 2.27 to 10.1%cv ) and 4.85%cv ( range of 2.71 to 7.30%cv ) , respectively . this % cv is acceptable , and please note that differences in absolute values of foi may differ among donors due to different levels of surface expression of sema4d . whole blood was collected in edta , acd , and cytochex bct blood collection tubes and spiked ex vivo with 100 ng / ml of vx15/2503 . samples were assayed on the day of collection and spiking ( baseline ) and at 24 h , 48 h , 72 h , and 96 h. samples were maintained under two conditions : ambient temperature ( 1822c ) or refrigerated ( 4c ) . the percent change from baseline at each time point and condition was calculated with the following formula : % change = time point baselinebaseline 100 . [ color figure can be viewed in the online issue , which is available at wileyonlinelibrary.com . ] whole blood collected from three apparently healthy volunteers was dosed ex vivo with varying concentrations of vx15/2503 . interassay imprecision was assessed using commercially available preserved whole blood material , immunotrol cells and immunotrol low cells ( table 2 ) . given that control material was not prespiked with vx15/2503 , it was not possible to evaluate the percent saturation of sema4d on peripheral t cells ; however , the foi for mab 2282biotin was evaluated . in addition , the apc gmfi from tubes 24 was evaluated ; the mean interassay imprecision values were 18.0%cv ( range of 13.6 to 22.3%cv ) for mab 2282biotin foi , 10%cv ( range of 9.84 to 10.2%cv ) for the apc gmfi of tube 2 , 15.1%cv ( range of 14.4 to 15.9%cv ) for the apc gmfi of tube 3 , and 9.43%cv ( range of 9.38 to 9.47% cv ) for the apc gmfi of tube 4 . for the apc gmfi of tube 5 , sema4d saturation assay interassay imprecision interinstrument variability was evaluated using the interassay imprecision experiment ( table 3 ) . for the apc gmfi of tube 2 , the interinstrument imprecision was 5.93%cv for qcl and 10.5%cv for qcn . similarly , the interinstrument precision for the apc gmfi of tube 3 was 0.388%cv for qcl and 3.67%cv for qcn . for the apc gmfi of tube 4 , the interinstrument precision was 4.04%cv for qcl and 11.1%cv for qcn . for the apc gmfi of tube 5 , the grand mean interassay imprecision was 4.99%cv for qcl and 4.03%cv for qcn . sema4d saturation assay interinstrument imprecision in order to assess longitudinal assay performance , two levels of quality control samples ( immunotrol & immunotrol low ) were included in each analytical run . mean values , sd and % cv ranges were initially established during method validation from the interassay imprecision data ( repeat testing over a course of multiple days , instruments , and technicians ) . mean values for each additional new lot of qc were determined and a fixed % cv applied . qc results were evaluated for acceptability and monitored for trending using biorad unity real time 2.0 statistical package , westgard statistical process control ( spc ) rules , and westgard advisor 22 . as described in the discussion of the interassay imprecision evaluation , the qc material was not prespiked with vx15/2503 , thus it was not possible to evaluate the percent saturation of sema4d on peripheral t cells during validation in monitoring assay performance in study . however , all other appropriate primary stains were added to the qc tubes and the apc gmfi for tubes 2 , 3 , 4 , and 5 were monitored . the assay performance was within 1sd of the established mean throughout the life of the lot number of the qc ( february april 2014 ) ( fig . qc performance ( level 1 immunol trol low , level 2 immunol trol normal ) of the apc gmfi of tube 2 on each date of testing . [ color figure can be viewed in the online issue , which is available at wileyonlinelibrary.com . ] the csema4d saturation assay provided saturation and ligand expression data which aided in evaluation of patients enrolled in the oncology study . patients in the first two dose cohorts were initially treated on day 14 , followed for two weeks , and then dosed again on day 0 and weekly thereafter . mg / kg ) all patients showed complete saturation following the initial dose at day 14 , and the csema4d saturation level declined during the two week safety evaluation period prior to the second dose ( fig . this was expected based on the dose delivered and level of drug achieved in the serum . when the level of drug in the serum fell below approximately 0.1 to 0.3 g / ml , complete t cell saturation was lost ( patnaik , a , et al . patients were dosed weekly thereafter and saturation results demonstrated that the assay was capable of detecting intermediate levels of t cell sema4d saturation as well as complete ligand desaturation . to note , in figure 4a , patients # 1 and 3 received their last dose on day 29 and patient # 2 also received doses on days 36 and 43 . analysis of the level of csema4d on the surface of the t cells ( fig . 4b ) demonstrated that the level of sema4d expressed on the cell surface decreased upon cellular saturation . levels returned to nearnormal after cells became completely unsaturated , as can be seen by the rise in csema4d expression prior to the second dose on day 1 . clinical data was also obtained using serum samples collected from subjects enrolled in the placebo controlled , singleascending dose ms study ( fig . saturation ( fig . 5a and 5c ) and cellular expression ( fig . 5b and 5d ) data from the 1 mg / kg dose cohort is shown . as shown , saturation levels drop off sooner in patient # 8 who is also the patient who had the highest level of antidrug antibodies . overall , the clinical data demonstrate the fidelity , sensitivity , and significance of the clinical saturation assay . patients from a select cohort in the phase 1 oncology trial were scheduled to be dosed with vx15/2503 at 0.3 mg / kg on day 14 , day 0 , and weekly thereafter ( dotted lines ) . to note , patients # 1 and 3 received their last dose on day 29 and patient # 2 also received doses on days 36 and 43 . blood was collected for cellular saturation analysis at several time points following the first dose and also pre and eoi following each of the weekly doses . ( a ) percent saturation values for three patients in the cohort ( 20% represent cells that are unsaturated and values 80% represent cells that are fully saturated ) . ( b ) cellular levels of sema4d expressed as fold expression of mab 2282 binding over isotype . patients from a select cohort in the phase 1 ms trial were dosed with vx15/2503 at 1 mg / kg ( n = 8) or placebo ( n = 2 ) on day 0 , and blood was collected for cellular saturation analysis at several time points through day 45 . percent saturation values are shown for individual patients ( a ) and the mean of the vx15/2503 or placebo groups ( b ) . cellular levels of sema4d , expressed as fold expression of mab 2282 binding over isotype , is also shown for individuals ( c ) and the mean of the vx15/2503 or placebo groups ( d ) . the csema4d saturation assay uses a peripheral whole blood matrix and a bound receptor design to calculate receptor saturation ( figs . 1a and 1b ) . the bound study drug , vx15/2503 , a human igg4 antisema4d mab , is detected with a fluorescently labeled antiigg4 monoclonal antibody . total available receptors were detected two ways ; the first in the same manner as above except with specimens spiked ex vivo with supersaturating levels of study drug . an advantage of this approach is that the bound drug and available receptors are detected with the same reagents , thus affinity differences and reagent competition do not confound the signal . in addition , a second measurement of total receptor is generated by using a proprietary , noncompeting antisema4d mab ( clone 2282 ) . the csema4d saturation assay uses fewer fluorochromes and more assay tubes to further minimize the effects of unintended antibody and fluorochrome interactions . assay specificity is increased by subtracting the geometric mean fluorescence intensity ( gmfi ) signal from control tubes with secondary reagents alone ( tube 1 ) from tubes containing both primary and secondary reagents ( tubes 2 and 3 ) . t cells were selected as the cell of interest as they express the highest levels of csema4d in human blood ( unpublished results , vaccinex ) . the csema4d saturation assay is a threecolor , fivetube , wash / lyse / wash flow cytometric assay designed to evaluate the level of sema4d and sema4d saturation by vx15/2503 on peripheral t cells . peripheral whole blood was collected in edta , acd , and cytochex bct blood collection tubes . samples were spiked with samples spiked ex vivo with 10 g / ml and 100 ng / ml of vx15/2503 . the three reportable results generated are : 1 the percent saturation of sema4d on peripheral t cells ; 2 the fold over isotype ( foi ) for vx15/2503 ; and 3 foi for mab 2282biotin . the geometric mean fluorescence intensity ( gmfi ) derived from the saapc fl4 for each tube was used to calculate percent saturation and foi using the following equations : percent saturation = | baca 100| vx15/2503 foi = ca mab 2282biotin foi = edwhere : a = fl4 gmfi generated from assay panel tube 1 which represents the background signal . b = fl4 gmfi generated from assay panel tube 2 which represents the unknown sample signal . c = fl4 gmfi generated from assay panel tube 3 which represents the vx15/2503 mab saturated ( maximum ) signal . d = fl4 gmfi generated from assay panel tube 4 which represents the mab 2269 isotype . e = fl4 gmfi generated from assay panel tube 5 which represents the mab 2282 saturated ( maximum ) signal . the assay was capable of detecting differences in receptor saturation in whole blood dosed ex vivo with varying levels of vx15/2503 . assay performance was comparable when the whole blood was collected in a variety of anticoagulants . samples spiked ex vivo with vx15/2503 were stable for up to 3 days in either edta held under refrigerated conditions or in acd or cytochex bct held at ambient temperature ( fig . 2 ) . cytochex bct was selected for further validation and clinical specimens , given that the variability up to day three was slightly less in cytochex bct ( 4.24%cv ) compared to acd ( 5.98%cv ) . the use of refrigerated samples in edta was not implemented due to logistical considerations and the expense of shipping refrigerated specimens . the mean intraassay imprecision for the samples spiked with low , mid , and high levels of vx15/2503 was 5.44%cv ( range of 3.84 to 7.24%cv ) ( table 1 ) . the mean intraassay imprecision values for vx15/2503 foi and for mab 2282biotin foi were 7.35%cv ( range of 2.27 to 10.1%cv ) and 4.85%cv ( range of 2.71 to 7.30%cv ) , respectively . this % cv is acceptable , and please note that differences in absolute values of foi may differ among donors due to different levels of surface expression of sema4d . whole blood was collected in edta , acd , and cytochex bct blood collection tubes and spiked ex vivo with 100 ng / ml of vx15/2503 . samples were assayed on the day of collection and spiking ( baseline ) and at 24 h , 48 h , 72 h , and 96 h. samples were maintained under two conditions : ambient temperature ( 1822c ) or refrigerated ( 4c ) . the percent change from baseline at each time point and condition was calculated with the following formula : % change = time point baselinebaseline 100 . [ color figure can be viewed in the online issue , which is available at wileyonlinelibrary.com . ] whole blood collected from three apparently healthy volunteers was dosed ex vivo with varying concentrations of vx15/2503 . interassay imprecision was assessed using commercially available preserved whole blood material , immunotrol cells and immunotrol low cells ( table 2 ) . given that control material was not prespiked with vx15/2503 , it was not possible to evaluate the percent saturation of sema4d on peripheral t cells ; however , the foi for mab 2282biotin was evaluated . in addition , the apc gmfi from tubes 24 was evaluated ; the mean interassay imprecision values were 18.0%cv ( range of 13.6 to 22.3%cv ) for mab 2282biotin foi , 10%cv ( range of 9.84 to 10.2%cv ) for the apc gmfi of tube 2 , 15.1%cv ( range of 14.4 to 15.9%cv ) for the apc gmfi of tube 3 , and 9.43%cv ( range of 9.38 to 9.47% cv ) for the apc gmfi of tube 4 . for the apc gmfi of tube 5 , sema4d saturation assay interassay imprecision interinstrument variability was evaluated using the interassay imprecision experiment ( table 3 ) . for the apc gmfi of tube 2 , the interinstrument imprecision was 5.93%cv for qcl and 10.5%cv for qcn . similarly , the interinstrument precision for the apc gmfi of tube 3 was 0.388%cv for qcl and 3.67%cv for qcn . for the apc gmfi of tube 4 , the interinstrument precision was 4.04%cv for qcl and 11.1%cv for qcn . for the apc gmfi of tube 5 , the grand mean interassay imprecision was 4.99%cv for qcl and 4.03%cv for qcn . in order to assess longitudinal assay performance , two levels of quality control samples ( immunotrol & immunotrol low ) were included in each analytical run . mean values , sd and % cv ranges were initially established during method validation from the interassay imprecision data ( repeat testing over a course of multiple days , instruments , and technicians ) . mean values for each additional new lot of qc were determined and a fixed % cv applied . qc results were evaluated for acceptability and monitored for trending using biorad unity real time 2.0 statistical package , westgard statistical process control ( spc ) rules , and westgard advisor 22 . as described in the discussion of the interassay imprecision evaluation , the qc material was not prespiked with vx15/2503 , thus it was not possible to evaluate the percent saturation of sema4d on peripheral t cells during validation in monitoring assay performance in study . however , all other appropriate primary stains were added to the qc tubes and the apc gmfi for tubes 2 , 3 , 4 , and 5 were monitored . the assay performance was within 1sd of the established mean throughout the life of the lot number of the qc ( february april 2014 ) ( fig . qc performance ( level 1 immunol trol low , level 2 immunol trol normal ) of the apc gmfi of tube 2 on each date of testing . [ color figure can be viewed in the online issue , which is available at wileyonlinelibrary.com . ] the csema4d saturation assay provided saturation and ligand expression data which aided in evaluation of patients enrolled in the oncology study . patients in the first two dose cohorts were initially treated on day 14 , followed for two weeks , and then dosed again on day 0 and weekly thereafter . 4 ) . even at the lowest dose level ( 0.3 mg / kg ) all patients showed complete saturation following the initial dose at day 14 , and the csema4d saturation level declined during the two week safety evaluation period prior to the second dose ( fig . this was expected based on the dose delivered and level of drug achieved in the serum . when the level of drug in the serum fell below approximately 0.1 to 0.3 g / ml , complete t cell saturation was lost ( patnaik , a , et al . patients were dosed weekly thereafter and saturation results demonstrated that the assay was capable of detecting intermediate levels of t cell sema4d saturation as well as complete ligand desaturation . to note , in figure 4a , patients # 1 and 3 received their last dose on day 29 and patient # 2 also received doses on days 36 and 43 . analysis of the level of csema4d on the surface of the t cells ( fig . 4b ) demonstrated that the level of sema4d expressed on the cell surface decreased upon cellular saturation . levels returned to nearnormal after cells became completely unsaturated , as can be seen by the rise in csema4d expression prior to the second dose on day 1 . clinical data was also obtained using serum samples collected from subjects enrolled in the placebo controlled , singleascending dose ms study ( fig . saturation ( fig . 5a and 5c ) and cellular expression ( fig . 5b and 5d ) data from the 1 mg / kg dose cohort is shown . as shown , saturation levels drop off sooner in patient # 8 who is also the patient who had the highest level of antidrug antibodies . overall , the clinical data demonstrate the fidelity , sensitivity , and significance of the clinical saturation assay . patients from a select cohort in the phase 1 oncology trial were scheduled to be dosed with vx15/2503 at 0.3 mg / kg on day 14 , day 0 , and weekly thereafter ( dotted lines ) . to note , patients # 1 and 3 received their last dose on day 29 and patient # 2 also received doses on days 36 and 43 . blood was collected for cellular saturation analysis at several time points following the first dose and also pre and eoi following each of the weekly doses . ( a ) percent saturation values for three patients in the cohort ( 20% represent cells that are unsaturated and values 80% represent cells that are fully saturated ) . ( b ) cellular levels of sema4d expressed as fold expression of mab 2282 binding over isotype . patients from a select cohort in the phase 1 ms trial were dosed with vx15/2503 at 1 mg / kg ( n = 8) or placebo ( n = 2 ) on day 0 , and blood was collected for cellular saturation analysis at several time points through day 45 . percent saturation values are shown for individual patients ( a ) and the mean of the vx15/2503 or placebo groups ( b ) . cellular levels of sema4d , expressed as fold expression of mab 2282 binding over isotype , is also shown for individuals ( c ) and the mean of the vx15/2503 or placebo groups ( d ) . sema4d is a multifunctional molecule with at least three known receptors , high affinity plexinb1 , intermediate affinity plexinb2 and low affinity cd72 3 . the blocking human igg4 monoclonal antibody vx15/2503 is being developed in a number of different indications . a phase 1 trial has been successfully completed in solid tumor patients ( patnaik a , et al . submitted clin can res ; clintrials.gov identifier nct01313065 ) , and a second singleascending dose phase 1 trial was completed in ms patients ( clintrials.gov identifier nct01764737 ) . a phase 2 study in huntington 's disease much is known about different potential mechanisms of action in these indications , but , in all cases , sema4d is blocked from binding to any of its receptors , inhibiting downstream physiological effects . the sema4d saturation assay described herein is a realtime flow cytometric method to measure the extent of this important initial blocking step . like many biomarker assays , the csema4d saturation assay was developed in parallel with vx15/2503 throughout the lifecycle of the compound from the drug discovery phase to the preclinical toxicology phase to clinical testing 23 . 6 ) the fitforpurpose , iterative approach was applied to the validation strategy in that , as the intended use of the data and the associated regulatory requirements changed , the assay was reoptimized and revalidated appropriately 24 , 25 , 26 . the prototype assay was developed during the drug discovery phase and initially used to detect binding of antisema4d candidates to cellular sema4d . during the evaluation of invivo murine models for both oncology and neurology applications , the csema4d saturation assay provided proofofconcept data confirming that the compound was binding as expected to murine splenocytes . for the murine models a mouse antimouse sema4d antibody ( mab 672 ) variant of the drug was used . vx15/2503 is a humanized version of 672 and both antibodies have very similar binding and functional characteristics ( fisher et al . the assay was then further optimized and appropriately validated to support formal glp toxicology studies in rats and cynomolgus monkeys ( using vx15/2503 ) . given that the toxicology studies required serial sampling , the assay was adapted to a whole blood matrix rather than splenocytes . for the primate studies , assay tubes 4 and 5 , which contain an additional isotype and mab 2282 , respectively , were incorporated in the panel . mab 2282 is another human anti sema4d antibody that recognizes primate sema4d even in the presence of bound vx15/2503 and thus provides the measurement for total cellassociated receptor . this reagent has the advantage of not being affected by antisema4d drug levels and can serve as an independent measure of antigen levels . next a clinical assay based on the primate assay was developed to support the firstinman trials . finally , the initial clinical assay was optimized and validated once more to make additional improvements to the process , and this final process is the one described here in the m&m . changes were made which allowed for improved assay performance yet allowed for a comparison of data generated with each method . these improvements included changing to a gmpgrade saapc as lottolot variability was observed in the nongmp grade reagent from another vendor . in the first iteration of the clinical assay , whole blood was prelysed in an ammonium chloride solution ; there were concerns that this step was too operator dependent and thus might introduce variability in the final data set . in the next generation of the assay , whole blood was first washed to remove free drug and serum ig prior to staining and finally a more robust lysefixation step was introduced . qc tubes ( immunotrol ) were added and cd45 staining was added to the final assay to further increase specificity as well as the time and debris gating strategy . the overall assay configuration and calculations were not modified so that data generated in both versions of the assay could be compared . progression of the csema4d saturation assay from discovery through clinical . highlighted list of assay modifications and improvements as the assay transitioned from preclinical to clinical development . flow cytometry , the premier technology for single cell analysis , is ideal for receptor occupancy measurements as it allows for multiparametric analysis . flow cytometry based saturation assays using whole blood provide a quick turnaround time for data ( a few days ) , compared to traditional pk batch testing in frozen samples ( up to a few months ) . thus insights into pk trends another advantage in using whole blood is the lack of manipulation as compared to frozen serum or plasma . in this case , sema4d is expressed in both cellular and soluble forms 8 , 9 and in assessing the level of target saturation , it is possible we could have chosen either form to analyze . the use of fresh whole blood and flow cytometry avoids elisabased issues with frozen samples , including dilutional or serummediated disruption of mabligand equilibrium kinetics , which can result in false positives and negatives 27 . flow cytometry also allows one to gate on specific populations when analyzing saturation , and allowed this assay to focus on t cells , which is the immune cell type in peripheral blood that expresses the highest level of sema4d . the information provided by the saturation assay has contributed to the vx15/2503 : sema4d data package including target biology , pd , and pk data that helped select dosing schedules for future trials . for example , it was determined employing this assay that cellular sema4d expression levels decrease after dosing . this pattern of expression confirmed preclinical data showing that vx15/2503 binding to cellular sema4d induced internalization of the complex ( fisher et al . also , the saturation data was critical to correlate clinical pk and pd results , demonstrating that approximately 0.1 to 0.3 g / ml vx15/2503 was the concentration of drug in serum below which complete t cell saturation was lost ( patnaik a , et al . the approximate concentration of drug in serum at which complete saturation was lost was 1 to 5 g / ml 28 . it is possible this difference from the clinical saturation threshold could be due to slightly different binding kinetics in humans vs. rats / monkeys , allometric scaling factors , the unavailability of appropriate samples needed at frequent time points to determine an accurate threshold , or slight differences in the clinical vs. nonclinical pk assays . this assay has also helped to provide realtime data regarding drug exposure ; for example patient # 3 in figure 4 and patient # 8 in figure 5 demonstrated decreases in saturation earlier than other patients in their respective cohorts , and these were the same two patients that demonstrated the higher titer antidrug antibody responses ( data not shown ) , indicating that the saturation assay is sensitive to immunogenic responses . in addition , the data from this assay has also supplied critical safety information . in the phase 1 oncology trial , a patient in the 1 mg / kg cohort had an infusion reaction following their second thru fourth injections of vx15/2503 . the patient was treated after each infusion to control the reaction but it was unknown whether the patient was indeed clearing the drug due to an antidrug response , in which case it would be logical to remove the patient from the study due to lack of exposure to drug . the saturation assay showed in realtime that the patient was becoming unsaturated between doses , and following the last infusion , even immediately after the dose was administered . the patient was appropriately removed , in part because the saturation data indicated that the patient was not being effectively exposed to test article due to a likely immunogenic response . this receptor occupancy assay has served to provide valuable information throughout the development of vx15/2503 . it has demonstrated that flow based assays can be validated and controlled in a manner suitable for clinical data collection and in some cases provide advantages over more traditional assay platforms . as immunebased targets and monoclonal antibody therapeutics continue to expand , successful validation and control of these flowbased assays will become increasingly important .

backgroundreceptor occupancy , or saturation , assays are often utilized in preclinical and clinical development programs to evaluate the binding of a biologic to a cellular target . these assays provide critical information regarding the dose of drug required to saturate the target as well as important pharmacodymamic ( pd ) data . a flow cytometric method was developed to measure the degree of semaphorin 4d ( sema4d ; cd100 ) saturation by vx15/2303 , an investigational monoclonal antibody specific for sema4d.methodsthe assay detects vx15/2503 , a human igg4 specific for sema4d , with an igg4specific monoclonal antibody.resultsdata generated allowed assessment of two related sema4dspecific pharmacodynamic ( pd ) markers : ( 1 ) the measurement of cellular sema4d ( csema4d ) saturation by vx15/2503 , and ( 2 ) the cell membrane expression levels of csema4d.conclusionsthis assay specifically and reproducibly measured csema4d saturation and expression levels . evaluation of the sema4dspecific pd markers were critical in determining the clinical saturation threshold of csema4d by vx15/2503 . 2015 he authors cytometry part b : clinical cytometry published by wiley periodicals , inc .

MATERIALS AND METHODS Reagents, Instrumentation, Software RESULTS cSEMA4D Saturation Assay Performance Monitoring Clinical Utility DISCUSSION

human igg4 isotype control 2269biotin ( clone 2269 ) , antisema4dbiotin ( clone 2282 ) and the study drug , vx15/2503 , were provided by vaccinex ( rochester , ny ) . after confirming that brightly stained cells would be within the linear range of the instrument , the brightest cs&t bead was used to create the corresponding target channels which were saved in worksheets as application settings . clinical specimens were obtained from patients enrolled in the firstinhuman study involving vx15/2503 entitled , safety , tolerability , pharmacokinetics and pharmacodynamics of vx15/2503 , a humanized igg4 antisema4d antibody , in a firstinhuman phase 1 study of patients with advanced solid tumors . clinical specimens were also obtained from the phase 1 ms study in humans involving vx15/2503 entitled , a phase 1 , multicenter , randomized , doubleblind , placebocontrolled , ascending singledose study of the safety , tolerability , and pharmacokinetics of intravenous vx15/2503 in patients with multiple sclerosis . method validation : samples for intraassay imprecision were created by spiking varying amounts of the compound vx15/2503 into peripheral whole blood collected from each of three apparently healthy volunteers . interassay imprecision was assessed by analyzing three replicates of immunotrol and immunotrol low ( described below ) in four analytical runs performed by two technicians and distributed over each of two instruments . for each sample , the grand mean ( mean of the daily means ) , sd ( of the daily means ) and % cv ( of all four runs ) were calculated . immunotrol reagents are commercial whole blood products intended to deliver consistent cell populations that can be repeatedly measured to verify reagent performance as well as method performance for staining and analysis . for the stability evaluation , peripheral whole blood was collected in ethylenediaminetetraacetic acid ( edta ) and acid citrate dextrose ( acd ) vacutainer tubes ( bectondickinson vacutainer systems , franklin lakes , nj ) as well as cytochex blood collection tube ( bct ) ( streck , inc . samples were spiked ex vivo with 100 ng / ml of vx15/2503 and held at ambient temperature ( 1822c ) or refrigerated ( 4c ) for 24 h , 48 h , 72 h , and 96 h. the percent change from baseline of the percent saturation of sema4d at each time point ( baseline , 24 , 48 , 72 , 96 h ) at a concentration of 100 ng / ml vx15/2503 mab was calculated using the following formula : % change = time point baselinebaseline 100 mouse antichicken iggbiotin ( clone g1 ) and mouse antihuman igg4biotin ( clone hp6025 ) were purchased from southern biotech ( birmingham , al ) . human igg4 isotype control 2269biotin ( clone 2269 ) , antisema4dbiotin ( clone 2282 ) and the study drug , vx15/2503 , were provided by vaccinex ( rochester , ny ) . clinical specimens were obtained from patients enrolled in the firstinhuman study involving vx15/2503 entitled , safety , tolerability , pharmacokinetics and pharmacodynamics of vx15/2503 , a humanized igg4 antisema4d antibody , in a firstinhuman phase 1 study of patients with advanced solid tumors . clinical specimens were also obtained from the phase 1 ms study in humans involving vx15/2503 entitled , a phase 1 , multicenter , randomized , doubleblind , placebocontrolled , ascending singledose study of the safety , tolerability , and pharmacokinetics of intravenous vx15/2503 in patients with multiple sclerosis . csema4d saturation assay : two ml of whole blood was washed with pbs and resuspended in one ml of staining buffer . method validation : samples for intraassay imprecision were created by spiking varying amounts of the compound vx15/2503 into peripheral whole blood collected from each of three apparently healthy volunteers . the resulting samples containing low , mid and high levels of vx15/2503 were then assayed in triplicate in a single analytical run . for each sample , the grand mean ( mean of the daily means ) , sd ( of the daily means ) and % cv ( of all four runs ) were calculated . immunotrol reagents are commercial whole blood products intended to deliver consistent cell populations that can be repeatedly measured to verify reagent performance as well as method performance for staining and analysis . for example , a particular lot number of immunotrol reagent may be expected to generate a value of 46% cd4 + cells . for the stability evaluation , peripheral whole blood was collected in ethylenediaminetetraacetic acid ( edta ) and acid citrate dextrose ( acd ) vacutainer tubes ( bectondickinson vacutainer systems , franklin lakes , nj ) as well as cytochex blood collection tube ( bct ) ( streck , inc . samples were spiked ex vivo with 100 ng / ml of vx15/2503 and held at ambient temperature ( 1822c ) or refrigerated ( 4c ) for 24 h , 48 h , 72 h , and 96 h. the percent change from baseline of the percent saturation of sema4d at each time point ( baseline , 24 , 48 , 72 , 96 h ) at a concentration of 100 ng / ml vx15/2503 mab was calculated using the following formula : % change = time point baselinebaseline 100 the csema4d saturation assay uses a peripheral whole blood matrix and a bound receptor design to calculate receptor saturation ( figs . the bound study drug , vx15/2503 , a human igg4 antisema4d mab , is detected with a fluorescently labeled antiigg4 monoclonal antibody . in addition , a second measurement of total receptor is generated by using a proprietary , noncompeting antisema4d mab ( clone 2282 ) . the csema4d saturation assay uses fewer fluorochromes and more assay tubes to further minimize the effects of unintended antibody and fluorochrome interactions . assay specificity is increased by subtracting the geometric mean fluorescence intensity ( gmfi ) signal from control tubes with secondary reagents alone ( tube 1 ) from tubes containing both primary and secondary reagents ( tubes 2 and 3 ) . t cells were selected as the cell of interest as they express the highest levels of csema4d in human blood ( unpublished results , vaccinex ) . the csema4d saturation assay is a threecolor , fivetube , wash / lyse / wash flow cytometric assay designed to evaluate the level of sema4d and sema4d saturation by vx15/2503 on peripheral t cells . peripheral whole blood was collected in edta , acd , and cytochex bct blood collection tubes . the mean intraassay imprecision for the samples spiked with low , mid , and high levels of vx15/2503 was 5.44%cv ( range of 3.84 to 7.24%cv ) ( table 1 ) . this % cv is acceptable , and please note that differences in absolute values of foi may differ among donors due to different levels of surface expression of sema4d . whole blood was collected in edta , acd , and cytochex bct blood collection tubes and spiked ex vivo with 100 ng / ml of vx15/2503 . given that control material was not prespiked with vx15/2503 , it was not possible to evaluate the percent saturation of sema4d on peripheral t cells ; however , the foi for mab 2282biotin was evaluated . sema4d saturation assay interinstrument imprecision in order to assess longitudinal assay performance , two levels of quality control samples ( immunotrol & immunotrol low ) were included in each analytical run . as described in the discussion of the interassay imprecision evaluation , the qc material was not prespiked with vx15/2503 , thus it was not possible to evaluate the percent saturation of sema4d on peripheral t cells during validation in monitoring assay performance in study . the csema4d saturation assay provided saturation and ligand expression data which aided in evaluation of patients enrolled in the oncology study . patients in the first two dose cohorts were initially treated on day 14 , followed for two weeks , and then dosed again on day 0 and weekly thereafter . mg / kg ) all patients showed complete saturation following the initial dose at day 14 , and the csema4d saturation level declined during the two week safety evaluation period prior to the second dose ( fig . this was expected based on the dose delivered and level of drug achieved in the serum . when the level of drug in the serum fell below approximately 0.1 to 0.3 g / ml , complete t cell saturation was lost ( patnaik , a , et al . patients were dosed weekly thereafter and saturation results demonstrated that the assay was capable of detecting intermediate levels of t cell sema4d saturation as well as complete ligand desaturation . analysis of the level of csema4d on the surface of the t cells ( fig . 5b and 5d ) data from the 1 mg / kg dose cohort is shown . overall , the clinical data demonstrate the fidelity , sensitivity , and significance of the clinical saturation assay . ( b ) cellular levels of sema4d expressed as fold expression of mab 2282 binding over isotype . patients from a select cohort in the phase 1 ms trial were dosed with vx15/2503 at 1 mg / kg ( n = 8) or placebo ( n = 2 ) on day 0 , and blood was collected for cellular saturation analysis at several time points through day 45 . cellular levels of sema4d , expressed as fold expression of mab 2282 binding over isotype , is also shown for individuals ( c ) and the mean of the vx15/2503 or placebo groups ( d ) . the bound study drug , vx15/2503 , a human igg4 antisema4d mab , is detected with a fluorescently labeled antiigg4 monoclonal antibody . in addition , a second measurement of total receptor is generated by using a proprietary , noncompeting antisema4d mab ( clone 2282 ) . the csema4d saturation assay uses fewer fluorochromes and more assay tubes to further minimize the effects of unintended antibody and fluorochrome interactions . t cells were selected as the cell of interest as they express the highest levels of csema4d in human blood ( unpublished results , vaccinex ) . the csema4d saturation assay is a threecolor , fivetube , wash / lyse / wash flow cytometric assay designed to evaluate the level of sema4d and sema4d saturation by vx15/2503 on peripheral t cells . the assay was capable of detecting differences in receptor saturation in whole blood dosed ex vivo with varying levels of vx15/2503 . cytochex bct was selected for further validation and clinical specimens , given that the variability up to day three was slightly less in cytochex bct ( 4.24%cv ) compared to acd ( 5.98%cv ) . the mean intraassay imprecision for the samples spiked with low , mid , and high levels of vx15/2503 was 5.44%cv ( range of 3.84 to 7.24%cv ) ( table 1 ) . this % cv is acceptable , and please note that differences in absolute values of foi may differ among donors due to different levels of surface expression of sema4d . whole blood was collected in edta , acd , and cytochex bct blood collection tubes and spiked ex vivo with 100 ng / ml of vx15/2503 . samples were assayed on the day of collection and spiking ( baseline ) and at 24 h , 48 h , 72 h , and 96 h. samples were maintained under two conditions : ambient temperature ( 1822c ) or refrigerated ( 4c ) . given that control material was not prespiked with vx15/2503 , it was not possible to evaluate the percent saturation of sema4d on peripheral t cells ; however , the foi for mab 2282biotin was evaluated . in addition , the apc gmfi from tubes 24 was evaluated ; the mean interassay imprecision values were 18.0%cv ( range of 13.6 to 22.3%cv ) for mab 2282biotin foi , 10%cv ( range of 9.84 to 10.2%cv ) for the apc gmfi of tube 2 , 15.1%cv ( range of 14.4 to 15.9%cv ) for the apc gmfi of tube 3 , and 9.43%cv ( range of 9.38 to 9.47% cv ) for the apc gmfi of tube 4 . as described in the discussion of the interassay imprecision evaluation , the qc material was not prespiked with vx15/2503 , thus it was not possible to evaluate the percent saturation of sema4d on peripheral t cells during validation in monitoring assay performance in study . qc performance ( level 1 immunol trol low , level 2 immunol trol normal ) of the apc gmfi of tube 2 on each date of testing . the csema4d saturation assay provided saturation and ligand expression data which aided in evaluation of patients enrolled in the oncology study . even at the lowest dose level ( 0.3 mg / kg ) all patients showed complete saturation following the initial dose at day 14 , and the csema4d saturation level declined during the two week safety evaluation period prior to the second dose ( fig . this was expected based on the dose delivered and level of drug achieved in the serum . when the level of drug in the serum fell below approximately 0.1 to 0.3 g / ml , complete t cell saturation was lost ( patnaik , a , et al . patients were dosed weekly thereafter and saturation results demonstrated that the assay was capable of detecting intermediate levels of t cell sema4d saturation as well as complete ligand desaturation . analysis of the level of csema4d on the surface of the t cells ( fig . 4b ) demonstrated that the level of sema4d expressed on the cell surface decreased upon cellular saturation . overall , the clinical data demonstrate the fidelity , sensitivity , and significance of the clinical saturation assay . ( b ) cellular levels of sema4d expressed as fold expression of mab 2282 binding over isotype . patients from a select cohort in the phase 1 ms trial were dosed with vx15/2503 at 1 mg / kg ( n = 8) or placebo ( n = 2 ) on day 0 , and blood was collected for cellular saturation analysis at several time points through day 45 . percent saturation values are shown for individual patients ( a ) and the mean of the vx15/2503 or placebo groups ( b ) . cellular levels of sema4d , expressed as fold expression of mab 2282 binding over isotype , is also shown for individuals ( c ) and the mean of the vx15/2503 or placebo groups ( d ) . the blocking human igg4 monoclonal antibody vx15/2503 is being developed in a number of different indications . submitted clin can res ; clintrials.gov identifier nct01313065 ) , and a second singleascending dose phase 1 trial was completed in ms patients ( clintrials.gov identifier nct01764737 ) . the sema4d saturation assay described herein is a realtime flow cytometric method to measure the extent of this important initial blocking step . like many biomarker assays , the csema4d saturation assay was developed in parallel with vx15/2503 throughout the lifecycle of the compound from the drug discovery phase to the preclinical toxicology phase to clinical testing 23 . 6 ) the fitforpurpose , iterative approach was applied to the validation strategy in that , as the intended use of the data and the associated regulatory requirements changed , the assay was reoptimized and revalidated appropriately 24 , 25 , 26 . the prototype assay was developed during the drug discovery phase and initially used to detect binding of antisema4d candidates to cellular sema4d . during the evaluation of invivo murine models for both oncology and neurology applications , the csema4d saturation assay provided proofofconcept data confirming that the compound was binding as expected to murine splenocytes . next a clinical assay based on the primate assay was developed to support the firstinman trials . finally , the initial clinical assay was optimized and validated once more to make additional improvements to the process , and this final process is the one described here in the m&m . in the first iteration of the clinical assay , whole blood was prelysed in an ammonium chloride solution ; there were concerns that this step was too operator dependent and thus might introduce variability in the final data set . in the next generation of the assay , whole blood was first washed to remove free drug and serum ig prior to staining and finally a more robust lysefixation step was introduced . qc tubes ( immunotrol ) were added and cd45 staining was added to the final assay to further increase specificity as well as the time and debris gating strategy . progression of the csema4d saturation assay from discovery through clinical . flow cytometry based saturation assays using whole blood provide a quick turnaround time for data ( a few days ) , compared to traditional pk batch testing in frozen samples ( up to a few months ) . flow cytometry also allows one to gate on specific populations when analyzing saturation , and allowed this assay to focus on t cells , which is the immune cell type in peripheral blood that expresses the highest level of sema4d . the information provided by the saturation assay has contributed to the vx15/2503 : sema4d data package including target biology , pd , and pk data that helped select dosing schedules for future trials . for example , it was determined employing this assay that cellular sema4d expression levels decrease after dosing . this pattern of expression confirmed preclinical data showing that vx15/2503 binding to cellular sema4d induced internalization of the complex ( fisher et al . it is possible this difference from the clinical saturation threshold could be due to slightly different binding kinetics in humans vs. rats / monkeys , allometric scaling factors , the unavailability of appropriate samples needed at frequent time points to determine an accurate threshold , or slight differences in the clinical vs. nonclinical pk assays . the saturation assay showed in realtime that the patient was becoming unsaturated between doses , and following the last infusion , even immediately after the dose was administered . the patient was appropriately removed , in part because the saturation data indicated that the patient was not being effectively exposed to test article due to a likely immunogenic response .

to date , no standard agents have been shown to improve healing of severely injured or transected muscle to support the effect on stress urinary incontinence ( sui ) , nor has this possibility been considered from theoretical or practical points of view [ 16 ] . to alternatively resolve the problem , we focused on sui and the stable gastric pentadecapeptide bpc 157 . interestingly , it is an antiulcer peptide that can not be degraded by 24-hour exposure to human gastric juice . interestingly , it is an antiulcer peptide that can not be degraded by 24-hour exposure to human gastric juice , with established safe therapy profile ( lethal dose not achieved even at 2 g / kg b.w . ) , efficient in inflammatory bowel disease ( pl 14736 ; for review see , e.g. ) , wound and collagen healing [ 912 ] , with particular effect on muscle healing [ 1317 ] , and failed lower esophageal sphincter and pyloric sphincter . therefore , its parenteral and per - oral application in rats after transabdominal urethrolysis ( tu ) and prolonged vaginal dilatation ( vd ) may be interesting . pharmacological treatment for sui is mostly focused on the use of nonselective alpha - agonists , which are often ineffective , and serotonin and norepinephrine reuptake inhibitors ( e.g. , duloxetine ) . in addition , particular combinations ( alpha2-adrenoceptor blockade and duloxetine ) and various other possibilities were suggested ( eg , angiotensin ii ) . most improvements of therapy ( e.g. , alpha - agonists ) aim to selectively ( eg , sub - type - selective alpha1-adrenoceptor agonists ) affect urethral pressure , peripherally , and most recently , centrally , and these therapies may affect the bladder or urethral properties of healthy subjects ( e.g. , selective , partial agonist at the human alpha1(a)-adrenoceptor ) , explaining the elevation in leak point pressure ( lpp ) . this approach did not offer the necessary degree of separation over cardiovascular events when assessed in in vivo models of cardiovascular function [ 46,20 ] . moreover , since a synergistically improved continence rate with a combination of physiotherapy and pharmacological treatment ( e.g. , with duloxetine ) , it is therefore more surprising that none of the standard agents was shown to improve the healing of severely injured or transected muscle to support the effect on sui . these were the reasons why we suggested the stable gastric pentadecapeptide bpc 157 to recover lpp in rat sui after tu and vd . namely , a particular rescuing effect on failed lpp , avoiding an effect in normal healthy rats , may be even more likely since this originally anti - ulcer stable gastric pentadecapeptide , bpc 157 , was shown to particularly heal sphincters in gastrointestinal tract , as well as both injured striated muscle and smooth muscle . providing that urethral cross section contains both smooth and striated muscle , bpc 157 s effect on both smooth muscle and striated muscle was assessed in proximal , middle and distal urethral segments , along with the effect on vessel density . intriguingly , as mentioned , previous studies have demonstrated this peptide s ability to rescue failed sphincter function in esophagitis rats , rapidly normalizing decreased pressure in lower esophageal and pyloric sphincters . however , gi and urinary tract dysfunctions ( e.g. , sui ) may present completely different mechanisms , and therefore it may be interesting to study sui rats , particularly after vd or tu . pressure - induced ischemia , pelvic floor injury , and dysfunction of the urethral continence mechanism seen in vd , as well as loss of anatomic urethral support , loss of innervations and muscle atrophy presented with tu , are commonly regarded as well reproducible sui features . taken together , these factors may correspond with sui complex pathophysiology , leading to a chronic functional syndrome . thus , tu - rats and vd - rats ( or healthy rats ) received bpc 157 in the regimens successfully used before , either intraperitoneally , or per - orally , in drinking water , during a 7-day period , . 1419 ( diagen , ljubljana , slovenia ) , a partial sequence of human gastric juice protein bpc , freely soluble in water at ph 7.0 and in saline , prepared with 99% high pressure liquid chromatography purity ( 1-des - gly peptide as impurity , biologically inactive ) ( without carrier or peptidase inhibitor ) for all treatment protocols , prepared as described before . female wistar albino rats ( n=7 in each group ) , retired breeders , weighing 310350 g , were used in this study . all experimental protocols were approved by the ethics committee at the medical faculty , university of zagreb and conformed to the international directive 2010/63/eu of the european parliament on the protection of animals used for scientific purposes . surgery was performed in deeply anesthetized rats : ketamine ( 20 mg / kg b.w . intraperitoneally ; ketanest , parke - davis gmbh , berlin , germany ) and diazepam ( 6 mg / kg b.w . intraperitoneally ; we utilized 2 well - established rat models [ 2426 ] : tu and vd . in brief , rats assigned to tu group were laparotomized and circumferential detachment of proximal and distal urethra from the anterior vaginal wall and pubic bone was carried out by sharp dissection of endopelvic fascia ; whereas sham - operated animals underwent laparotomy and bladder manipulation with forceps , but the urethra and bladder neck were untouched . the vd group was subjected to sustained 2-hr inflation ( 5 ml of saline ) of a modified ( tip cut off ) 12 fr foley catheter ( rsch inc . , duluth , georgia , usa ) inserted into the vagina and secured into place with one 3/0 silk suture , and the sham group had a catheter inserted and secured , but not inflated . bpc 157 was given throughout the 7-day regimen in healthy rats or after tu or vd ; intraperitoneally ( dissolved in saline [ 2 g or 2 ng / ml ] ) in a single daily dose of the 10 g / kg or 10 ng / kg b.w . , first administration 30 min after surgery , last 24 hr before the lpp testing and sacrifice ; or 10 g / kg / day , dissolved in tap water ( 0.16 g / ml , 12 ml / rat / day ) . controls received only drinking water or an equal volume of saline ( 5 ml / kg b.w . all operated ( tu and vd ) and sham - operated animals and 7 healthy rats underwent lpp assessment using a methodology based on previously described techniques [ 2428 ] . under urethane anesthesia ( 1.2 mg / kg intraperitoneally ; sigma - aldrich chemie gmbh , steinheim , germany ) , used in order to maintain physiologic urethral responses , the bladder was exposed via midline abdominal incision and manually emptied . a 24 g transvesical catheter , connected both to an infusion pump ( green stream vo - p argus 414 , argus medical ag , heimberg , switzerland ) and a monitor with an invasive pressure transducer module ( model 90309 , spacelabs medical inc . , redmond , washington , usa ) via a 3-way stop cock , was inserted and secured into the bladder dome , and the abdominal wall was temporarily closed with sutures . intravesical pressures [ mmhg ] were referenced to air pressure at the level of the bladder and were observed continuously as the bladder was subsequently filled with room - temperature saline at the rate of 5 ml / hr . at half bladder capacity ( 0.4 ml ) , infusion was stopped and gentle pressure was applied increasingly over the bladder until the first drop of fluid was seen on the urethral meatus ; the recorded intravesical pressure at that point was regarded as the lpp . by definition , sui occurs in the absence of bladder contractions . therefore , if a bladder contraction occurred during lpp measurement ( e.g. , void is triggered ; easily distinguished from leaks ) those data were omitted , the bladder was drained , refilled and the lpp test restarted . the average of 3 consecutively measured lpps was taken as a data point for each animal . the anesthetized animals were killed by exsanguination immediately after completing lpp measurements and the whole bladder and urethra were harvested by removing the symphysis pubis , thus preserving the entire urethral segment . the specimens were fixed in 10% neutral buffered formalin overnight and embedded in paraffin , semi - sequentially cut ( 5 m thickness ; proximal , middle and distal urethra ) , and stained with haematoxylin - eosin and with mallory s trichrome for morphometry assessment of muscle / connective tissue ratio in mid - urethral segment . additionally , for all 3 urethral segments , immunohistochemistry studies were carried out for desmin ( delineates a striated organization of this filamentous protein identical in the external sphincter and the skeletal muscle ) , smooth muscle actin ( sma ; smooth muscle cell marker ) and cd34 ( angiogenic marker ) ( 1:50 ; dako denmark a / s , glostrup , denmark ) , in order to assess striated and smooth muscular layer thickness and blood vessel count . each set of slides used for immunohistochemical study was accompanied by control sections known to contain cells positive for the examined antigen . morphometrical analysis was done using sform and issa computer programs ( vams tec d.o.o . , zagreb , croatia ) five high power fields were randomly selected for analysis . for analysis we used the software statistica 7.1 . subsequently , if normal distribution occurred , one - way anova with post hoc newman - keuls was performed . 1419 ( diagen , ljubljana , slovenia ) , a partial sequence of human gastric juice protein bpc , freely soluble in water at ph 7.0 and in saline , prepared with 99% high pressure liquid chromatography purity ( 1-des - gly peptide as impurity , biologically inactive ) ( without carrier or peptidase inhibitor ) for all treatment protocols , prepared as described before . female wistar albino rats ( n=7 in each group ) , retired breeders , weighing 310350 g , were used in this study . all experimental protocols were approved by the ethics committee at the medical faculty , university of zagreb and conformed to the international directive 2010/63/eu of the european parliament on the protection of animals used for scientific purposes . surgery was performed in deeply anesthetized rats : ketamine ( 20 mg / kg b.w . intraperitoneally ; ketanest , parke - davis gmbh , berlin , germany ) and diazepam ( 6 mg / kg b.w . we utilized 2 well - established rat models [ 2426 ] : tu and vd . in brief , rats assigned to tu group were laparotomized and circumferential detachment of proximal and distal urethra from the anterior vaginal wall and pubic bone was carried out by sharp dissection of endopelvic fascia ; whereas sham - operated animals underwent laparotomy and bladder manipulation with forceps , but the urethra and bladder neck were untouched . the vd group was subjected to sustained 2-hr inflation ( 5 ml of saline ) of a modified ( tip cut off ) 12 fr foley catheter ( rsch inc . , duluth , georgia , usa ) inserted into the vagina and secured into place with one 3/0 silk suture , and the sham group had a catheter inserted and secured , but not inflated . bpc 157 was given throughout the 7-day regimen in healthy rats or after tu or vd ; intraperitoneally ( dissolved in saline [ 2 g or 2 ng / ml ] ) in a single daily dose of the 10 g / kg or 10 ng / kg b.w . , first administration 30 min after surgery , last 24 hr before the lpp testing and sacrifice ; or 10 g / kg / day , dissolved in tap water ( 0.16 g / ml , 12 ml / rat / day ) . controls received only drinking water or an equal volume of saline ( 5 ml / kg b.w . , on the 7 postoperative day all operated ( tu and vd ) and sham - operated animals and 7 healthy rats underwent lpp assessment using a methodology based on previously described techniques [ 2428 ] . under urethane anesthesia ( 1.2 mg / kg intraperitoneally ; sigma - aldrich chemie gmbh , steinheim , germany ) , used in order to maintain physiologic urethral responses , the bladder was exposed via midline abdominal incision and manually emptied . a 24 g transvesical catheter , connected both to an infusion pump ( green stream vo - p argus 414 , argus medical ag , heimberg , switzerland ) and a monitor with an invasive pressure transducer module ( model 90309 , spacelabs medical inc . , redmond , washington , usa ) via a 3-way stop cock , was inserted and secured into the bladder dome , and the abdominal wall was temporarily closed with sutures . intravesical pressures [ mmhg ] were referenced to air pressure at the level of the bladder and were observed continuously as the bladder was subsequently filled with room - temperature saline at the rate of 5 ml / hr . at half bladder capacity ( 0.4 ml ) , infusion was stopped and gentle pressure was applied increasingly over the bladder until the first drop of fluid was seen on the urethral meatus ; the recorded intravesical pressure at that point was regarded as the lpp . by definition therefore , if a bladder contraction occurred during lpp measurement ( e.g. , void is triggered ; easily distinguished from leaks ) those data were omitted , the bladder was drained , refilled and the lpp test restarted . the average of 3 consecutively measured lpps was taken as a data point for each animal . the anesthetized animals were killed by exsanguination immediately after completing lpp measurements and the whole bladder and urethra were harvested by removing the symphysis pubis , thus preserving the entire urethral segment . the specimens were fixed in 10% neutral buffered formalin overnight and embedded in paraffin , semi - sequentially cut ( 5 m thickness ; proximal , middle and distal urethra ) , and stained with haematoxylin - eosin and with mallory s trichrome for morphometry assessment of muscle / connective tissue ratio in mid - urethral segment . additionally , for all 3 urethral segments , immunohistochemistry studies were carried out for desmin ( delineates a striated organization of this filamentous protein identical in the external sphincter and the skeletal muscle ) , smooth muscle actin ( sma ; smooth muscle cell marker ) and cd34 ( angiogenic marker ) ( 1:50 ; dako denmark a / s , glostrup , denmark ) , in order to assess striated and smooth muscular layer thickness and blood vessel count . each set of slides used for immunohistochemical study was accompanied by control sections known to contain cells positive for the examined antigen . morphometrical analysis was done using sform and issa computer programs ( vams tec d.o.o . , zagreb , croatia ) subsequently , if normal distribution occurred , one - way anova with post hoc newman - keuls was performed . likewise , sham operations did not produce any changes ( figure 1 ) . on the other hand , tu produced lpp values markedly decreased a week after surgery . bpc 157 therapy , regardless of the given dose - regimen or mode of administration , completely counteracted the decrease of lpp values . on some occasions , bpc 157 values reached those originally noted in healthy rats . likewise , in the vd group , at the end of the week all control rats exhibited markedly decreased lpp values . again , all of the used bpc 157 dose regimens or routes of administration completely opposed the decrease of lpp values , with some subgroups reaching values originally noted in healthy animals ( figure 2 ) . the histological findings agree with the lpp studies , finding no particular changes after sham operations . morphometrical histological studies with mallory s trichrome staining revealed marked post - operative decrease in muscle tissue content in the mid - urethral segment in all operated animals , but with much more pronounced muscle loss found in control groups . this loss yielded statistically significant differences in muscle / connective tissue ratio between treated and control animals in tu and vd groups ( figures 3 and 4 ) . additionally , higher proportion of sma ( figure 5 ) and desmin ( figures 6 and 7 ) positivity was regularly observed in the urethral wall of all bpc 157-treated vd and tu animals , and a noticeable increase in vessel density within all segments of the urethra ( immunohistochemical staining for cd34 ) ( figure 8) . accordingly , while the values in bpc 157 approached the values noted in healthy or sham operated rats , vd and tu controls consistently exhibited markedly lower values . likewise , sham operations did not produce any changes ( figure 1 ) . on the other hand , tu produced lpp values markedly decreased a week after surgery . bpc 157 therapy , regardless of the given dose - regimen or mode of administration , completely counteracted the decrease of lpp values . on some occasions , bpc 157 values reached those originally noted in healthy rats . likewise , in the vd group , at the end of the week all control rats exhibited markedly decreased lpp values . again , all of the used bpc 157 dose regimens or routes of administration completely opposed the decrease of lpp values , with some subgroups reaching values originally noted in healthy animals ( figure 2 ) . the histological findings agree with the lpp studies , finding no particular changes after sham operations . morphometrical histological studies with mallory s trichrome staining revealed marked post - operative decrease in muscle tissue content in the mid - urethral segment in all operated animals , but with much more pronounced muscle loss found in control groups . this loss yielded statistically significant differences in muscle / connective tissue ratio between treated and control animals in tu and vd groups ( figures 3 and 4 ) . additionally , higher proportion of sma ( figure 5 ) and desmin ( figures 6 and 7 ) positivity was regularly observed in the urethral wall of all bpc 157-treated vd and tu animals , and a noticeable increase in vessel density within all segments of the urethra ( immunohistochemical staining for cd34 ) ( figure 8) . accordingly , while the values in bpc 157 approached the values noted in healthy or sham operated rats , vd and tu controls consistently exhibited markedly lower values . as we hypothesized , based on bpc 157 s particular beneficial effect on damaged muscle healing [ 1317 ] and rescue of failed lower esophageal sphincter and pyloric sphincter , failed lpp after tu or vd was successfully recovered , and this was regularly achieved in all bpc 157-treated rats , either with intraperitoneal ( both g and ng dose ) or per - oral application , along with microscopic / immunohistochemical improvement involving both striated and smooth muscle , specifically shown in all urethral segments . unlike the standard therapy that may affect the bladder or urethral pressure of normal rats , and consequently explain the elevation in lpp ( e.g. , alpha adrenergic agonism ) , lpp values in healthy rats were not changed by bpc 157 medication , and thereby , failed lpp was specifically recovered . unlike poor control tu or vd presentation ( which corresponds to those regularly noted in other sui studies ) , the recovered function in bpc 157-tu or bpc 157-vd rats , as commonly observed in muscle injury studies [ 1317 ] , may be consequent to an enhanced healing process , and vice versa ; the recovered function by itself promotes healing , and such functional recovery could be rather complete , and also dose - dependent , with lpp reaching values originally noted in healthy animals . a marked attenuation of both striated and smooth muscle layers in all urethral segments of control animals was alleviated toward nearly normal values with bpc 157 regimens , thus the function of both striated and smooth muscle was improved to rescue failure of lpp . from the viewpoint of the standard therapy , consistency in severely injured and/or transected muscle healing to support the corresponding effect on sui [ 1317 ] , established by bpc 157 , has thus far not been commonly encountered with other agents [ 16 ] , therefore the noted importance of such recovery of failed lpp is better founded in bpc 157-tu-/vd rats . accordingly , providing the greatest histological evidence of urethral damage is especially obvious in the skeletal muscle layer . improved microscopy / immunochemistry and restored function ( that had to be regularly , definitively debilitated ) seen with transected or crushed muscle is congruous with increased desmin positivity noted in all urethral segments of bpc 157 rats after tu and vd , and lpp values fully recovered . bpc 157-induced muscle healing implies modulation of the same events ; fostering of myocyte regeneration , thus shortening the healing period and avoiding excessive scar formation , again analogous to the preserved muscle / connective tissue ratio in treated rat urethras observed in this study . additionally , with the same regimens of bpc 157 therapy , along with the previous findings on injured striated muscle , healing effect on peripheral nerve , and on smooth muscle of gastrointestinal tract and sphincters [ 15,1719 ] , and angiogenesis , may be particularly relevant to the noted recovery of the failed lpp in rats after tu and vd . providing that the procedures used , vd and tu , also directly damaged smooth muscle function , eliminating some of their functions for a while , after gi tract massive resection the remaining part more vigorously adapts in bpc 157 rats , and overwhelms the lack of the removed part . since both smooth and striated muscle contribute to urethral pressure during filling phase , with accompanying fast twitch fibers contraction reflex that further elevate urethral tone when intraabdominal pressure rises , the restoration of their integrity and functions likely contributed to bpc 157 anti - sui mechanisms . likewise ( even if gi and urinary tract dysfunctions ( e.g. , sui ) may present completely different mechanisms ) , sphincter failure match esophagitis rats and vd rats , with the methods of prolonged dilation , the fairly analogous muscle stretch , and the definitive sphincter failure , did not spontaneously recover until the end of the experiments . in either case , the effect of the bpc 157 is particularly evident in animals with failed sphincter function and obtained using both parenteral and per - oral applications . considering that pudendal nerve damage is also implicated in sui pathophysiology , as demonstrated in some other sui rat model studies ( crush , transection ) , it is important to point out that bpc 157 , besides having muscle healing potential [ 1319 ] , exhibited significant neuroprotective capabilities ( e.g. , directly improving the transected sciatic nerve healing ) , thus contributing to regained function after major injury . of note , loss of innervations and muscle atrophy exists in both vd and tu models as well . since damaged / transected muscle healing commonly requires regeneration of damaged intramuscular nerve branches , it may be that in successful recovery bpc 157 course these parallel healing processes promote each other . likewise , it was clearly demonstrated that vd results in decreased blood flow to , and hypoxia of , the bladder , urethra and vagina , supportive of hypoxic injury as a possible mechanism of injury leading to sui . thus , the observed increase in urethral vessel density , which parallels lpp recovery after tu or vd in bpc 157 treated animals , specifically implies this peptide s previously documented angiogenic effect to be potentially accountable for rapid restoration of urethral function . noticeably , it is along with a new vascular shift toward the left as shown in different models , particularly in muscle healing , even in corticosteroid - aggravated conditions , and also in hypovascular tissues ( e.g. , tendon ) . this demonstrates the prominent up - regulation of vascular endothelial growth factor ( vegf ) , likely with particular effect on connective tissue healing , such as the expression of early growth response 1 ( egr-1 ) gene and its repressor , nerve growth factor 1-a binding protein-2 ( nab2 ) , resulting in early extracellular matrix ( collagen ) formation . of note , an enhanced angiogenesis ( ie , initial angiogenesis phase followed by accelerated vegf , cd34 and fviii ) always correlated with increased biomechanical healing rate . also , bpc 157 may have a direct effect on myocytes , probably analogous to that on tendon fibroblasts throughout the fak - paxillin pathway . the evidence was compelling in all of the models used , providing the consistently recovered lpp after vd and tu with all bpc 157 regimens , suggesting that these beneficial effects may be selectively applied to the urethra and treatment of sui . however , due to the potential limitations of the study , the evidence that bpc 157 ameliorates the sui is inconclusive . namely , although the 7-day course obviously is very short compared to clinical situations in which urethral injury usually occurred decades ago , the relevance of the commonly used methods ( and also therapies proposed ) has been established in a period as short as 4 days . this is not surprising , considering common understanding that the earliest course its aggravation or attenuation may be the most relevant for the final ( even long - term ) positive or negative injury outcome . in other relevant muscle and nerve bpc 157 studies , however , very long injury periods models were also used [ 1319,30,32 ] . in other words , even if this study is about prevention rather than reversal of sui , this does not diminish the relevant value of the obtained beneficial effects of bpc 157 application in rats that underwent tu and vd , since from the results it is obvious that bpc 157 therapy benefit has a long - term and sustained effect , providing the recovered lpp in tu- and vd - treated rats when the last administration had been at 24 hr before assessment ( intraperitoneal regimen ) . bpc 157 could be easily administered ( e.g. , also per - orally ; in drinking water ) . furthermore , considering the cardiovascular effects that may be a common problem with standard sui therapy , in vivo models of cardiovascular function showed that bpc 157 does not affect normal blood pressure or heart rhythm [ 4244 ] , but it did reduce l - name hypertension , counteract no system failure by nos - blockade in different models [ 4345 ] ( no - synthesis is directly related to muscle injury healing ) , doxorubicine chronic heart failure and digitalis overdose arrhythmias , and in toxicology studies a lethal dose could be not achieved and no adverse effects were noted in clinical trials . finally , regardless of the critically assessed standard sui therapy , duloxetine is still of particular importance , while imipramine was also suggested . bpc 157 given peripherally may selectively affect regional serotonin synthesis in the rat brain and improve behavioral response in porsolt s test ( vs. imipramine ) , with particular counteraction of pargyline- and l - tryptophan - induced serotonin syndrome , implicated in therapy with specific serotonin ( norepinephrine ) reuptake inhibitors and triptans . pentadecapeptide bpc 157 applied parenterally or per - orally appears to ameliorate the sui in rat models , improving the otherwise detrimental course of healing after vd and tu , which may be analogous to human injury .

backgroundsince an originally anti - ulcer stable gastric pentadecapeptide bpc 157 ( pl 14736 ) was shown to promote healing of injured striated muscle and smooth muscle in the gastrointestinal tract , we explored its therapeutic potentials for leak point pressure ( lpp ) recovery in rat stress urinary incontinence ( sui ) after transabdominal urethrolysis ( tu ) and prolonged vaginal dilatation ( vd).material / methodsduring a 7-day period , tu - rats and vd - rats ( or healthy rats ) received bpc 157 , either ( i ) intraperitoneally , 10 g / kg or 10 ng / kg , once daily ( first administration 30 min after surgery , last 24 h before lpp - testing and sacrifice ) , or ( ii ) per - orally , 10 g / kg in drinking water ( 0.16 g / ml , 12 ml / rat / day ) . vesicourethral segments were harvested for immunohistochemical evaluation.resultsall bpc 157 regimens counteracted decrease of lpp values in tu - rats and vd - rats . additionally , bpc 157-tu rats ( g - intraperitoneally or per - orally ) and bpc 157-vd rats ( g intraperitoneally ) reached lpp values originally noted in healthy rats . conversely , in healthy rats , bpc 157 did not alter lpp . immunohistochemical studies revealed higher desmin ( delineates striated organization of skeletal muscle ) , smooth muscle actin , and cd34 ( angiogenic marker ) positivity within the urethral wall in bpc 157-treated rats vs. controls , as well as overall preserved muscle / connective tissue ratio assessed with mallory s trichrome staining.conclusionspentadecapeptide bpc 157 , applied parenterally or per - orally , appears to ameliorate the sui in rat models , improving the otherwise detrimental course of healing after vd and tu , which may be analogous to human injury . these beneficial effects may possibly be selectively used in future strategies for treatment of sui .

Background Material and Methods Pentadecapeptide BPC 157 Animals Surgery BPC 157 treatment regimens LPP testing Histological studies Statistical analysis Results LPP testing Histological studies Discussion Conclusions

to date , no standard agents have been shown to improve healing of severely injured or transected muscle to support the effect on stress urinary incontinence ( sui ) , nor has this possibility been considered from theoretical or practical points of view [ 16 ] . to alternatively resolve the problem , we focused on sui and the stable gastric pentadecapeptide bpc 157 . interestingly , it is an antiulcer peptide that can not be degraded by 24-hour exposure to human gastric juice , with established safe therapy profile ( lethal dose not achieved even at 2 g / kg b.w . ) therefore , its parenteral and per - oral application in rats after transabdominal urethrolysis ( tu ) and prolonged vaginal dilatation ( vd ) may be interesting . pharmacological treatment for sui is mostly focused on the use of nonselective alpha - agonists , which are often ineffective , and serotonin and norepinephrine reuptake inhibitors ( e.g. in addition , particular combinations ( alpha2-adrenoceptor blockade and duloxetine ) and various other possibilities were suggested ( eg , angiotensin ii ) . , selective , partial agonist at the human alpha1(a)-adrenoceptor ) , explaining the elevation in leak point pressure ( lpp ) . , with duloxetine ) , it is therefore more surprising that none of the standard agents was shown to improve the healing of severely injured or transected muscle to support the effect on sui . these were the reasons why we suggested the stable gastric pentadecapeptide bpc 157 to recover lpp in rat sui after tu and vd . namely , a particular rescuing effect on failed lpp , avoiding an effect in normal healthy rats , may be even more likely since this originally anti - ulcer stable gastric pentadecapeptide , bpc 157 , was shown to particularly heal sphincters in gastrointestinal tract , as well as both injured striated muscle and smooth muscle . providing that urethral cross section contains both smooth and striated muscle , bpc 157 s effect on both smooth muscle and striated muscle was assessed in proximal , middle and distal urethral segments , along with the effect on vessel density . intriguingly , as mentioned , previous studies have demonstrated this peptide s ability to rescue failed sphincter function in esophagitis rats , rapidly normalizing decreased pressure in lower esophageal and pyloric sphincters . , sui ) may present completely different mechanisms , and therefore it may be interesting to study sui rats , particularly after vd or tu . pressure - induced ischemia , pelvic floor injury , and dysfunction of the urethral continence mechanism seen in vd , as well as loss of anatomic urethral support , loss of innervations and muscle atrophy presented with tu , are commonly regarded as well reproducible sui features . thus , tu - rats and vd - rats ( or healthy rats ) received bpc 157 in the regimens successfully used before , either intraperitoneally , or per - orally , in drinking water , during a 7-day period , . female wistar albino rats ( n=7 in each group ) , retired breeders , weighing 310350 g , were used in this study . intraperitoneally ; we utilized 2 well - established rat models [ 2426 ] : tu and vd . bpc 157 was given throughout the 7-day regimen in healthy rats or after tu or vd ; intraperitoneally ( dissolved in saline [ 2 g or 2 ng / ml ] ) in a single daily dose of the 10 g / kg or 10 ng / kg b.w . , first administration 30 min after surgery , last 24 hr before the lpp testing and sacrifice ; or 10 g / kg / day , dissolved in tap water ( 0.16 g / ml , 12 ml / rat / day ) . controls received only drinking water or an equal volume of saline ( 5 ml / kg b.w . all operated ( tu and vd ) and sham - operated animals and 7 healthy rats underwent lpp assessment using a methodology based on previously described techniques [ 2428 ] . under urethane anesthesia ( 1.2 mg / kg intraperitoneally ; sigma - aldrich chemie gmbh , steinheim , germany ) , used in order to maintain physiologic urethral responses , the bladder was exposed via midline abdominal incision and manually emptied . at half bladder capacity ( 0.4 ml ) , infusion was stopped and gentle pressure was applied increasingly over the bladder until the first drop of fluid was seen on the urethral meatus ; the recorded intravesical pressure at that point was regarded as the lpp . the specimens were fixed in 10% neutral buffered formalin overnight and embedded in paraffin , semi - sequentially cut ( 5 m thickness ; proximal , middle and distal urethra ) , and stained with haematoxylin - eosin and with mallory s trichrome for morphometry assessment of muscle / connective tissue ratio in mid - urethral segment . additionally , for all 3 urethral segments , immunohistochemistry studies were carried out for desmin ( delineates a striated organization of this filamentous protein identical in the external sphincter and the skeletal muscle ) , smooth muscle actin ( sma ; smooth muscle cell marker ) and cd34 ( angiogenic marker ) ( 1:50 ; dako denmark a / s , glostrup , denmark ) , in order to assess striated and smooth muscular layer thickness and blood vessel count . female wistar albino rats ( n=7 in each group ) , retired breeders , weighing 310350 g , were used in this study . intraperitoneally ; ketanest , parke - davis gmbh , berlin , germany ) and diazepam ( 6 mg / kg b.w . we utilized 2 well - established rat models [ 2426 ] : tu and vd . bpc 157 was given throughout the 7-day regimen in healthy rats or after tu or vd ; intraperitoneally ( dissolved in saline [ 2 g or 2 ng / ml ] ) in a single daily dose of the 10 g / kg or 10 ng / kg b.w . , first administration 30 min after surgery , last 24 hr before the lpp testing and sacrifice ; or 10 g / kg / day , dissolved in tap water ( 0.16 g / ml , 12 ml / rat / day ) . controls received only drinking water or an equal volume of saline ( 5 ml / kg b.w . , on the 7 postoperative day all operated ( tu and vd ) and sham - operated animals and 7 healthy rats underwent lpp assessment using a methodology based on previously described techniques [ 2428 ] . under urethane anesthesia ( 1.2 mg / kg intraperitoneally ; sigma - aldrich chemie gmbh , steinheim , germany ) , used in order to maintain physiologic urethral responses , the bladder was exposed via midline abdominal incision and manually emptied . at half bladder capacity ( 0.4 ml ) , infusion was stopped and gentle pressure was applied increasingly over the bladder until the first drop of fluid was seen on the urethral meatus ; the recorded intravesical pressure at that point was regarded as the lpp . the specimens were fixed in 10% neutral buffered formalin overnight and embedded in paraffin , semi - sequentially cut ( 5 m thickness ; proximal , middle and distal urethra ) , and stained with haematoxylin - eosin and with mallory s trichrome for morphometry assessment of muscle / connective tissue ratio in mid - urethral segment . additionally , for all 3 urethral segments , immunohistochemistry studies were carried out for desmin ( delineates a striated organization of this filamentous protein identical in the external sphincter and the skeletal muscle ) , smooth muscle actin ( sma ; smooth muscle cell marker ) and cd34 ( angiogenic marker ) ( 1:50 ; dako denmark a / s , glostrup , denmark ) , in order to assess striated and smooth muscular layer thickness and blood vessel count . on the other hand , tu produced lpp values markedly decreased a week after surgery . bpc 157 therapy , regardless of the given dose - regimen or mode of administration , completely counteracted the decrease of lpp values . on some occasions , bpc 157 values reached those originally noted in healthy rats . likewise , in the vd group , at the end of the week all control rats exhibited markedly decreased lpp values . again , all of the used bpc 157 dose regimens or routes of administration completely opposed the decrease of lpp values , with some subgroups reaching values originally noted in healthy animals ( figure 2 ) . morphometrical histological studies with mallory s trichrome staining revealed marked post - operative decrease in muscle tissue content in the mid - urethral segment in all operated animals , but with much more pronounced muscle loss found in control groups . this loss yielded statistically significant differences in muscle / connective tissue ratio between treated and control animals in tu and vd groups ( figures 3 and 4 ) . additionally , higher proportion of sma ( figure 5 ) and desmin ( figures 6 and 7 ) positivity was regularly observed in the urethral wall of all bpc 157-treated vd and tu animals , and a noticeable increase in vessel density within all segments of the urethra ( immunohistochemical staining for cd34 ) ( figure 8) . accordingly , while the values in bpc 157 approached the values noted in healthy or sham operated rats , vd and tu controls consistently exhibited markedly lower values . on the other hand , tu produced lpp values markedly decreased a week after surgery . bpc 157 therapy , regardless of the given dose - regimen or mode of administration , completely counteracted the decrease of lpp values . on some occasions , bpc 157 values reached those originally noted in healthy rats . likewise , in the vd group , at the end of the week all control rats exhibited markedly decreased lpp values . again , all of the used bpc 157 dose regimens or routes of administration completely opposed the decrease of lpp values , with some subgroups reaching values originally noted in healthy animals ( figure 2 ) . morphometrical histological studies with mallory s trichrome staining revealed marked post - operative decrease in muscle tissue content in the mid - urethral segment in all operated animals , but with much more pronounced muscle loss found in control groups . this loss yielded statistically significant differences in muscle / connective tissue ratio between treated and control animals in tu and vd groups ( figures 3 and 4 ) . additionally , higher proportion of sma ( figure 5 ) and desmin ( figures 6 and 7 ) positivity was regularly observed in the urethral wall of all bpc 157-treated vd and tu animals , and a noticeable increase in vessel density within all segments of the urethra ( immunohistochemical staining for cd34 ) ( figure 8) . accordingly , while the values in bpc 157 approached the values noted in healthy or sham operated rats , vd and tu controls consistently exhibited markedly lower values . as we hypothesized , based on bpc 157 s particular beneficial effect on damaged muscle healing [ 1317 ] and rescue of failed lower esophageal sphincter and pyloric sphincter , failed lpp after tu or vd was successfully recovered , and this was regularly achieved in all bpc 157-treated rats , either with intraperitoneal ( both g and ng dose ) or per - oral application , along with microscopic / immunohistochemical improvement involving both striated and smooth muscle , specifically shown in all urethral segments . unlike the standard therapy that may affect the bladder or urethral pressure of normal rats , and consequently explain the elevation in lpp ( e.g. , alpha adrenergic agonism ) , lpp values in healthy rats were not changed by bpc 157 medication , and thereby , failed lpp was specifically recovered . unlike poor control tu or vd presentation ( which corresponds to those regularly noted in other sui studies ) , the recovered function in bpc 157-tu or bpc 157-vd rats , as commonly observed in muscle injury studies [ 1317 ] , may be consequent to an enhanced healing process , and vice versa ; the recovered function by itself promotes healing , and such functional recovery could be rather complete , and also dose - dependent , with lpp reaching values originally noted in healthy animals . a marked attenuation of both striated and smooth muscle layers in all urethral segments of control animals was alleviated toward nearly normal values with bpc 157 regimens , thus the function of both striated and smooth muscle was improved to rescue failure of lpp . from the viewpoint of the standard therapy , consistency in severely injured and/or transected muscle healing to support the corresponding effect on sui [ 1317 ] , established by bpc 157 , has thus far not been commonly encountered with other agents [ 16 ] , therefore the noted importance of such recovery of failed lpp is better founded in bpc 157-tu-/vd rats . accordingly , providing the greatest histological evidence of urethral damage is especially obvious in the skeletal muscle layer . improved microscopy / immunochemistry and restored function ( that had to be regularly , definitively debilitated ) seen with transected or crushed muscle is congruous with increased desmin positivity noted in all urethral segments of bpc 157 rats after tu and vd , and lpp values fully recovered . bpc 157-induced muscle healing implies modulation of the same events ; fostering of myocyte regeneration , thus shortening the healing period and avoiding excessive scar formation , again analogous to the preserved muscle / connective tissue ratio in treated rat urethras observed in this study . additionally , with the same regimens of bpc 157 therapy , along with the previous findings on injured striated muscle , healing effect on peripheral nerve , and on smooth muscle of gastrointestinal tract and sphincters [ 15,1719 ] , and angiogenesis , may be particularly relevant to the noted recovery of the failed lpp in rats after tu and vd . providing that the procedures used , vd and tu , also directly damaged smooth muscle function , eliminating some of their functions for a while , after gi tract massive resection the remaining part more vigorously adapts in bpc 157 rats , and overwhelms the lack of the removed part . since both smooth and striated muscle contribute to urethral pressure during filling phase , with accompanying fast twitch fibers contraction reflex that further elevate urethral tone when intraabdominal pressure rises , the restoration of their integrity and functions likely contributed to bpc 157 anti - sui mechanisms . , sui ) may present completely different mechanisms ) , sphincter failure match esophagitis rats and vd rats , with the methods of prolonged dilation , the fairly analogous muscle stretch , and the definitive sphincter failure , did not spontaneously recover until the end of the experiments . in either case , the effect of the bpc 157 is particularly evident in animals with failed sphincter function and obtained using both parenteral and per - oral applications . considering that pudendal nerve damage is also implicated in sui pathophysiology , as demonstrated in some other sui rat model studies ( crush , transection ) , it is important to point out that bpc 157 , besides having muscle healing potential [ 1319 ] , exhibited significant neuroprotective capabilities ( e.g. , directly improving the transected sciatic nerve healing ) , thus contributing to regained function after major injury . of note , loss of innervations and muscle atrophy exists in both vd and tu models as well . since damaged / transected muscle healing commonly requires regeneration of damaged intramuscular nerve branches , it may be that in successful recovery bpc 157 course these parallel healing processes promote each other . thus , the observed increase in urethral vessel density , which parallels lpp recovery after tu or vd in bpc 157 treated animals , specifically implies this peptide s previously documented angiogenic effect to be potentially accountable for rapid restoration of urethral function . noticeably , it is along with a new vascular shift toward the left as shown in different models , particularly in muscle healing , even in corticosteroid - aggravated conditions , and also in hypovascular tissues ( e.g. this demonstrates the prominent up - regulation of vascular endothelial growth factor ( vegf ) , likely with particular effect on connective tissue healing , such as the expression of early growth response 1 ( egr-1 ) gene and its repressor , nerve growth factor 1-a binding protein-2 ( nab2 ) , resulting in early extracellular matrix ( collagen ) formation . also , bpc 157 may have a direct effect on myocytes , probably analogous to that on tendon fibroblasts throughout the fak - paxillin pathway . the evidence was compelling in all of the models used , providing the consistently recovered lpp after vd and tu with all bpc 157 regimens , suggesting that these beneficial effects may be selectively applied to the urethra and treatment of sui . however , due to the potential limitations of the study , the evidence that bpc 157 ameliorates the sui is inconclusive . in other relevant muscle and nerve bpc 157 studies , however , very long injury periods models were also used [ 1319,30,32 ] . in other words , even if this study is about prevention rather than reversal of sui , this does not diminish the relevant value of the obtained beneficial effects of bpc 157 application in rats that underwent tu and vd , since from the results it is obvious that bpc 157 therapy benefit has a long - term and sustained effect , providing the recovered lpp in tu- and vd - treated rats when the last administration had been at 24 hr before assessment ( intraperitoneal regimen ) . , also per - orally ; in drinking water ) . furthermore , considering the cardiovascular effects that may be a common problem with standard sui therapy , in vivo models of cardiovascular function showed that bpc 157 does not affect normal blood pressure or heart rhythm [ 4244 ] , but it did reduce l - name hypertension , counteract no system failure by nos - blockade in different models [ 4345 ] ( no - synthesis is directly related to muscle injury healing ) , doxorubicine chronic heart failure and digitalis overdose arrhythmias , and in toxicology studies a lethal dose could be not achieved and no adverse effects were noted in clinical trials . bpc 157 given peripherally may selectively affect regional serotonin synthesis in the rat brain and improve behavioral response in porsolt s test ( vs. imipramine ) , with particular counteraction of pargyline- and l - tryptophan - induced serotonin syndrome , implicated in therapy with specific serotonin ( norepinephrine ) reuptake inhibitors and triptans . pentadecapeptide bpc 157 applied parenterally or per - orally appears to ameliorate the sui in rat models , improving the otherwise detrimental course of healing after vd and tu , which may be analogous to human injury .

malaria has attracted global attention as one of the world 's leading major diseases due to its high morbidity and mortality . the world health organization ( who ) estimated that about 3.3 billion people are at risk of malaria because they live in malaria endemic areas and about 300500 million malaria cases malaria is also responsible for almost a million deaths yearly [ 2 , 3 ] . the disease is strongly associated with poverty because it disengages patients from carrying out meaningful economic activities during attacks and it also consumes huge expenditure budgets . it is estimated that as high as 40% of public health expenditure is spent on malaria alone in endemic countries . malaria is a febrile disease caused by parasites of the genus plasmodium which are transmitted to susceptible persons through the bite of infected female anopheles mosquitoes . the burden of malaria is greatest in sub - sahara africa where approximately 80% of the global malaria cases as well as 90% of fatalities occur . children less than 5 years are the most severely affected accounting for 86% of the deaths . to this extent , malaria has been recognized as an impediment to achievement of the united nation 's millennium development goal ( mdg ) 4 which targets at the reduction of child mortality . therefore , without effective prevention and control of malaria ( which is the mdg 6 ) , it will be difficult to achieve the mdg 4 . in order to minimise the effects of malaria , prompt clinical diagnosis and effective treatment are needed [ 79 ] beside the preventive measures such as use of insecticide treated nets ( itn ) and indoor residual spraying ( irs ) with insecticides . however , prompt malaria treatment efforts are being threatened by widespread problem of antimalaria drug resistance [ 1 , 6 , 10 ] . for instance , plasmodium falciparum had developed resistance to chloroquine which was the most affordable and readily available antimalaria drug in africa . as a result , the who recommended artemisinin - based combination therapy ( act ) for treatment of uncomplicated falciparum malaria [ 11 , 12 ] . this type of treatment makes use of combination of an artemisinin derivative with a partner drug . the artemisinin derivatives are fast acting drugs capable of rapid clearing of the falciparum parasites during treatment [ 12 , 13 ] but have short half - lives which render them almost unsuitable for use as single therapeutic drugs because of risk for drug resistance which increases as plasma levels fall . following the who recommendation , most sub - sahara african countries have changed their antimalaria drug policy to the use of the acts . artemether - lumefantrine ( al ) is one of the acts commonly used as first - line drug for treatment of uncomplicated falciparum malaria in the sub - sahara african countries . another act however , dp is not widely used in sub - sahara africa compared to the use of al . the half - lives of the partner drugs in the acts vary across the different act drugs available see table 1 . the partner drug in al , lumefantrine , has a half - life of ~5 days while the partner drug in dp , piperaquine , has a half - life of ~5 weeks , the longest of all the acts . as a result , it is being speculated that dp could be the act with greatest posttreatment prophylactic efficacy due to the longer half - life and sustained plasma levels of piperaquine compared to other acts such as al . however , little is known about the relative extent to which these two acts exert their prophylactic effect after treatment . it is not yet clear whether the sheer longer half - life of piperaquine in dp could translate into a better prophylaxis and to what extent compared to lumefantrine in al . no review had comprehensively compared these two acts head - to - head to determine their relative prophylactic effectiveness . olliaro and colleagues found that recurrent new infections ( rnis ) were associated with higher risk for development of symptomatic disease ( malaria ) than recrudescence parasitaemia . in contrast , other researchers had found that rather recrudescence parasitaemia carries greater risk for worse haematological outcome for patients . these two studies were not conducted on acts but the most important issue at stake here is that both recrudescence and new infections are associated with various risks to patients ; hence , prevention of their occurrence should be of much concern . this current review aimed at synthesizing available primary studies conducted in sub - sahara africa to determine whether use of dp reduces incidence of new falciparum infections ( rnis ) and treatment failure more than use of al within 28 and 42 days after treatment . a clear knowledge in this regard would offer a better guidance to country antimalaria drug policies in sub - sahara africa . the main review question is , does the use of dp in treating uncomplicated falciparum malaria reduce the risk for recurrent new falciparum infections and treatment failure more than al ? the review tested the null hypothesis that there is no difference between the two drugs . for purposes of this review , recurrent new falciparum infection ( rni ) is defined as new infections which patients acquired by day 28 or 42 following treatment for uncomplicated falciparum malaria using dp or al . total treatment failure ( ttf ) refers to recrudescence recorded by day 28 or day 42 ( tables 5 and 6 ) after treatment , where recrudescence refers to the old or the original parasites for which the antimalaria treatment was initiated but could not be cleared by the antimalaria drug and the disease ( symptomatic malaria ) has returned . the new infections must have been differentiated from the old infections in the primary studies by use of polymerase chain reaction ( pcr ) . prophylactic effectiveness used in this review refers to the ability of the act drug ( dp or al ) to prevent the occurrence of new falciparum infections within 28 or 42 days after treatment with either drug ( tables 7 and 8) . studies were included in the review if they were randomized controlled trial ( rct ) conducted in sub - sahara africa and had compared the two drugs ( dp and al ) head - to - head for treatment of uncomplicated falciparum monoinfections only . all patients recruited for the primary studies must have been confirmed through laboratory investigation to be infected with p. falciparum only and must not have shown signs of severe malaria or be suffering from co - febrile infection at the time of enrolment . the trial must be conducted according to the who protocols for antimalaria drug efficacy study . studies which included pregnant women were excluded . an electronic search to locate relevant published studies was conducted in four main databases , namely , embase , medline , cochrane library , and global health , using the search strategy presented in table 2 . the final database search for the review was conducted in the selected databases on june 2 , 2013 , as follows : embase : from 1980 to 2013 week 22 ; medline : from 1946 to may 2013 week 4 ; global health : from 1973 to 2013 week 21 . the main terms in the review question were structured into the pico ( population , intervention , comparison , and outcome ) format and all corresponding synonyms identified . all main terms in the review question were searched under medical subject heading ( mesh terms ) and also as free texts . all other terms ( synonyms of the main terms ) were searched as free texts . search results of terms under the same category were brought together using the boolean operator or , while the boolean operator and was used finally ( once ) to bring all or results together . the final search result after use of the boolean operator and was limited to studies conducted on humans and have been published in english language see table 3 . only terms in the population , intervention , and comparison categories were used during the search because the reviewers were of the view that further inclusion of terms in the outcome category in the search might lead to exclusion of some important studies . all search results were reviewed by wa and cross - checked by ep and studies that met the inclusion criteria selected . due to the limited time available for the review , no trial author was contacted for further clarification on the trials and , as a result , articles with instances of unclear or missing data were excluded . the preferred reporting items for systematic reviews and meta - analysis ( prisma ) flow diagram has been used to summarize study selection process see figure 1 . the methodological quality of the selected studies was assessed using the cochrane collaboration 's tool for assessing risk of bias . these domains include sequence generation , allocation concealment , blinding of participants or outcome assessors , incomplete outcome data , selective outcome reporting , and other sources of bias . all data were extracted as binary data in the form of number of participants who experienced the event of interest and total number of participants in each study group . the outcomes of interest included recurrent new falciparum infections ( rnis ) and total treatment failure ( ttf ) reported by day 28 and 42 . data on new infection and treatment failure were extracted using the differentiated pcr results recorded in the studies . data extraction was done by author wa and figures were cross - checked by author ep for accuracy . instances of disagreement on the accuracy of the extracted figures were resolved by extensive discussions and explanations and then agreement is finally reached on which figure is accurate . data analysis was done using the comprehensive meta - analysis programme ( cmap ) version 2.0 . effect sizes were calculated in risk ratio ( rr ) at 95% confidence interval ( ci ) . results of dp group relative to al group were regarded as statistically significant if the 95% ci did not include number of no effect , which is 1 . absolute relative difference ( ard ) and number needed to treat ( nnt ) were calculated . the grading of recommendations assessment , development , and evaluation ( grade ) system ( table 9 ) was used to assess level of evidence quality [ 25 , 26 ] . this model operates on the premise that each trial included in the analysis has estimated an effect size peculiar to its study population and that any difference in effect estimation across the studies is due to both random error and heterogeneity . statistical heterogeneity was assessed to determine i - squared ( i ) , the q - value , and the accompanying p values [ 27 , 28 ] . the i values were interpreted as follows : i values less than 50% were regarded as low , values 50% were regarded as moderate , and an i above 75% was interpreted as high or substantial heterogeneity [ 24 , 28 ] . a total of 1,673 records were retrieved from all the database searches combined and 1,631 records ( 97.5% ) were excluded initially because they have irrelevant study titles . abstract of the remaining 42 publications ( 2.5% ) were read , after which 11 ( 26.2% ) were further selected and subjected to thorough full text reading and scrutiny . the remaining four [ 15 , 3638 ] were excluded for various reasons ( see table 3 for reasons for exclusion ) . table 4 contains general description of selected studies . sequence generation was done adequately in all the studies and was judged to pose a low risk of bias . four of the trials used computer generated random list done by an off - site investigator . one trial reported use of block randomisation while the remaining two [ 32 , 34 ] used stratified random lists generated by off - site investigators ( figure 2 ) . five trials [ 29 , 30 , 32 , 33 , 35 ] used adequate allocation concealment methods and were judged to be at low risk for selection bias because the researchers used sequentially numbered , sealed opaque envelops to obscure treatment group before allocation . the concealment process was judged to pose an unclear risk of bias in only two of the studies [ 31 , 34 ] for lack of clear information . blinding was judged to be adequate ( low risk of bias ) in six studies [ 3035 ] because all laboratory personnel and trial investigators involved in assessing outcome measures were blinded to the treatment allocation of the study participants . this was deemed to pose low risk of bias to parasitological outcome measures for treatment failure and rate of new infections . one study did not report on blinding and was judged to pose high risk of measurement and performance bias . there was no blinding for participants and nurses in four studies and this was considered to pose rather high risk of bias to the outcome measures of adverse clinical events ( side effects of the drugs ) which were not the focus of this review . all the trials selected addressed incomplete outcome data and were rated to be of low risk for attrition bias . more than 90% of randomised participants were included in final analysis in all the trials and this was deemed adequate . result on total treatment failure ( ttf ) due to recrudescence was obtained from six studies and involved a total of 3,172 patients . of this , 1,861 participants received dp , out of which 107 ( 5.7% ) experienced treatment failure at day 28 . on the other hand , the pooled rr yielded 0.453 , 95% ci : [ 0.203 to 1.012 , p = 0.05 ] . this implies that there was an average of 55% reduction in risk for ttf at day 28 in favour of dp treatment ; largest plausible reduction possible was 80% ; however , the upper boundary of the 95% ci includes the number of no effect ( 1 ) and a risk for harm of 1.2% . extent of heterogeneity was 47% ( i = 47 , p = 0.09 , q - value = 9.5 , df = 5 ) and this level of heterogeneity was considered to be low see figure 3 . the pooled ard was 0.016 , 95% ci [ 0.0300.002 , p = 0.02 ] , which means that for every 63 patients treated with dp , one case of treatment failure was prevented , which would have occurred if such patients had received al treatment . data on ttf at day 42 was extracted from four studies involving 2 , 662 participants , out of which treatment failure was reported in 274 participants representing 10.3% . a total of 1,598 participants were those treated with dp and 161 of them ( 10.1% ) experienced treatment failure . those who received al treatment were 1,064 participants out of which 113(10.6% ) experienced failure , an indication that dp was associated with a marginal reduction in treatment failure compared to al . the pooled estimate for the rr was 0.560 with 95% ci of [ 0.275 to 1.140 , p = 0.1 ] . this implies that there was an average reduction in risk of treatment failure of 44% in favour of dp treatment compared to al , with the highest plausible reduction in risk of up to 73% . however , the upper boundary of the 95% ci includes number of no effect ( 1 ) and includes 14% harm of failure . test for heterogeneity in random effect model shows high statistical heterogeneity of 71% across the studies ( q = 10 , df ( q ) = 3 , i = 71% , p = 0.02)see figure 4 . results from four studies involving a total of 2,340 patients showed that 104 of the patients ( representing 4.4% ) acquired new falciparum infections within the 28 day period after treatment . out of the total number , 1,433 participants received dp , of which 28 patients representing 2% had new infections . in the al group , 907 patients received the treatment and 76 ( 8% ) acquired new infections an indication that rate of new falciparum infection was higher among those treated with al than those treated with dp . pooled rr was 0.207 , 95% ci [ 0.136 to 0.315 , p < 0.001 ] . this indicates that risk associated with a patient getting new infections at day 28 was significantly reduced averagely by 79% in favour of dp treatment . the lowest plausible reduction was 69% and highest was 86% ; the difference was statistically significant ( p < 0.001)figure 5 . results extracted from four studies involving a total of 2,662 patients indicated that 455 patients representing 17% experienced new infections with falciparum parasites . those treated with dp were 1,598 , out of which 218 ( 14% ) acquired new falciparum infections . on the other hand , a total of 1,064 received al , of which 237 ( 22% ) experienced new infections . this means that the risk of a patient acquiring new falciparum infection at 42 day following treatment was averagely reduced by 44% in favour of dp compared to al the 95% ci was 0.342 to 0.908 , suggesting that the actual effect could be anywhere between 9 and 66% ; the difference was statistically significant , p = 0.02 . there was a substantial heterogeneity ( extent was 83% ) associated with this result ( i = 83 , q - value = 18 , df = 3 , p < 0.001 ) . this review compared two acts dihydroartemisinin - piperaquine ( dp ) and artemether - lumefantrine ( al)to determine which has the greatest effect in reducing recurrent new falciparum infections ( rnis ) and treatment failure . the null hypothesis which was tested was that there is no difference between the two drugs in reducing risk of treatment failure and recurrent new falciparum infections at days 28 and 42 . however , the overall evidence gathered suggests that patients who were treated with dp experienced less total treatment failure ( ttf ) and less recurrent new falciparum infections ( rnis ) than those who received al . majority of studies included in this review were conducted in east africa and only few were carried out in west africa and therefore findings and conclusion are more applicable to countries in the east africa region . the evidence synthesized indicates that both dp and al are effective in preventing treatment failure at days 28 and 42 after treatment . however , dp reduced treatment failure higher than al at 28 and 42 days but the difference in magnitude of failure reduction between the two drugs is clinically marginal and not statistically significant . this finding concurs with that of earlier review conducted on studies done in asia , america , and africa , in which dp was found to be associated with an appreciable efficacy of cure rate compared to non - act and other act drugs . the average percentage of risk reduction in favour of dp compared to al in this review decreased from 55% at day 28 to 44% at day 42 . this confirms the assertion by yeka and colleagues that as length of followup increases , difference between dp and al in their ability to prevent treatment failure in high endemic areas becomes insignificant . this observation is attributed to the overwhelming rate of new infections in such areas which may have outweighed the efficacy of piperaquine coupled with the decreasing concentration of piperaquine in the blood stream over time . it has been speculated that dp could offer a greater posttreatment prophylaxis ( ptp ) than other acts due to its longer half - life by which it could exert longer efficacy against newly infecting parasites and reduce risk for development of both clinical malaria and resistance parasite strains . however , the extent to which dp could offer prophylaxis ( ptp ) by preventing clinical malaria due to new infections has not been clearly specified compared to other acts such as al . result on prevention of recurrent new falciparum infections ( rnis ) in this review demonstrates that dp offered greater and significant ptp for patients against rni than al . average percentage reduction in risk for rni was up to 79% at day 28 in favour of dp and up to 44% at day 42 in favour of dp . there is , however , substantially significant statistical heterogeneity ( extent was 83% ) associated with pooled result on rni at day 42 in this review . the possible source of this heterogeneity is attributed to the variability in malaria infection transmission rate of the various study sites ; some of the studies were conducted in settings where rate of malaria transmission intensity was very high while other studies were conducted in areas of relatively low malaria transmission intensity . thus , study settings with very high transmission intensity are likely to be associated with higher rates of anopheles mosquito bites which will result in recurrent new infections more than settings with low transmission intensities . even though participants were provided with insecticide treated nets , it can not be ascertained whether patients actually slept in the nets to prevent mosquito bites after treatment and this could also affect the proportion of patient who experienced recurrent new infections . the superiority of dp in offering this higher level of posttreatment prophylaxis ( ptp ) is attributable to the longer half - life of piperaquine ( which is ~5 weeks ) compared to the shorter half - life ( of about 4 to 5 days ) of lumefantrine in al . the higher ptp of dp will be of significant importance for areas of higher malaria transmission intensities more than for areas of low transmission intensities which are associated with lower frequency of acquiring malaria due to new infections . however , in spite of the benefit of higher ptp of dp it must also be pointed out that the longer half - life of piperaquine is likely to pose high risk for faster development of drug resistant strains of the falciparum parasites to dp . this is thought to be possible because the new infecting parasites will be exposed to subtherapeutic ( low ) concentrations of piperaquine over time . this low concentration will not be able to eliminate the parasites completely and will provide an opportunity for development of resistant strains . therefore , any attempt to promote more utilisation of dp for the benefit associated with its higher ptp must be weighed against the possible risk for development of resistant strains because of potential public health dangers associated with drug resistant parasites . notwithstanding , the risk for development of drug resistant parasites could be minimized if more than one act drug is used as a first - line drug for treatment of uncomplicated malaria in a particular area . the use of more than one drug as a first - line act may prevent the possibility of the parasites becoming adjusted to one overused - first - line act . there is , therefore , the need for encouraging the use of multiple first - line drugs and constant monitoring of efficacy of the act drugs to determine development of resistant strains at early stages . using the grade criteria for rating evidence quality , the quality of evidence obtained regarding superiority of dp over al in preventing ttf at day 28 was rated moderate quality while that of day 42 was rated low quality . evidence obtained on prophylactic superiority of dp over al in preventing rni at day 28 was rated high quality while that of day 42 was rated moderate quality . high quality grade evidence rating means that the authors are very confident that the true effect lies close to the average estimated while moderate quality rating means that authors ' confidence in the estimate is moderate which means that the true effect size is likely to be close to the average estimate , but there is also likelihood that there could be substantial difference . it is worth indicating that treatment failure ( ttf ) and recurrent new infections ( rnis ) are of great importance and concern to patients due to the negative socioeconomic impact of clinical malaria on patients . malaria imposes high economic burden on patients by preventing them from working when the disease attacks them and this slows work force productivity . malaria is highly prevalent in sub - sahara africa and it is undeniable that patients acquiring frequent recurrent new infections contribute to the high number of cases . therefore , preventing rnis means that an appreciable proportion of the population would remain healthier for productive economic activities and this could help reduce the malaria expenditure burden on the healthcare system and on the continent as well as on donor agencies . considering these reasons above and level of relative reduction in risk for ttf and rni in favour of dp together with the quality of the evidence obtained , all studies included in the review were rcts which is appropriate for answering clinical intervention questions . the methodological quality of the selected studies was generally high and had been rated to pose low risk of bias . meta - analysis was done in random effects model to integrate extracted data for better interpretation and all these are considered as strengths of the review . data extracted from the various studies were done by aw and cross - checked by ep to ensure accuracy and prevent individual bias . there was no attempt to locate studies in the grey literature and other sources apart from those indexed in the four databases searched . search results were limited to studies published in english language alone and therefore excluded other equally valuable articles which might have been published in other languages such as french . all of these carry a potential risk of selection bias which could undermine the completeness of the review data and weaken findings and conclusion . also , majority of the included studies were conducted in countries in east africa which weakens generalisation of findings to other parts of sub - sahara africa ; hence this review result and conclusion are most valid for countries in east africa . there is high statistical heterogeneity associated with some estimates ; hence each pooled effect estimate must be seen as an average representing different estimates which are peculiar to each study population , bearing in mind , however , that effect directions were similar and , in most cases , favoured dp ( see forest plots in figures 36 ) . this systematic review compared dihydroartemisinin - piperaquine ( dp ) and artemether - lumefantrine ( al ) and aimed at identifying which one has greater ability to reduce total treatment failure ( ttf ) and incidence of recurrent new falciparum infections ( rnis ) in high transmission areas in sub - sahara africa . the results showed that participants treated with dp compared to al experienced lesser risk for ttf at days 28 and 42 . on the other hand , dp offers a significant posttreatment prophylaxis against recurrent new falciparum infections superior to that of al . the average percentage reduction in risk for the incidence of rni was up to 79% at day 28 in favour of dp [ rr , 0.21 ; 95% ci : 0.14 to 0.32 , p < 0.001 ] and 44% in favour of dp at day 42 [ rr , 0.56 ; 95% ci : 0.34 to 0.90 ; p = 0.02 ] . it is , therefore , concluded that treatment of uncomplicated falciparum malaria using dihydroartemisinin - piperaquine ( dp ) in high transmission areas in sub - sahara africa ( especially east africa ) could result in an average reduction in risk for recurrent new falciparum infections of up to 79% and 44% within 28 and 42 days , respectively , compared to artemether - lumefantrine ( al ) . and this implies that use of dp can help reduce burden of malaria in such areas more than al . however , both dp and al have similar effectiveness in preventing treatment failure though dp has marginal benefit over al . it is recommended that the antimalaria drug policy in countries especially in east africa should be streamlined to include use of dihydroartemisinin - piperaquine alongside artemether - lumefantrine and other acts in countries where it is found to be effective but this must be done bearing in mind the potential risk for development of resistant falciparum strains . more studies should be conducted in other parts of the sub - sahara africa such as west africa to determine stronger evidence which will be more applicable to countries in that area . it has been identified that the current study protocol by the who regarding antimalaria drug efficacy research does not incorporate specific outcome measure on recurrent new infections . it is , therefore , recommended that measurement of rate of recurrent new infections should be incorporated into future guidelines , and future trial investigators should make direct assessment on it .

malaria contributes significantly to the global disease burden . the world health organization recommended the use of artemisinin - based combination therapies ( acts ) for treatment of uncomplicated falciparum malaria a decade ago in response to problems of drug resistance . this review compared two of the acts dihydroartemisinin - piperaquine ( dp ) and artemether - lumefantrine ( al ) to provide evidence which one has the ability to offer superior posttreatment prophylaxis at 28 and 42 days posttreatment . four databases ( medline , embase , cochrane database and global health ) were searched on june 2 , 2013 and a total of seven randomized controlled trials conducted in sub - sahara africa were included . results involving 2 , 340 participants indicates that reduction in risk for recurrent new falciparum infections ( rnis ) was 79% at day 28 in favour of dp [ rr , 0.21 ; 95% ci : 0.14 to 0.32 , p < 0.001 ] , and at day 42 was 44% favouring dp [ rr , 0.56 ; 95% ci : 0.34 to 0.90 ; p = 0.02 ] . no significant difference was seen in treatment failure rates between the two drugs at days 28 and 42 . it is concluded that use of dp offers superior posttreatment prophylaxis compared to al in the study areas . hence dp can help reduce malaria cases in such areas more than al .

1. Introduction 2. Materials and Methods 3. Search Result 4. Findings 5. Discussion 6. Conclusion 7. Recommendation

malaria has attracted global attention as one of the world 's leading major diseases due to its high morbidity and mortality . the world health organization ( who ) estimated that about 3.3 billion people are at risk of malaria because they live in malaria endemic areas and about 300500 million malaria cases malaria is also responsible for almost a million deaths yearly [ 2 , 3 ] . the burden of malaria is greatest in sub - sahara africa where approximately 80% of the global malaria cases as well as 90% of fatalities occur . as a result , the who recommended artemisinin - based combination therapy ( act ) for treatment of uncomplicated falciparum malaria [ 11 , 12 ] . the artemisinin derivatives are fast acting drugs capable of rapid clearing of the falciparum parasites during treatment [ 12 , 13 ] but have short half - lives which render them almost unsuitable for use as single therapeutic drugs because of risk for drug resistance which increases as plasma levels fall . following the who recommendation , most sub - sahara african countries have changed their antimalaria drug policy to the use of the acts . artemether - lumefantrine ( al ) is one of the acts commonly used as first - line drug for treatment of uncomplicated falciparum malaria in the sub - sahara african countries . another act however , dp is not widely used in sub - sahara africa compared to the use of al . the half - lives of the partner drugs in the acts vary across the different act drugs available see table 1 . as a result , it is being speculated that dp could be the act with greatest posttreatment prophylactic efficacy due to the longer half - life and sustained plasma levels of piperaquine compared to other acts such as al . it is not yet clear whether the sheer longer half - life of piperaquine in dp could translate into a better prophylaxis and to what extent compared to lumefantrine in al . olliaro and colleagues found that recurrent new infections ( rnis ) were associated with higher risk for development of symptomatic disease ( malaria ) than recrudescence parasitaemia . this current review aimed at synthesizing available primary studies conducted in sub - sahara africa to determine whether use of dp reduces incidence of new falciparum infections ( rnis ) and treatment failure more than use of al within 28 and 42 days after treatment . a clear knowledge in this regard would offer a better guidance to country antimalaria drug policies in sub - sahara africa . the main review question is , does the use of dp in treating uncomplicated falciparum malaria reduce the risk for recurrent new falciparum infections and treatment failure more than al ? the review tested the null hypothesis that there is no difference between the two drugs . for purposes of this review , recurrent new falciparum infection ( rni ) is defined as new infections which patients acquired by day 28 or 42 following treatment for uncomplicated falciparum malaria using dp or al . total treatment failure ( ttf ) refers to recrudescence recorded by day 28 or day 42 ( tables 5 and 6 ) after treatment , where recrudescence refers to the old or the original parasites for which the antimalaria treatment was initiated but could not be cleared by the antimalaria drug and the disease ( symptomatic malaria ) has returned . the new infections must have been differentiated from the old infections in the primary studies by use of polymerase chain reaction ( pcr ) . prophylactic effectiveness used in this review refers to the ability of the act drug ( dp or al ) to prevent the occurrence of new falciparum infections within 28 or 42 days after treatment with either drug ( tables 7 and 8) . studies were included in the review if they were randomized controlled trial ( rct ) conducted in sub - sahara africa and had compared the two drugs ( dp and al ) head - to - head for treatment of uncomplicated falciparum monoinfections only . an electronic search to locate relevant published studies was conducted in four main databases , namely , embase , medline , cochrane library , and global health , using the search strategy presented in table 2 . the final database search for the review was conducted in the selected databases on june 2 , 2013 , as follows : embase : from 1980 to 2013 week 22 ; medline : from 1946 to may 2013 week 4 ; global health : from 1973 to 2013 week 21 . the main terms in the review question were structured into the pico ( population , intervention , comparison , and outcome ) format and all corresponding synonyms identified . all main terms in the review question were searched under medical subject heading ( mesh terms ) and also as free texts . all other terms ( synonyms of the main terms ) were searched as free texts . the final search result after use of the boolean operator and was limited to studies conducted on humans and have been published in english language see table 3 . only terms in the population , intervention , and comparison categories were used during the search because the reviewers were of the view that further inclusion of terms in the outcome category in the search might lead to exclusion of some important studies . the outcomes of interest included recurrent new falciparum infections ( rnis ) and total treatment failure ( ttf ) reported by day 28 and 42 . data on new infection and treatment failure were extracted using the differentiated pcr results recorded in the studies . results of dp group relative to al group were regarded as statistically significant if the 95% ci did not include number of no effect , which is 1 . absolute relative difference ( ard ) and number needed to treat ( nnt ) were calculated . a total of 1,673 records were retrieved from all the database searches combined and 1,631 records ( 97.5% ) were excluded initially because they have irrelevant study titles . abstract of the remaining 42 publications ( 2.5% ) were read , after which 11 ( 26.2% ) were further selected and subjected to thorough full text reading and scrutiny . one trial reported use of block randomisation while the remaining two [ 32 , 34 ] used stratified random lists generated by off - site investigators ( figure 2 ) . five trials [ 29 , 30 , 32 , 33 , 35 ] used adequate allocation concealment methods and were judged to be at low risk for selection bias because the researchers used sequentially numbered , sealed opaque envelops to obscure treatment group before allocation . the concealment process was judged to pose an unclear risk of bias in only two of the studies [ 31 , 34 ] for lack of clear information . blinding was judged to be adequate ( low risk of bias ) in six studies [ 3035 ] because all laboratory personnel and trial investigators involved in assessing outcome measures were blinded to the treatment allocation of the study participants . this was deemed to pose low risk of bias to parasitological outcome measures for treatment failure and rate of new infections . there was no blinding for participants and nurses in four studies and this was considered to pose rather high risk of bias to the outcome measures of adverse clinical events ( side effects of the drugs ) which were not the focus of this review . more than 90% of randomised participants were included in final analysis in all the trials and this was deemed adequate . result on total treatment failure ( ttf ) due to recrudescence was obtained from six studies and involved a total of 3,172 patients . of this , 1,861 participants received dp , out of which 107 ( 5.7% ) experienced treatment failure at day 28 . on the other hand , the pooled rr yielded 0.453 , 95% ci : [ 0.203 to 1.012 , p = 0.05 ] . this implies that there was an average of 55% reduction in risk for ttf at day 28 in favour of dp treatment ; largest plausible reduction possible was 80% ; however , the upper boundary of the 95% ci includes the number of no effect ( 1 ) and a risk for harm of 1.2% . extent of heterogeneity was 47% ( i = 47 , p = 0.09 , q - value = 9.5 , df = 5 ) and this level of heterogeneity was considered to be low see figure 3 . the pooled ard was 0.016 , 95% ci [ 0.0300.002 , p = 0.02 ] , which means that for every 63 patients treated with dp , one case of treatment failure was prevented , which would have occurred if such patients had received al treatment . data on ttf at day 42 was extracted from four studies involving 2 , 662 participants , out of which treatment failure was reported in 274 participants representing 10.3% . a total of 1,598 participants were those treated with dp and 161 of them ( 10.1% ) experienced treatment failure . those who received al treatment were 1,064 participants out of which 113(10.6% ) experienced failure , an indication that dp was associated with a marginal reduction in treatment failure compared to al . the pooled estimate for the rr was 0.560 with 95% ci of [ 0.275 to 1.140 , p = 0.1 ] . this implies that there was an average reduction in risk of treatment failure of 44% in favour of dp treatment compared to al , with the highest plausible reduction in risk of up to 73% . however , the upper boundary of the 95% ci includes number of no effect ( 1 ) and includes 14% harm of failure . test for heterogeneity in random effect model shows high statistical heterogeneity of 71% across the studies ( q = 10 , df ( q ) = 3 , i = 71% , p = 0.02)see figure 4 . results from four studies involving a total of 2,340 patients showed that 104 of the patients ( representing 4.4% ) acquired new falciparum infections within the 28 day period after treatment . in the al group , 907 patients received the treatment and 76 ( 8% ) acquired new infections an indication that rate of new falciparum infection was higher among those treated with al than those treated with dp . pooled rr was 0.207 , 95% ci [ 0.136 to 0.315 , p < 0.001 ] . this indicates that risk associated with a patient getting new infections at day 28 was significantly reduced averagely by 79% in favour of dp treatment . the lowest plausible reduction was 69% and highest was 86% ; the difference was statistically significant ( p < 0.001)figure 5 . results extracted from four studies involving a total of 2,662 patients indicated that 455 patients representing 17% experienced new infections with falciparum parasites . on the other hand , a total of 1,064 received al , of which 237 ( 22% ) experienced new infections . this means that the risk of a patient acquiring new falciparum infection at 42 day following treatment was averagely reduced by 44% in favour of dp compared to al the 95% ci was 0.342 to 0.908 , suggesting that the actual effect could be anywhere between 9 and 66% ; the difference was statistically significant , p = 0.02 . there was a substantial heterogeneity ( extent was 83% ) associated with this result ( i = 83 , q - value = 18 , df = 3 , p < 0.001 ) . this review compared two acts dihydroartemisinin - piperaquine ( dp ) and artemether - lumefantrine ( al)to determine which has the greatest effect in reducing recurrent new falciparum infections ( rnis ) and treatment failure . the null hypothesis which was tested was that there is no difference between the two drugs in reducing risk of treatment failure and recurrent new falciparum infections at days 28 and 42 . however , the overall evidence gathered suggests that patients who were treated with dp experienced less total treatment failure ( ttf ) and less recurrent new falciparum infections ( rnis ) than those who received al . majority of studies included in this review were conducted in east africa and only few were carried out in west africa and therefore findings and conclusion are more applicable to countries in the east africa region . the evidence synthesized indicates that both dp and al are effective in preventing treatment failure at days 28 and 42 after treatment . however , dp reduced treatment failure higher than al at 28 and 42 days but the difference in magnitude of failure reduction between the two drugs is clinically marginal and not statistically significant . this finding concurs with that of earlier review conducted on studies done in asia , america , and africa , in which dp was found to be associated with an appreciable efficacy of cure rate compared to non - act and other act drugs . the average percentage of risk reduction in favour of dp compared to al in this review decreased from 55% at day 28 to 44% at day 42 . this confirms the assertion by yeka and colleagues that as length of followup increases , difference between dp and al in their ability to prevent treatment failure in high endemic areas becomes insignificant . this observation is attributed to the overwhelming rate of new infections in such areas which may have outweighed the efficacy of piperaquine coupled with the decreasing concentration of piperaquine in the blood stream over time . result on prevention of recurrent new falciparum infections ( rnis ) in this review demonstrates that dp offered greater and significant ptp for patients against rni than al . average percentage reduction in risk for rni was up to 79% at day 28 in favour of dp and up to 44% at day 42 in favour of dp . there is , however , substantially significant statistical heterogeneity ( extent was 83% ) associated with pooled result on rni at day 42 in this review . the possible source of this heterogeneity is attributed to the variability in malaria infection transmission rate of the various study sites ; some of the studies were conducted in settings where rate of malaria transmission intensity was very high while other studies were conducted in areas of relatively low malaria transmission intensity . even though participants were provided with insecticide treated nets , it can not be ascertained whether patients actually slept in the nets to prevent mosquito bites after treatment and this could also affect the proportion of patient who experienced recurrent new infections . the superiority of dp in offering this higher level of posttreatment prophylaxis ( ptp ) is attributable to the longer half - life of piperaquine ( which is ~5 weeks ) compared to the shorter half - life ( of about 4 to 5 days ) of lumefantrine in al . the higher ptp of dp will be of significant importance for areas of higher malaria transmission intensities more than for areas of low transmission intensities which are associated with lower frequency of acquiring malaria due to new infections . however , in spite of the benefit of higher ptp of dp it must also be pointed out that the longer half - life of piperaquine is likely to pose high risk for faster development of drug resistant strains of the falciparum parasites to dp . notwithstanding , the risk for development of drug resistant parasites could be minimized if more than one act drug is used as a first - line drug for treatment of uncomplicated malaria in a particular area . the use of more than one drug as a first - line act may prevent the possibility of the parasites becoming adjusted to one overused - first - line act . there is , therefore , the need for encouraging the use of multiple first - line drugs and constant monitoring of efficacy of the act drugs to determine development of resistant strains at early stages . using the grade criteria for rating evidence quality , the quality of evidence obtained regarding superiority of dp over al in preventing ttf at day 28 was rated moderate quality while that of day 42 was rated low quality . evidence obtained on prophylactic superiority of dp over al in preventing rni at day 28 was rated high quality while that of day 42 was rated moderate quality . high quality grade evidence rating means that the authors are very confident that the true effect lies close to the average estimated while moderate quality rating means that authors ' confidence in the estimate is moderate which means that the true effect size is likely to be close to the average estimate , but there is also likelihood that there could be substantial difference . it is worth indicating that treatment failure ( ttf ) and recurrent new infections ( rnis ) are of great importance and concern to patients due to the negative socioeconomic impact of clinical malaria on patients . malaria is highly prevalent in sub - sahara africa and it is undeniable that patients acquiring frequent recurrent new infections contribute to the high number of cases . considering these reasons above and level of relative reduction in risk for ttf and rni in favour of dp together with the quality of the evidence obtained , all studies included in the review were rcts which is appropriate for answering clinical intervention questions . there was no attempt to locate studies in the grey literature and other sources apart from those indexed in the four databases searched . also , majority of the included studies were conducted in countries in east africa which weakens generalisation of findings to other parts of sub - sahara africa ; hence this review result and conclusion are most valid for countries in east africa . this systematic review compared dihydroartemisinin - piperaquine ( dp ) and artemether - lumefantrine ( al ) and aimed at identifying which one has greater ability to reduce total treatment failure ( ttf ) and incidence of recurrent new falciparum infections ( rnis ) in high transmission areas in sub - sahara africa . the results showed that participants treated with dp compared to al experienced lesser risk for ttf at days 28 and 42 . on the other hand , dp offers a significant posttreatment prophylaxis against recurrent new falciparum infections superior to that of al . the average percentage reduction in risk for the incidence of rni was up to 79% at day 28 in favour of dp [ rr , 0.21 ; 95% ci : 0.14 to 0.32 , p < 0.001 ] and 44% in favour of dp at day 42 [ rr , 0.56 ; 95% ci : 0.34 to 0.90 ; p = 0.02 ] . it is , therefore , concluded that treatment of uncomplicated falciparum malaria using dihydroartemisinin - piperaquine ( dp ) in high transmission areas in sub - sahara africa ( especially east africa ) could result in an average reduction in risk for recurrent new falciparum infections of up to 79% and 44% within 28 and 42 days , respectively , compared to artemether - lumefantrine ( al ) . and this implies that use of dp can help reduce burden of malaria in such areas more than al . it is recommended that the antimalaria drug policy in countries especially in east africa should be streamlined to include use of dihydroartemisinin - piperaquine alongside artemether - lumefantrine and other acts in countries where it is found to be effective but this must be done bearing in mind the potential risk for development of resistant falciparum strains . more studies should be conducted in other parts of the sub - sahara africa such as west africa to determine stronger evidence which will be more applicable to countries in that area . it is , therefore , recommended that measurement of rate of recurrent new infections should be incorporated into future guidelines , and future trial investigators should make direct assessment on it .

to achieve an accurate pathological evaluation in toxicity studies , it is particularly important to know the background histopathology , that is , to be familiar with pictures of incidental findings . as abundant studies using rats are common in laboratories , toxicological pathologists know the background data of spontaneous lesions in rats and can visualize these histological figures easily . meanwhile with monkeys , only a few pathologists in limited laboratories have experience with studies using nonhuman primates . therefore , most pathologists can not help but depend on the literature or textbooks to participate in the evaluation of monkey studies as a study pathologist or reviewing pathologist . in the present state , however , relatively few background data or pictures of incidental findings in monkeys have been published due to the lack of understanding of the importance of publishing incidental findings , which are pathologically or toxicologically insignificant spontaneous lesions with no sign of disease . therefore , in this report , we provide pictures of spontaneous lesions in cynomolgus monkeys ( macaca fascicularis ) that were detected in background data collection studies and ordinary toxicity studies in our laboratory . the figures are grouped and arranged according to the cardiovascular , lymphoid , respiratory , alimentary , urinary , reproductive , endocrine , nervous , musculoskeletal and integumentary systems . a total of 660 cynomolgus monkeys ( 332 males and 328 females ) were subjected to background data collection studies and ordinary toxicity studies conducted in our laboratory from 1998 to 2011 . they were imported from the philippines ( 44 males and 50 females ) , indonesia ( 7 males and 7 females ) , malaysia ( 10 females ) , vietnam ( 271 males and 251 females ) and china ( 10 males and 10 females ) . the animals were housed individually in metal cages ( 680 608 770 mm or 680 658 770 mm ) in conventional rooms air - conditioned at 23 c to 29 c with 35% to 75% relative humidity and a 12-hour light/12-hour dark cycle . they were provided with 100 g of commercially available food ( cmk-1 , cmk-1 or cmk-2 , clea japan , inc . ) the animals were cared for according to the principles outlined in the guides for the care and use of laboratory animals prepared by the japanese association for laboratory animal science and our institution . organs fixed in 10% neutral phosphate - buffered formalin were embedded in paraffin , and the sections were made and stained with hematoxylin and eosin ( he ) for microscopic examination . selected sections were stained with warthin - starry , periodic acid - schiff ( pas ) and periodic acid - methenamine - silver ( pam ) . we chose 195 typical findings or rare lesions of the cynomolgus monkeys from the background data collection studies and ordinary toxicity studies conducted in our laboratory , and they are shown as follows . focal infiltration of inflammatory cells mainly consisting of lymphocytes is frequently seen in the myocardium . 1 . heart : focal inflammatory cell infiltration in the myocardium heart : focal myocardial necrosis . focal myocardial necrosis occurs occasionally , but a large focus like this case is rare . this lesion must be distinguished from artifacts because a similar figure is also formed due to inadequate handling of the heart during necropsy and tissue preparation . 2 . heart : focal myocardial necrosis heart : proliferation and squamous metaplasia of the epicardial mesothelium . focal proliferation of single layered mesothelial cells occurs occasionally in the atrium ( atrial auricle ) . squamous metaplasia also occurs in the same area , but the incidence is low . they are thought to be reactive changes to some physical irritation . 3 . heart : proliferation and squamous metaplasia of the epicardial mesothelium heart : hemorrhage in the endocardium . focal and mild hemorrhage in the endocardium and subendocardium is considered mainly to be an agonal change . intimal and medial thickening of the arterial wall with duplication of the elastic lamina is rare in the coronary and mural arteries . 5 ) , presumably because of ischemia by arterial stenosis accompanied by a thickening of the wall . fig . heart : infarction ( elastica - van gieson ) artery : arteritis ( figs . 712 ) the term of arteritis refers to various inflammatory changes of the arterial wall . endarteritis , periarteritis and panarteritis are used to describe the affected part , and polyarteritis is used for multiple lesions . various adjectives are added to describe the morphological characteristics such as acute arteritis , necrotizing arteritis , polyarteritis nodosa or granulomatous arteritis . arteritis is usually observed in one or a few organs / tissues , and systemic arteritis is rare in cynomolgus monkeys . inflammatory cells infiltrate focally in the tunica intima and adventitia of the mural artery . artery : arteritis in the mural artery artery : arteritis in the coronary artery . panarteritis consists of intimal thickening and infiltration of lymphocytes from the tunica intima to the adventitia . the incidence and grade of arteritis in coronary arteries are remarkably lower in cynomolgus monkeys than in beagles . artery : arteritis in the coronary artery artery : arteritis in the kidney . panarteritis nodosa is seen relatively frequently in the intrarenal arteries , especially the arcuate arteries , in cynomolgus monkeys . fig . arteritis in the submucosa of the intestinal tract is characterized by fibrinoid necrosis of the tunica media and mild inflammatory cell infiltration in the adventitia . artery : arteritis in the intestinal artery artery : arteritis in the bronchial artery . arteritis in the bronchial artery is characterized by thickening of the tunica intima and inflammatory cell infiltration in the tunica intima and adventitia . artery : arteritis in the bronchial artery artery : arteritis in the epididymis . artery : arteritis in the epididymis mesenteric lymph node : increased number of pigment - laden macrophages in the sinuses . accumulation of a large number of histiocytes containing yellow - brown pigment is common in the sinuses of the mesenteric lymph nodes . mesenteric lymph node : increased number of pigment - laden macrophages in the sinuses thymus : involution ( physiological atrophy ) . decreases in lymphoid cells from both the cortex and medulla cause a reduction in organ size and weight . the thymus generally starts to involute from about 4 or 5 years old in cynomolgus monkeys . the cysts are lined by a partially ciliated cuboidal epithelium and contain eosinophilic and proteinic substances that are the same as those in the parathyroid cysts ( fig . as the parathyroid and thymus share the same primordium , the third pharyngeal pouch , it is comprehensible that the thymic tissue contains a mass of parathyroid cells . germinal center formation in the thymic medulla in cynomolgus monkeys is seen less frequently than in beagles . increases in the number of follicles above the normal variance may suggest an immunologic failure or overresponse . thymus : lymph follicle formation in the medulla thymus : proliferation of the thymic epithelium . proliferation of the thymic epithelium is probably associated with thymic atrophy ( involution ) . thymus : proliferation of the thymic epithelium thymus : thymoma ( mixed thymoma ) . this thymoma was found as a mass with a diameter of 1.5 cm in the thymus . thymus : thymoma ( mixed thymoma ) spleen : nodular hyperplasia ( lymphoid follicle ) . there is no capsule around the lesion , but the lesion slightly compresses the normal splenic tissues . this change consists of lymphocytic follicular hyperplasia with / without other splenic components . spleen : nodular hyperplasia ( lymphoid follicle ) spleen : nodular hyperplasia ( red pulp ) . grossly , the lesion is detected as a protruded white patch in the subcapsular part . the hyperplastic area is composed of proliferated cells of plural types consisting of normal red pulp . spleen : nodular hyperplasia ( red pulp ) spleen : hyalinization of the germinal center . hyalinization ( deposition of eosinophilic substances ) is frequently seen in germinal centers of follicles in cynomolgus monkeys . spleen : hyalinization of the germinal center spleen : pigment deposition in the red pulp . brown or black pigments occasionally deposit in the red pulp . for the most part , brown pigments are hemosiderin ( left ) , and black ones are unidentified ( right ) . we have confirmed that the unidentified pigment is not melanin , lipofuscin , malaria pigment or formalin pigment using some special stains ( unpublished data ) . spleen : pigment deposition in the red pulp bone marrow : lymph follicle formation . accumulation of yellow - brown pigments occurs occasionally in the perivascular area of bone marrow tissue . these pigments are confirmed to be hemosiderin by special staining or reactions on the slide . the lymphoid tissue prominently developed in the nasal septa in this case , although lymphocytic infiltration is always seen in the lamina propria of the septal or turbinate mucosa . nasal cavity : hyperplasia of the lymph follicle nasal cavity : mucosal erosion of the turbinate . slight erosion , focal inflammation and/or focal squamous metaplasia of the respiratory epithelium are occasionally seen in the nasal septum and turbinate and are caused by an inserted nasogastric catheter used for the intragastric administration . nasal cavity : mucosal erosion of the turbinate lung : focal hemorrhage in the alveolus . hemorrhage without inflammatory cell infiltration may be related to certain aspects of death . lung : focal hemorrhage in the alveolus lung : accumulation of pigment - laden macrophages . accumulation of macrophages containing yellow - brown pigments ( probably hemosiderin ) is rarely seen in the perivascular interstitium or alveolar wall . these hemosiderin depositions are presumably due to extravasation of blood or congestion caused by some abnormality of vessels , cardiac function or blood coagulation . fig . 30 . lung : accumulation of pigment - laden macrophages lung : anthracotic pigment deposition ( anthracosis ) . fine black pigments are deposited in the interstitium around the bronchi in almost all cynomolgus monkeys . thrombus occasionally occurs in the pulmonary vessels , especially under the influence of intravenous injection . lung : accumulation of foam cells in the alveolus lung : focal infiltration of macrophages in the alveolus . lung : focal infiltration of macrophages in the alveolus lung : foreign body granuloma . this change may have resulted from aspiration of stomach contents or food particles . focal epithelial hyperplasia with fibrous thickening of the alveolar wall is rarely seen . microscopically , characteristic findings such as dilatation of the bronchial or bronchiolar lumen , thickening of the bronchial wall and inflammatory cell infiltration around the bronchus , lymphoid hyperplasia and brown pigment deposition strongly suggest pulmonary acariasis even if there is no acarid . osseous metaplasia presumably caused by differentiation from fibroblasts to osteoblasts occurs occasionally in the lung alveolar wall . the incidence in cynomolgus monkeys is lower than that in other laboratory animals . lymphoplasmacytic infiltration is usually seen in the lamina propria , especially in the lingual papillae . these changes are characterized by swelling of prickle cells , intercellular edema and cell infiltration . most likely , it is a stage of the degenerative change such as erosion . foreign body granuloma caused by a fragment of hair stuck on the tongue occasionally occurs in the lamina propria or muscle layer . focal regenerative changes of muscle fibers occur occasionally in the lateral tongue muscle layer . tongue : regeneration of the muscle fiber esophagus : focal inflammatory cell infiltration in the lamina propria . esophagus : focal inflammatory cell infiltration in the lamina propria stomach : inflammatory cell infiltration in the lamina propria . lymphoplasmacytic infiltration in the lamina propria of the fundus and pylorus , so - called gastritis , is frequently seen in cynomolgus monkeys and is seen accompanied by regeneration of the mucosal epithelium . stomach : inflammatory cell infiltration in the lamina propria stomach : infection by helicobacter pylori in the gastric mucosa . many fine bacteria are seen in mucosal crypts in a warthin - starry - stained section with inflammatory changes . the detailed morphology of helicobacter pylori and relation between the gastritis and helicobacter infection have been reported ( j toxicol pathol . stomach : infection by helicobacter pylori in the gastric mucosa stomach : infection by helicobacter heilmannii in the gastric mucosa . some fine bacteria are detected in parietal cells in a warthin - starry - stained section without any inflammatory changes in the infected mucosa . stomach : infection by helicobacter heilmannii in the gastric mucosa stomach : erosion . erosion accompanied by gastritis occurs even in monkeys kept under nonstressed conditions , but usually the lesions are small . brown pigment - laden macrophages accumulate in the lamina propria at the top of the villi . slight pigmentation is usually seen in the duodenal lamina propria of cynomolgus monkeys , but an increase in the number of pigment macrophages might also occur following administration of certain pigmented compounds . ectopic pancreatic tissue occurs occasionally in the submucosa near the major papilla of vater . focal muscle fiber necrosis occurs occasionally in the whole intestinal tract , especially in the duodenum . duodenum : necrosis of the muscle layer ileum ( jejunum ) : increase in goblet cells . the population of jejunal and ileal goblet cells varies among individuals . fig . 52 . ileum ( jejunum ) : increase in goblet cells rectum : dilatation of the crypt . dilatation of a crypt filled with / without cell debris or infiltrated cells is frequently seen . this change is localized in not only the rectum but other parts of the intestinal tract . focal lymphoplasmacytic infiltration , occasionally accompanied by lymph follicle formation , is common around the ducts or acini . fig . similar changes are caused by dilatation of the endoplasmic reticulum filled with low electron density amorphous substances . salivary gland : hydropic degeneration of acinar cells salivary gland : salivary calculus and inflammatory cell infiltration . small mineralized calculus occurs occasionally in a duct of a relatively large caliber and is frequently accompanied by lymphoplasmacytic infiltration in the surrounding parenchyma . salivary gland : salivary calculus and inflammatory cell infiltration liver : eosinophilic inclusion bodies in hepatocytes . these bodies are considered to be blood plasma due to rise of the internal pressure in the sinusoids or increased permeability of the hepatic cell membrane liver : eosinophilic inclusion bodies in hepatocytes liver : accumulation of glycogen in hepatocytes . this change is characterized by a clear appearance of the hepatic cytoplasm after formalin fixation . liver : accumulation of glycogen in hepatocytes liver : diffuse fatty change of hepatocytes . small lipid droplets ( clear vacuoles ) liver : diffuse fatty change of hepatocytes liver : focal fatty change of hepatocytes . this change tends to occur in the border of the right and left central lobes , and it is thought to be tension lipidosis . liver : focal fatty change of hepatocytes liver : focal necrosis of hepatocytes . a necrotic focus of hepatocytes with inflammatory cell infiltration occurs occasionally without any apparent cause . microgranuloma consists of accumulation of inflammatory cells , mainly of macrophages , lymphocytes and small number of neutrophils , and may be associated with minute necrosis of hepatocytes . microgranulomas are of much lower incidence in cynomolgus monkeys than in other laboratory animals . fig . this inflammatory focus consists of lymphoplasmacytes and is mainly accumulated in the glisson s sheath or around the central vein . if the inflammatory cells are predominantly eosinophils , parasitic infection might be considered as one of the causes . brown pigments are presumably hemosiderin ( left ) , and black ones ( right ) are unidentified pigments like those described in the spleen . fig . liver : pigment deposition in kupffer cells gall bladder : inflammatory cell infiltration in the lamina propria . development of the lymphoid tissue in the lamina propria occurs occasionally , and slight lymphoplasmacytic infiltration is constant in almost all cynomolgus monkeys . gall bladder : inflammatory cell infiltration in the lamina propria pancreas : ectopic splenic tissue . splenic tissue consisting of red and white pulp is contained in the pancreatic parenchyma without a fibrous capsule . lobular atrophy consisting of shrinkage and loss of acini with interstitial fibrosis can be seen occasionally in the exocrine pancreas . pancreas : lobular atrophy of the acinus pancreas : hydropic degeneration of acinar cells . a decrease in zymogen granules and accumulation of pale eosinophilic substances in acinar cells are rare . similar changes are caused by dilatation of the endoplasmic reticulum filled with low electron density amorphous substances . pancreas : hydropic degeneration of acinar cells pancreas : apoptosis of acinar cells . apoptosis of acinar cells is relatively rare in the pancreas of cynomolgus monkeys , in contrast to the larger number and higher frequency in beagles . although a decrease of zymogen granules is thought to be a stress - related change , it rarely occurs in animals under normal conditions . the histopathologic features of chronic pancreatitis consist of necrosis and loss of acinar cells , dense fibrosis and lymphoplasmacytic infiltration in the exocrine pancreas , although the islets usually remain intact in the lesions . on autopsy , many ductal structures are lined by cuboidal epithelial cells proliferating within a densely fibrotic area . focal lymphoplasmacytic infiltration occurs occasionally in the interstitium of the pancreas , especially around the pancreatic duct . it has been described that similar angiectasis was induced in cynomolgus monkeys treated with an antineoplastic immunomodulator . focal hemorrhage is rare in islets , presumably due to some focal damage of the islet cells or vessel wall . the islet tissue is lobulated into multiple compartments by fibrous septa , presumably due to some focal damage of the islets such as in fig . 78 . weakly eosinophilic amorphous material deposits around the capillaries of an islet with loss of a fairly large number of islet cells . islet amyloidosis occurs occasionally in aged cynomolgus monkeys but is rare in young ones . embryologically , both primordia of the renal and adrenal tissues are adjacent to each other . kidney : ectopic adrenocortical tissue kidney : focal inflammatory cell infiltration in the interstitium . focal lymphoplasmacytic infiltration is common in the interstitium ; usually the lesion size is small . kidney : focal inflammatory cell infiltration in the interstitium kidney : regeneration of the tubular epithelium . focal regenerative changes of damaged tubules are detected as basophilic tubules with a high nuclear density in the renal cortex or outer medulla . kidney : regeneration of the tubular epithelium kidney : brown pigment deposition in the tubular epithelium . brown pigment deposition is frequently seen in the tubular epithelium , mainly in the henle s tubules and straight portions of proximal tubules . small mineralization is usually seen in the interstitium of the renal papilla and also occurs occasionally in the cortex and outer medulla . renal mineralization is found in various laboratory animals but to a lesser extent in cynomolgus monkeys . kidney : osseous metaplasia kidney : crystal deposition.a yellowish lucent crystal formed in the tubule causes epithelial damage and inflammatory cell infiltration . oxalate is contained in various plants eaten by wild cynomolgus monkeys , and oxalate crystals are common among them but are very rare in animals bred for laboratory use . kidney : crystal deposition kidney : focal hyperplasia / hypertrophy of the tubular epithelium . this figure shows single cell layered renal tubular hyperplasia , which shows an increase in the number of epithelial cells and hypertrophy of nuclei and cytoplasm . kidney : focal hyperplasia / hypertrophy of the tubular epithelium kidney : atypical hyperplasia of the tubular epithelium . the pathogenesis of this lesion is probably the same as that described in fig . there is no evidence to suggest that these lesions ever progress to renal tumor . kidney : atypical hyperplasia of the tubular epithelium kidney : scar ( nephrosclerotic lesion ) . a renal scar consists of obsolescent glomeruli , interstitial fibrosis and inflammatory cell infiltration . fig . 90 . kidney : scar ( nephrosclerotic lesion ) kidney : multinucleated epithelial cells in the collecting tubules . this change occurs in collecting tubules of the renal papilla in some cynomolgus monkeys , although its pathological meaning and pathogenesis are unknown . kidney : multinucleated epithelial cells in the collecting tubules kidney : edema of the renal papilla . interstitial edema without inflammation is frequently seen in the renal papilla but is restricted to a small area . inflammatory cells rarely infiltrate any mucosal regions of the pelvis and papillary interstitium with tubular damage and regeneration . fig . focal lesions distributed along the nephron suggest ascending spread of inflammatory lesions from the pelvis . chronic pyelonephritis is tubulointerstitial nephritis consisting of fibrosis and inflammatory cell infiltration in the interstitium , tubular atrophy and thyroid - like appearance , extra - afferent fibrosis of the bowman s capsule and obsolescent glomeruli . capillary formation is indistinct , whereas epithelial cells are prominent in an immature glomerulus . immature glomerulus is seen more frequently in the outer layer of the cortex , reflecting the delayed maturation of the glomeruli in the outer cortex than in the deep layer . 97102 ) these lesions are usually seen in cynomolgus monkeys but are solitary ( one or two abnormal glomeruli in one section ) . the details of these changes are not understood because every one of the lesions is stained eosinophilic in he sections . a segmental eosinophilic nodule containing some mesangial cells is frequently seen in one or a few glomeruli in an he section . pam staining makes it easy to demonstrate marked proliferation of mesangial cells and increase in mesangial matrix and the collapse of capillary loops . kidney : segmental sclerosis of the glomerulus ( he and pam ) kidney : global sclerosis of the glomerulus ( he and pam ) . global proliferation of mesangial cells with increased matrix is prominent along the glomerular tuft , and capillary loops are not visible presumably because of mesangial proliferation into the capillary loops . fig . kidney : global sclerosis of the glomerulus ( he and pam ) kidney : obsolescent glomerulus ( he and pas ) . one or a few obsolescent glomeruli in a section of the kidney are frequently seen in cynomolgus monkeys . this lesion is commonly accompanied by interstitial fibrosis and tubular atrophy , suggesting local ischemia as the pathogenic cause . kidney : obsolescent glomerulus ( he and pas ) kidney : hyalinosis of the glomerulus ( he and pas ) . so - called hyalinosis is characterized by the eosinophilic substance consisting of a serum glycoprotein that reacts positively for pas reaction and negatively for pam stain . kidney : hyalinosis of the glomerulus ( he and pas ) kidney : angiectasis of the glomerular capillary ( he and pas ) . the change is usually seen , but the pathogenesis is unknown . fig . kidney : angiectasis of the glomerular capillary ( he and pas ) kidney : fatty metaplasia of the glomerulus . the histopathologic feature differs from the glomerular lipidosis in beagles in some respects such as the lack of closely packed fine vacuoles and eosinophilic droplets . kidney : fatty metaplasia of the glomerulus kidney : mesangial proliferative glomerulonephritis ( he ) . kidney : mesangial proliferative glomerulonephritis ( he ) kidney : mesangial proliferative glomerulonephritis ( pam ) . proliferation of mesangial cells along the glomerular tufts and thickening of the bowman s capsule are revealed with pam stain . kidney : mesangial proliferative glomerulonephritis ( pam ) kidney : mesangiocapillary glomerulonephritis ( he ) . the glomeruli are strikingly enlarged and show lobulation due to the proliferation of mesangial cells and increase in mesangial matrix toward the peripheral area in an he section . kidney : mesangiocapillary glomerulonephritis ( he ) kidney : mesangiocapillary glomerulonephritis ( pam ) . the characteristic features of mesangiocapillary glomerulonephritis , such as the double contour ( arrows ) of the basement membranes caused by mesangial interposition , are observed in a pam - stained section . though membranoproliferative glomerulonephritis is generally used to refer to this lesion kidney : mesangiocapillary glomerulonephritis ( pam ) kidney ( pelvis ) : eosinophilic droplets in the transitional epithelium . eosinophilic droplets in transitional epithelium occur occasionally in the renal pelvis and bladder . kidney ( pelvis ) : eosinophilic droplets in the transitional epithelium urinary bladder : focal inflammatory cell infiltration in the lamina propria . urinary bladder : focal inflammatory cell infiltration in the lamina propria testis : immature ( grade 3 ) . testes of relatively young monkeys used in toxicologic studies show various stages of maturation with some variability between individuals . seminiferous tubules consisting of sertoli cells , spermatogonia and a few spermatocytes are grade 3 ( fig . 109 ) , which is the most usual grade for animals 3 years of age or much younger . the features of grade 4 , the most immature grade , consist of only sertoli cells and undifferentiated spermatogonia . fig . 109 . testis : immature ( grade 3 ) testis : immature ( grade 2 ) . in most seminiferous tubules testis : immature ( grade 2 ) testis : immature ( grade 1 ) . the number of spermatozoa is very low , whereas development up to spermatids completes in almost all seminiferous tubules . this stage is the most usual in 4- to 5-year - old animals . testis : immature ( grade 1 ) testis : segmental dilatation of the seminiferous tubules . seminiferous tubules consisting of sertoli cells , spermatogonia , and spermatocytes are occasionally dilated segmentally in the mature testis . testis : segmental dilatation of the seminiferous tubules testis : prepubertal testis . occasional multinucleated giant cell formation and/or aggregation of round spermatogonia occur in the seminiferous tubules in the prepubertal testis . it may be difficult to distinguish these spontaneously occurring changes from some chemical- or drug - induced changes in the seminiferous tubules . testis : prepubertal testis testis : swelling with eosinophilic changes of the sertoli cells . swelling with eosinophilic changes of sertoli cells testis : swelling with eosinophilic changes of the sertoli cells testis : spermatocele . this change is caused by disturbance of spermatozoa flow due to degenerative changes of the seminiferous tubule . this body is thought to be derived from degenerated cells or secretions containing protein , and it does not have any important pathological significance . the change can be seen in all phases of maturation but can not be seen in the mature testis . maturation of the seminal vesicle also closely correlates with the degree of testes maturation . the glandular epithelia are small and flat , and lumens are narrow because of no prostatic secretion . maturation of the prostate also closely correlates with the degree of testes maturation . embryologically , both primordia of the reproductive organ and adrenocortical tissue are adjacently located to each other . focal accumulation of spermatozoa ( spermatocele ) occurs occasionally in the seminal vesicle of monkeys after genital maturation , and the accumulated spermatozoa might often be mineralized . prostate : focal inflammatory cell infiltration cyclic changes in female reproductive system ( figures 125130 ) . uterus , ovary ( containing dominant follicle ) , ovary ( containing involuting corpus luteum ) , vagina fig . female reproductive system : early follicular phase female reproductive system : follicular phase . uterus , ovary ( containing dominant follicle ) , ovary ( containing involuting corpus luteum ) , vagina fig . uterus , ovary ( containing dominant follicle ) , ovary ( containing involuting corpus luteum ) , vagina fig . uterus , ovary ( containing postovulatory corpus luteum ) , ovary ( containing involuting corpus luteum ) , vagina fig . female reproductive system : early luteal phase female reproductive system : luteal phase . uterus , ovary ( containing functional corpus luteum ) , ovary ( containing involuting corpus luteum ) , vagina fig . uterus , ovary ( containing dominant follicle ) , ovary ( containing menstrual corpus luteum ) , vagina fig . immature female reproductive tissues are characterized by a thin endometrium ( less than 50% of the mature uterine basal layer ) and thin myometrium in the uterus , a small ovary containing a lot of primordial follicle and a few graafian follicles and thin or no keratinized squamous epithelium in the vagina . it is necessary to distinguish this immature figure from atrophy of the female reproductive system because similar figures are also seen under cachectic conditions . they consist of characteristic luteal cells with vesicular nuclei and deeply stained cytoplasm ( eosinophilic or basophilic cytoplasm ) and abundant capillaries . the luteal cells are positive for inhibin ( right ) . we think that the acl may have a role in controlling the number of follicles by inhibin secretion for a single ovulation in monkeys . similar features are well - known as generally occurring changes in women during or after pregnancy . fig . various numbers of mineralized foci are frequently seen in the ovarian cortex lined by many primordial follicles , especially in young cynomolgus monkeys . these cysts arise from remnants of the paroophoron . dilated ducts lined by cuboidal or columnar ciliated epithelia occur occasionally in peripheral tissue of the ovaries . it differs from a paroophoritic cyst in the existence of developed smooth muscle in the pericystic layer . remnants of the rete ovarii are frequently seen in the hilum of the ovary and occasionally undergo hyperplastic changes . multiple cysts lined by mucinous epithelia resembling the cervical canal epithelia in the ovarian parenchyma . ovary : mucinous cystadenoma ovary : ectopic ovarian tissue in the uterus or urinary bladder . ovarian tissue consisting of primordial follicles , primary follicles , preantral follicles and surrounding stromal cells occurs occasionally in the muscle layer or serosa of the uterus ( left ) but rarely in the urinary bladder ( right ) . ovary : ectopic ovarian tissue in the uterus or urinary bladder uterus : adenomyosis ( endometriosis interna ) . endometrial tissue consisting of endometrial glands and stromal cells occurs occasionally in the uterine muscle layer . endometriosis externa in the intestinal wall , greater omentum and mesenterium has been reported in older cynomolgus monkeys , and we have never observed it in young animals . melanin pigments occur occasionally in the endometrium . melanin pigment deposition ( melanosis ) can be seen in various organs in cynomolgus monkeys . uterus : melanin pigment deposition uterus : focal inflammatory cell infiltration in the endometrium . this lesion differs from estrus cycle - related changes , although some inflammatory cells generally infiltrate into the apical endometrial tissue in the late luteal phase ( ischemic phase of uterus ) . uterus : focal inflammatory cell infiltration in the endometrium uterus : dilatation of the endometrial gland . many endometrial glands dilate excessively , so this lesion differs from physiological dilatation during the follicular phase . melanin pigments occur occasionally in the muscle layer and/or serosa . melanin pigment deposition ( melanosis ) can be seen in various organs in cynomolgus monkeys . pituitary cysts are frequently seen in each lobe such as the anterior ( left ) , intermediate ( middle ) and posterior ( right ) lobes but are often found in the anterior lobe . the left figure is the intermediate lobe , and the right figure is the posterior lobe . thyroid ( parathyroid ) : ectopic salivary gland thyroid ( parathyroid ) : ectopic thymic tissue . because the parathyroid , thyroid and thymus derive from the same primordium , the pharyngeal cavity , it is comprehensible that the thyroid and/or parathyroid tissue contain thymic tissue . thyroid ( parathyroid ) : ectopic thymic tissue thyroid : cystic dilatation of the follicle . huge follicles containing weakly basophilic substances occur occasionally . the pathologic meaning and significance of this change thyroid : cystic dilatation of the follicle thyroid : focal c - cell hyperplasia . generally , c - cells are not easily recognizable in the thyroid of cynomolgus monkeys , in contrast to those in beagles . in this figure , the c - cell hyperplastic focus is small , but even a small focus appears very rarely in cynomolgus monkeys . thyroid : focal c - cell hyperplasia thyroid : infiltration of macrophages in the follicles . focal follicular atrophy , desquamation of follicular epithelia and infiltration of macrophages in follicles are frequently seen in the thyroid of cynomolgus monkeys . fig . 153 . thyroid : infiltration of macrophages in the follicles thyroid : focal inflammatory cell infiltration . focal lymphoplasmacytic infiltration , often with lymph follicle formation in the interstitium , is frequently seen . the incidence and degree of this lesion are lower in cynomolgus monkeys than in beagles . thyroid : focal inflammatory cell infiltration thyroid : hydropic degeneration of follicular cells . similar changes are caused by dilatation of the endoplasmic reticulum filled with low electron density amorphous substances . thyroid : hydropic degeneration of follicular cells thyroid and parathyroid : fatty infiltration . adipose cells infiltrate focally or entirely into the interstitium of the thyroid and/or parathyroid . thyroid and parathyroid : fatty infiltration parathyroid : cyst ( krsteiner s cyst ) . cysts are lined by cuboidal epithelia , occasionally ciliated , and contain proteinic substances in the lumens . this cyst is a remnant of the embryonal duct connecting the parathyroid - thymus tissue in the iii and iv pharyngeal pouches . parathyroid : cyst ( krsteiner s cyst ) parathyroid : increase in oxyphil cells . the number of oxyphil cells in the parathyroid tends to increase with advancing age ; however , this change is rarely seen at a young age . parathyroid : increase in oxyphil cells parathyroid : focal hypertrophy of chief cells . parathyroid : focal hypertrophy of chief cells parathyroid : focal inflammatory cell infiltration . parathyroid : focal inflammatory cell infiltration adrenal : accessory adrenocortical tissue . the aberration of adrenocortical tissue separated from the original body and surrounding capsule is called accessory adrenocortical tissue . fig . 161 . adrenal : accessory adrenocortical tissue adrenal : normal variance of cortical cells ( 1 ) . generally , each of the three adrenocortical zones is obviously distinguishable in cynomolgus monkeys because cells of the zona fasciculata contain numerous fine vacuoles causing a very clear appearance of the cytoplasm . partial replacement of the zona fasciculata by glomerulosa - like cells occurs occasionally as a normal variance . adrenal : normal variance of cortical cells ( 1 ) adrenal : normal variance of cortical cells ( 2 ) . eosinophilic and hypertrophic cells of the zona fasciculata are focally seen without compressing the adjacent tissue as a normal variance . adrenal : normal variance of cortical cells ( 2 ) adrenal : high magnification of fig . the hypertrophic cortical cells have eosinophilic cytoplasm containing basophilic granules instead of lipid droplets . basophilic granules are thought to be rough endoplasmic reticulum , and these changes are the same as for hypertrophic cortical cells under stress conditions . a focus consisting of proliferative and hypertrophic eosinophilic cells appears mainly in the zona fasciculata and reticularis . eosinophilic cells in the focus have few lipid droplets in the cytoplasm . fig . adrenal : nodular hyperplasia of cortical cells ( eosinophilic ) adrenal : nodular hyperplasia of cortical cells ( vacuolated ) . a focus consisting of proliferative and hypertrophic vacuolated cells containing numerous fine lipid droplets originates mainly in the zona fasciculata . adrenal : nodular hyperplasia of cortical cells ( vacuolated ) adrenal : decreased lipid droplets in cortical cells . if the lipid droplets decrease due to stress , each of the adrenocortical zones is indistinct . sometimes , cells of the zona fasciculata contain a little fewer lipid droplets under normal conditions . adrenal : decreased lipid droplets in cortical cells adrenal : mineralization in the corticomedullary junction . the change may be dystrophic mineralization presumably caused by preexisting involution , hemorrhage and/or fibrosis in the inner fetal layer during postnatal development . adrenal : mineralization in the corticomedullary junction adrenal : pigment deposition in the corticomedullary junction . adrenal : pigment deposition in the corticomedullary junction adrenal : pigment deposition in the cells of the zona reticularis . unlike fig . 169 , yellow - brown pigments in cells of the zona reticularis are mostly lipofuscin . fig adrenal : pigment deposition in the cells of the zona reticularis adrenal : adrenohepatic fusion . grossly , the right adrenal is attached to the right lobe of the liver . the hepatic tissue occasionally occupies a part of the cortex of the right adrenal . focal and slight lymphoplasmacytic infiltration occurs occasionally in the zona reticularis ( left ) or medulla ( right ) . adrenal : focal inflammatory cell infiltration brain ( spinal cord ) : melanin pigment deposition in the meninx and vascular wall ( perivascular tissue ) . brain ( spinal cord ) : melanin pigment deposition in the meninx and vascular wall ( perivascular tissue ) brain : hemosiderin pigment deposition in the meninx or cerebral cortex . extravasation of erythrocytes is most likely the cause of the change , although no changes suggesting the preceding hemorrhage or congestion are evident . brain : hemosiderin pigment deposition in the meninx or cerebral cortex brain : focal inflammatory cell infiltration in the meninx . brain : focal inflammatory cell infiltration in the meninx brain : focal inflammatory cell infiltration in the choroid plexus . brain : focal inflammatory cell infiltration in the choroid plexus brain : focal perivascular inflammatory cell infiltration . focal and slight brain : focal perivascular inflammatory cell infiltration spinal cord : mineralization in the meninx . fig . 178 . spinal cord : mineralization in the meninx spinal cord : mineralization in the arterial wall . mineralization in the arterial wall occurs occasionally in the meninx . fig . 179 . spinal cord : mineralization in the arterial wall spinal cord : pigment deposition in neuronal cells . large neuronal cells ( motor ventral horn cells ) occasionally contain yellow - brown pigments . fig . 180 . spinal cord : pigment deposition in neuronal cells sciatic nerve : renaut body . the irregular fine fibrous structure in the nerve fiber bundles is called the renaut body . its function is thought to be protection of the peripheral nerve fibers from pressure damage . focal interfiber fibrosis with decreased nerve fibers is thought to be an end - stage change of peripheral nerve damage . sciatic nerve : focal fibrosis eye : focal inflammatory cell infiltration in the conjunctiva . eye : focal inflammatory cell infiltration in the conjunctiva eye : focal inflammatory cell infiltration in the ciliary body . eye : focal inflammatory cell infiltration in the ciliary body eye : cataract . swelling and irregular arrangement of lens fibers are characteristic changes of cataract . the causes of this lesion may be congenital failures of optic fissure closure ( retinal coloboma ) , dysplasia of retinal structures or focal damage of the retina in the fetal developmental stage . eye : disarrangement of retinal structures lacrimal gland : focal inflammatory cell infiltration . lacrimal gland : focal inflammatory cell infiltration lacrimal gland : melanin pigment deposition . lacrimal gland : melanin pigment deposition skeletal muscle : focal inflammatory cell infiltration . fig . 190 . skeletal muscle : focal inflammatory cell infiltration skeletal muscle : focal fibrosis . focal interfiber fibrosis is probably a result of the degenerative and/or inflammatory lesions such as those shown in fig . fig . 191 . skeletal muscle : focal fibrosis skin : focal inflammatory cell infiltration in the dermis . accumulation of lymphoplasmacytes is commonly localized but frequently seen in the dermis , especially in the perivascular area . skin : focal inflammatory cell infiltration in the dermis stress - induced lesions ( figs . 193 - 195 ) some stress - induced lesions are observed in moribund or dead cynomolgus monkeys . lymphoid atrophy , acinar atrophy in the exocrine glands , gelatinous atrophy of the adipose tissue and diffuse hypertrophy of the adrenocortical cells of the zona fasciculata are known to be discriminative . the figures show thymic atrophy ( figs . 193 ) , gelatinous atrophy of the bone marrow adipose tissue ( figs . 194 ) and diffuse hypertrophy of the adrenocortical cells of the zona fasciculata ( figs . in veterinary pathology and toxicological pathology , many textbooks have been published , and information concerning the disease pathology in rodents and dogs is readily available . however , the publications referring to spontaneous lesions are substantially fewer in cynomolgus monkeys than those in other laboratory animals . furthermore , publications showing pictures of spontaneous nonneoplastic lesions exhaustively as a histopathology atlas can not be found except for a color atlas of diseases in nonhuman primates . therefore , we provided many pictures of spontaneous lesions in cynomolgus monkeys that were detected in background data collection studies and ordinary toxicity studies in our laboratory . the common lesions of the heart , such as focal inflammatory cell infiltration in the myocardium ( fig . arteritis in cynomolgus monkeys is usually observed in one or a few organs / tissues , and systemic arteritis is rare . morphologically , the features of arteritis in the previous report are similar to those in figs . 7 , 8 , 9 , 10 , 11 . testicular spermatogenesis of cynomolgus monkeys was reported in detail by dreef et al .. the female monkey genital system needs to be understood morphologically because the cyclic changes are different from those of other laboratory animals . cyclic changes in the ovaries of nonhuman primates during the menstrual cycle were reported in detail by koering , and common lesions in the female reproductive system such as deciduosis ( fig . ovarian tumors occasionally occur in monkeys , and some case reports refer to those in cynomolgus monkeys , including a report of mucinous cystadenoma ( fig . concerning the common lesions of cynomolgus monkeys , we introduced the reports of multinucleated epithelial cells in collecting tubules ( fig . 107 ) , adenohepatic fusion ( fig . 171 ) and focal inflammatory cell infiltration in the ciliary body ( fig . concerning the rare lesions of young cynomolgus monkeys , we also introduced the reports of thymoma ( fig . these background lesions may not have an effect on the results of toxicity studies but should be taken into account along with their potential to influence safety assessment in drug administration .

the purpose of our publication is to widely communicate pictures of spontaneous findings occurring in cynomolgus monkeys . focal lymphoplasmacytic infiltration is commonly seen in the general organs . the frequency and severity of these lesions may be influenced by the administration of drugs with an effect on the immune system . lymphoplasmacytic infiltration in the lamina propria of the stomach is also frequently seen in cynomolgus monkeys , and it is caused mainly by a helicobacter pylori infection . various degrees of brown pigments are observed in various organs , and it is possible to distinguish the material of the pigments by its morphological features and site . a focal / segmental glomerular lesion is occasionally seen in a section of the kidney , and the minimal lesion has no influence on the urinalysis . we showed the common glomerular lesions in he - stained sections , as well as in pam- or pas - stained sections , for understanding the details . young and pubertal monkeys are usually used in toxicity studies ; therefore , understanding various maturation stages of the genital system is important . in particular , the female genital system needs to be understood in the morphology , because their cyclic changes are different from other laboratory animals . thus , we present the normal features of the cyclic changes of the female genital organs . furthermore , we provide more information on spontaneous findings in cynomolgus monkeys for exact diagnoses in toxicity studies .

Introduction Materials and Methods Results Discussion

to achieve an accurate pathological evaluation in toxicity studies , it is particularly important to know the background histopathology , that is , to be familiar with pictures of incidental findings . therefore , most pathologists can not help but depend on the literature or textbooks to participate in the evaluation of monkey studies as a study pathologist or reviewing pathologist . in the present state , however , relatively few background data or pictures of incidental findings in monkeys have been published due to the lack of understanding of the importance of publishing incidental findings , which are pathologically or toxicologically insignificant spontaneous lesions with no sign of disease . therefore , in this report , we provide pictures of spontaneous lesions in cynomolgus monkeys ( macaca fascicularis ) that were detected in background data collection studies and ordinary toxicity studies in our laboratory . the animals were cared for according to the principles outlined in the guides for the care and use of laboratory animals prepared by the japanese association for laboratory animal science and our institution . we chose 195 typical findings or rare lesions of the cynomolgus monkeys from the background data collection studies and ordinary toxicity studies conducted in our laboratory , and they are shown as follows . focal infiltration of inflammatory cells mainly consisting of lymphocytes is frequently seen in the myocardium . heart : focal inflammatory cell infiltration in the myocardium heart : focal myocardial necrosis . this lesion must be distinguished from artifacts because a similar figure is also formed due to inadequate handling of the heart during necropsy and tissue preparation . heart : proliferation and squamous metaplasia of the epicardial mesothelium heart : hemorrhage in the endocardium . intimal and medial thickening of the arterial wall with duplication of the elastic lamina is rare in the coronary and mural arteries . arteritis is usually observed in one or a few organs / tissues , and systemic arteritis is rare in cynomolgus monkeys . panarteritis nodosa is seen relatively frequently in the intrarenal arteries , especially the arcuate arteries , in cynomolgus monkeys . arteritis in the submucosa of the intestinal tract is characterized by fibrinoid necrosis of the tunica media and mild inflammatory cell infiltration in the adventitia . arteritis in the bronchial artery is characterized by thickening of the tunica intima and inflammatory cell infiltration in the tunica intima and adventitia . accumulation of a large number of histiocytes containing yellow - brown pigment is common in the sinuses of the mesenteric lymph nodes . germinal center formation in the thymic medulla in cynomolgus monkeys is seen less frequently than in beagles . grossly , the lesion is detected as a protruded white patch in the subcapsular part . hyalinization ( deposition of eosinophilic substances ) is frequently seen in germinal centers of follicles in cynomolgus monkeys . spleen : hyalinization of the germinal center spleen : pigment deposition in the red pulp . for the most part , brown pigments are hemosiderin ( left ) , and black ones are unidentified ( right ) . accumulation of yellow - brown pigments occurs occasionally in the perivascular area of bone marrow tissue . these pigments are confirmed to be hemosiderin by special staining or reactions on the slide . the lymphoid tissue prominently developed in the nasal septa in this case , although lymphocytic infiltration is always seen in the lamina propria of the septal or turbinate mucosa . slight erosion , focal inflammation and/or focal squamous metaplasia of the respiratory epithelium are occasionally seen in the nasal septum and turbinate and are caused by an inserted nasogastric catheter used for the intragastric administration . accumulation of macrophages containing yellow - brown pigments ( probably hemosiderin ) is rarely seen in the perivascular interstitium or alveolar wall . fine black pigments are deposited in the interstitium around the bronchi in almost all cynomolgus monkeys . the incidence in cynomolgus monkeys is lower than that in other laboratory animals . lymphoplasmacytic infiltration is usually seen in the lamina propria , especially in the lingual papillae . most likely , it is a stage of the degenerative change such as erosion . foreign body granuloma caused by a fragment of hair stuck on the tongue occasionally occurs in the lamina propria or muscle layer . focal regenerative changes of muscle fibers occur occasionally in the lateral tongue muscle layer . tongue : regeneration of the muscle fiber esophagus : focal inflammatory cell infiltration in the lamina propria . esophagus : focal inflammatory cell infiltration in the lamina propria stomach : inflammatory cell infiltration in the lamina propria . lymphoplasmacytic infiltration in the lamina propria of the fundus and pylorus , so - called gastritis , is frequently seen in cynomolgus monkeys and is seen accompanied by regeneration of the mucosal epithelium . stomach : inflammatory cell infiltration in the lamina propria stomach : infection by helicobacter pylori in the gastric mucosa . many fine bacteria are seen in mucosal crypts in a warthin - starry - stained section with inflammatory changes . some fine bacteria are detected in parietal cells in a warthin - starry - stained section without any inflammatory changes in the infected mucosa . brown pigment - laden macrophages accumulate in the lamina propria at the top of the villi . slight pigmentation is usually seen in the duodenal lamina propria of cynomolgus monkeys , but an increase in the number of pigment macrophages might also occur following administration of certain pigmented compounds . similar changes are caused by dilatation of the endoplasmic reticulum filled with low electron density amorphous substances . small mineralized calculus occurs occasionally in a duct of a relatively large caliber and is frequently accompanied by lymphoplasmacytic infiltration in the surrounding parenchyma . these bodies are considered to be blood plasma due to rise of the internal pressure in the sinusoids or increased permeability of the hepatic cell membrane liver : eosinophilic inclusion bodies in hepatocytes liver : accumulation of glycogen in hepatocytes . this change is characterized by a clear appearance of the hepatic cytoplasm after formalin fixation . this change tends to occur in the border of the right and left central lobes , and it is thought to be tension lipidosis . microgranuloma consists of accumulation of inflammatory cells , mainly of macrophages , lymphocytes and small number of neutrophils , and may be associated with minute necrosis of hepatocytes . microgranulomas are of much lower incidence in cynomolgus monkeys than in other laboratory animals . brown pigments are presumably hemosiderin ( left ) , and black ones ( right ) are unidentified pigments like those described in the spleen . liver : pigment deposition in kupffer cells gall bladder : inflammatory cell infiltration in the lamina propria . development of the lymphoid tissue in the lamina propria occurs occasionally , and slight lymphoplasmacytic infiltration is constant in almost all cynomolgus monkeys . gall bladder : inflammatory cell infiltration in the lamina propria pancreas : ectopic splenic tissue . apoptosis of acinar cells is relatively rare in the pancreas of cynomolgus monkeys , in contrast to the larger number and higher frequency in beagles . the histopathologic features of chronic pancreatitis consist of necrosis and loss of acinar cells , dense fibrosis and lymphoplasmacytic infiltration in the exocrine pancreas , although the islets usually remain intact in the lesions . focal lymphoplasmacytic infiltration occurs occasionally in the interstitium of the pancreas , especially around the pancreatic duct . it has been described that similar angiectasis was induced in cynomolgus monkeys treated with an antineoplastic immunomodulator . kidney : ectopic adrenocortical tissue kidney : focal inflammatory cell infiltration in the interstitium . focal lymphoplasmacytic infiltration is common in the interstitium ; usually the lesion size is small . kidney : focal inflammatory cell infiltration in the interstitium kidney : regeneration of the tubular epithelium . focal regenerative changes of damaged tubules are detected as basophilic tubules with a high nuclear density in the renal cortex or outer medulla . brown pigment deposition is frequently seen in the tubular epithelium , mainly in the henle s tubules and straight portions of proximal tubules . small mineralization is usually seen in the interstitium of the renal papilla and also occurs occasionally in the cortex and outer medulla . renal mineralization is found in various laboratory animals but to a lesser extent in cynomolgus monkeys . oxalate is contained in various plants eaten by wild cynomolgus monkeys , and oxalate crystals are common among them but are very rare in animals bred for laboratory use . this change occurs in collecting tubules of the renal papilla in some cynomolgus monkeys , although its pathological meaning and pathogenesis are unknown . interstitial edema without inflammation is frequently seen in the renal papilla but is restricted to a small area . chronic pyelonephritis is tubulointerstitial nephritis consisting of fibrosis and inflammatory cell infiltration in the interstitium , tubular atrophy and thyroid - like appearance , extra - afferent fibrosis of the bowman s capsule and obsolescent glomeruli . 97102 ) these lesions are usually seen in cynomolgus monkeys but are solitary ( one or two abnormal glomeruli in one section ) . the details of these changes are not understood because every one of the lesions is stained eosinophilic in he sections . one or a few obsolescent glomeruli in a section of the kidney are frequently seen in cynomolgus monkeys . the characteristic features of mesangiocapillary glomerulonephritis , such as the double contour ( arrows ) of the basement membranes caused by mesangial interposition , are observed in a pam - stained section . kidney ( pelvis ) : eosinophilic droplets in the transitional epithelium urinary bladder : focal inflammatory cell infiltration in the lamina propria . urinary bladder : focal inflammatory cell infiltration in the lamina propria testis : immature ( grade 3 ) . the features of grade 4 , the most immature grade , consist of only sertoli cells and undifferentiated spermatogonia . seminiferous tubules consisting of sertoli cells , spermatogonia , and spermatocytes are occasionally dilated segmentally in the mature testis . it may be difficult to distinguish these spontaneously occurring changes from some chemical- or drug - induced changes in the seminiferous tubules . this change is caused by disturbance of spermatozoa flow due to degenerative changes of the seminiferous tubule . this body is thought to be derived from degenerated cells or secretions containing protein , and it does not have any important pathological significance . the change can be seen in all phases of maturation but can not be seen in the mature testis . focal accumulation of spermatozoa ( spermatocele ) occurs occasionally in the seminal vesicle of monkeys after genital maturation , and the accumulated spermatozoa might often be mineralized . immature female reproductive tissues are characterized by a thin endometrium ( less than 50% of the mature uterine basal layer ) and thin myometrium in the uterus , a small ovary containing a lot of primordial follicle and a few graafian follicles and thin or no keratinized squamous epithelium in the vagina . it is necessary to distinguish this immature figure from atrophy of the female reproductive system because similar figures are also seen under cachectic conditions . various numbers of mineralized foci are frequently seen in the ovarian cortex lined by many primordial follicles , especially in young cynomolgus monkeys . remnants of the rete ovarii are frequently seen in the hilum of the ovary and occasionally undergo hyperplastic changes . ovarian tissue consisting of primordial follicles , primary follicles , preantral follicles and surrounding stromal cells occurs occasionally in the muscle layer or serosa of the uterus ( left ) but rarely in the urinary bladder ( right ) . endometriosis externa in the intestinal wall , greater omentum and mesenterium has been reported in older cynomolgus monkeys , and we have never observed it in young animals . melanin pigment deposition ( melanosis ) can be seen in various organs in cynomolgus monkeys . uterus : focal inflammatory cell infiltration in the endometrium uterus : dilatation of the endometrial gland . melanin pigment deposition ( melanosis ) can be seen in various organs in cynomolgus monkeys . pituitary cysts are frequently seen in each lobe such as the anterior ( left ) , intermediate ( middle ) and posterior ( right ) lobes but are often found in the anterior lobe . generally , c - cells are not easily recognizable in the thyroid of cynomolgus monkeys , in contrast to those in beagles . in this figure , the c - cell hyperplastic focus is small , but even a small focus appears very rarely in cynomolgus monkeys . focal follicular atrophy , desquamation of follicular epithelia and infiltration of macrophages in follicles are frequently seen in the thyroid of cynomolgus monkeys . focal lymphoplasmacytic infiltration , often with lymph follicle formation in the interstitium , is frequently seen . the incidence and degree of this lesion are lower in cynomolgus monkeys than in beagles . this cyst is a remnant of the embryonal duct connecting the parathyroid - thymus tissue in the iii and iv pharyngeal pouches . generally , each of the three adrenocortical zones is obviously distinguishable in cynomolgus monkeys because cells of the zona fasciculata contain numerous fine vacuoles causing a very clear appearance of the cytoplasm . basophilic granules are thought to be rough endoplasmic reticulum , and these changes are the same as for hypertrophic cortical cells under stress conditions . the change may be dystrophic mineralization presumably caused by preexisting involution , hemorrhage and/or fibrosis in the inner fetal layer during postnatal development . grossly , the right adrenal is attached to the right lobe of the liver . brain : focal inflammatory cell infiltration in the meninx brain : focal inflammatory cell infiltration in the choroid plexus . sciatic nerve : focal fibrosis eye : focal inflammatory cell infiltration in the conjunctiva . the causes of this lesion may be congenital failures of optic fissure closure ( retinal coloboma ) , dysplasia of retinal structures or focal damage of the retina in the fetal developmental stage . skeletal muscle : focal fibrosis skin : focal inflammatory cell infiltration in the dermis . accumulation of lymphoplasmacytes is commonly localized but frequently seen in the dermis , especially in the perivascular area . skin : focal inflammatory cell infiltration in the dermis stress - induced lesions ( figs . 193 - 195 ) some stress - induced lesions are observed in moribund or dead cynomolgus monkeys . lymphoid atrophy , acinar atrophy in the exocrine glands , gelatinous atrophy of the adipose tissue and diffuse hypertrophy of the adrenocortical cells of the zona fasciculata are known to be discriminative . however , the publications referring to spontaneous lesions are substantially fewer in cynomolgus monkeys than those in other laboratory animals . furthermore , publications showing pictures of spontaneous nonneoplastic lesions exhaustively as a histopathology atlas can not be found except for a color atlas of diseases in nonhuman primates . therefore , we provided many pictures of spontaneous lesions in cynomolgus monkeys that were detected in background data collection studies and ordinary toxicity studies in our laboratory . the common lesions of the heart , such as focal inflammatory cell infiltration in the myocardium ( fig . arteritis in cynomolgus monkeys is usually observed in one or a few organs / tissues , and systemic arteritis is rare . morphologically , the features of arteritis in the previous report are similar to those in figs . testicular spermatogenesis of cynomolgus monkeys was reported in detail by dreef et al .. the female monkey genital system needs to be understood morphologically because the cyclic changes are different from those of other laboratory animals . cyclic changes in the ovaries of nonhuman primates during the menstrual cycle were reported in detail by koering , and common lesions in the female reproductive system such as deciduosis ( fig . ovarian tumors occasionally occur in monkeys , and some case reports refer to those in cynomolgus monkeys , including a report of mucinous cystadenoma ( fig . concerning the common lesions of cynomolgus monkeys , we introduced the reports of multinucleated epithelial cells in collecting tubules ( fig . concerning the rare lesions of young cynomolgus monkeys , we also introduced the reports of thymoma ( fig . these background lesions may not have an effect on the results of toxicity studies but should be taken into account along with their potential to influence safety assessment in drug administration .

evasion of apoptosis or programmed cell death , a key regulator of physiological growth control and regulation of tissue homeostasis , is a hallmark of cancer and a contributor to the emergence of resistance to current therapies . the b - cell lymphoma-2 ( bcl-2 ) family of proteins regulate the intrinsic ( mitochondrial ) pathway of apoptosis through a network of protein protein interactions between pro- and antiapoptotic members . twenty - five known members of the family can be grouped functionally according to their pro- and antiapoptotic effects as well as structurally according to their bcl-2 homology ( bh ) regions . the bcl-2 antiapoptotic proteins , consisting of bcl-2 , bcl - xl , bcl - b , bcl - w , mcl-1 , and a1 , share up to four bh domains which form the hydrophobic bh3-binding groove for binding their cognate partners . the pro - apoptotic proteins are divided into two groups : ( a ) multidomain proteins including bax and bak with bh1bh4 domains and ( b ) bh3-only proteins including bad , bid , bim , noxa , and puma , which share homology only in the bh3 -helical domain . the bh3 domain possesses four conserved hydrophobic residues that are involved in the interaction with bh3-binding groove of the pro - survival bcl-2 family members , which results in the sequestering and blocking of the function of pro - death members . overexpression of bcl-2 survival members is observed in different types of human tumor samples and cancer cell lines , and much effort has been focused on developing therapeutics against this family of proteins for the treatment of cancers . selective and potent small - molecule inhibitors have been successfully developed against bcl-2 , bcl - xl , and bcl-2/bcl - xl with navitoclax ( abt-263 ) , currently in phase i / ii clinical trials . the efficacy of abt-263 as a single agent has been demonstrated in tumors with low levels of the pro - survival mcl-1 . several studies have shown that resistance to abt-263 and its analogue abt-737 is linked to high expression levels of mcl-1 , and in many instances this resistance can be overcome by treatment with agents that downregulate , destabilize , or inactivate mcl-1 . the biological significance of mcl-1 protein expression in support of cell survival has been well documented in a number of cell systems , including human myeloblastic leukemia , myeloma , b - lymphoma , nonsmall cell lung , melanoma , pancreatic , and prostate . it has been shown that mcl-1 down - regulation is important toward making multiple myeloma cells susceptible to bh3-only proteins and therefore to mitochondrial disruption . such down - regulation can increase the sensitivity to rituximab - mediated killing of chronic and acute lymphoid leukemia ( cll and all ) . antisense strategies targeting mcl-1 in vitro and in vivo have provided promising results in sensitizing human melanoma and pancreatic cancer . these data suggest that therapies specifically targeting mcl-1 , either as a single agent or in combination , can be effective in the treatment of different human cancers . recently , several groups including us have reported on small molecules and stapled peptides as mcl-1 inhibitors . herein we disclose a novel series of small - molecule mcl-1 inhibitors discovered through high - throughput screening ( hts ) followed by the utilization of structure - based design to develop structure activity relationships ( sar ) around lead compound 1 ( figure 1a ) . docking studies and two - dimensional h n heteronuclear single quantum coherence spectroscopy ( hsqc ) nmr studies were employed to provide information about its binding mode which was used for the design and synthesis of additional analogues . a sar was developed which resulted in the identification of several selective small - molecule mcl-1 inhibitors with improved binding . selected analogues were then tested in a series of complementary biochemical , biophysical , functional , and cellular assays to evaluate their potency , specificity , and mechanism of action . ( a ) structure of the hts lead compound 1 . ( b ) putative binding mode of 1 to mcl-1(pdb i d : 2nla ) . significant shift ( > 0.09 ppm ) is represented with purple , moderate shift ( 0.03 and 0.09 ppm ) represented with pink . h hsqc spectra of mcl-1 ( red ) and in the presence of 1 ( mcl-1:1 ratio of 1:2 ) ( black ) , ( mcl-1:1 ratio of 1:1 ) ( purple ) . ( d ) plot of chemical shift changes of mcl-1 amide upon addition of 1 ( mcl-1:1 ratio of 1:2 ) as a function of mcl-1 residue numbers . hts of a 53.3k small - molecule library was performed using a fluorescence polarization ( fp ) binding assay based on the interaction between recombinant human mcl-1 and fluorescently labeled bid bh3 peptide ( flu - bid ) . compound 1 ( figure 1a ) was one of the validated hits , which was also previously identified as a proteasome inhibitor . compound 1 was resynthesized and its binding to mcl-1 was confirmed with a ki of 1.55 0.18 m . compound 1 exhibits similar potency as mim1 ( ic50 = 4.72 m ) , mcl-1 small - molecule inhibitor with a thiazolyl substituted core , which was also discovered with high - throughput competitive fp screen approach . it is known that the binding efficiency index ( bei ) is important in identifying leads that exhibit good potency relative to their size . the bei of compound 1 , calculated as a ratio between pki and molecular weight , is 14.7 , which encouraged us to further pursue with modifications of this scaffold . to explore the binding mode of 1 with mcl-1 , in silico induced fit docking ( ifd ) studies were performed using the crystal structure of mcl-1 in complex with mnoxa bh3 peptide ( pdb i d : 2nla ) . the predicted binding model of 1 in complex with mcl-1 revealed that the thiophene and the naphthalene rings of 1 occupy two hydrophobic pockets , h2 and h3 , in the mcl-1 protein , mimicking two conserved hydrophobic residues in the bh3 binding motif represented by leu 78 and ile 81 , respectively , in mnoxab ( figure 1b ) . the importance of the two conserved hydrophobic residues , leu and ile , in the bh3 binding motif for the high affinity interaction and selective binding to mcl-1 has been demonstrated with structural studies of bims2a , a highly selective peptide derived from bim bh3-only protein , as well as from recently reported selective small - molecule inhibitor in complex with mcl-1 . the carboxylic acid group of 1 forms a network of hydrogen bonds with arg 263 and asn 260 of mcl-1 , mimicking the conserved asp in bh3 peptides ( asp 83 in mnoxa ) . the predicted binding model also suggests that the phenolic group forms a hydrogen bond with his 224 , which is one of the residues composing the h3 pocket of mcl-1 . a recent report on the conformational flexibility of mcl-1 and its binding hotspots identified his 224 as an acidic hotspot in the h3 site of mcl-1 , supporting the predicted hydrogen bonding in this region of mcl-1 . to validate the computationally predicted binding site and confirm the binding of 1 to the bh3 groove of mcl-1 protein , hsqc nmr spectroscopy studies were performed . for this purpose , the assignment of the backbone amides of apo human mcl-1 was based on the work by liu et al . , and the hsqc spectra were of good quality with well - dispersed peaks , and concentration - dependent perturbations of residues were observed ( figure 1c ) , indicating that 1 binds mcl-1 specifically and causes dose - dependent perturbations of backbone amides . the chemical shift changes in the presence of a 2-fold excess of 1 were mapped and plotted against mcl-1 residues ( figure 1d ) . compound 1 caused moderate to significant chemical shift perturbations for residues of met 231 , met 250 , leu 267 , and phe 270 , which constitute the h2 and h3 pockets and which are predicted to be occupied by the thiophene and naphthalene rings of 1 , respectively . moderate chemical shift perturbations of arg 263 and his 224 were also observed , which are predicted to form hydrogen bonds with the thioacidic acid and phenolic moieties of 1 , respectively . additionally , the residues in the vicinity of the predicted binding pose ( leu 232 , val 243 , arg 248 ) and the ones located on an unstructured loop connecting -helix 3 to -helix 4 ( lys 234 , leu 235 , lys 238 , asn 239 ) were also perturbed . overall analysis of the chemical shift changes of the compound 1 in complex with mcl-1 showed that 1 affects the residues forming the bh3-binding groove and provided conclusive evidence that 1 binds mcl-1 protein at the same site that the conserved bh3 peptides interact with mcl-1 protein . therefore , on the basis of our modeling and nmr results , the substituted - n-(4-hydroxynaphthalen-1-yl)arylsulfonamide represents a promising class for further optimization . a structure - based design approach was undertaken , and a focused library of analogues of 1 was designed and synthesized to improve the potency of this series . except for analogues 48 , 15 , and 39 , which were commercially available , all analogues were synthesized through a novel , modular route ( scheme 1 ) , which differs from that recently reported by ge et al . our route allows for facile access to a variety of analogues with variations at r1 , r2 , and r3 and the linker region ( x ) . it starts with an electrophilic aromatic substitution of 1-methoxy-4-nitronaphthalene with n - iodosuccinamide to provide aryl iodide ( 47 ) . this was subjected to pd - catalyzed c s or c c cross - coupling using conditions previously reported , or developed in our lab based on recent literature , to provide several desired intermediates ( 48 ) . the nitro function was reduced with iron or via catalytic hydrogenation to provide the corresponding amines . reaction of the amines with appropriate sulfonyl or acyl chlorides or 3-methyl-1-((4-phenylpiperazin-1-yl)sulfonyl)-1h - imidazol-3-ium provided the penultimate compounds ( 49 ) , which were demethylated with bbr3 followed by purification by trituration or reverse - phase hplc to afford the target compounds ( 13 , 914 , 1623 , 2526 , 2829 , 3138 ) of > 95% purity ( tables 13 ) . in the case of analogues with an ester side chain , the bbr3 step provided a convenient way to concomitantly hydrolyze the ester in a single pot . to preserve the methyl ester of analogue 32 ( table 3 ) , analogue 41 ( table 4 ) was subjected to bbr3 conditions followed by a quench with meoh . for the synthesis of 49o , intermediate 49k underwent suzuki miyaura coupling with phenyl boronic acid to provide the desired compound which was subjected to bbr3 to provide 17 ( table 1 ) . aminolysis of 41 and 49s with nh4oh provided carboxamides 42 ( table 4 ) and 49aa , respectively . the terminal amide of 42 and 49aa were converted by a known procedure to tetrazoles 49cc and 49dd , which after bbr3 demethylation provided 36 and 37 ( table 3 ) , respectively . the thioacidic acid analogues with a methoxy at r3 ( 40 , 4344 ) ( table 4 ) were obtained via lioh hydrolysis of esters 41 , 49n , and 49o , respectively . acetylation of 10 ( table 1 ) with acetyl chloride provided 46 ( table 4 ) . analogue 24 ( table 1 ) with a phenyl scaffold in place of naphthalene was synthesized using conditions similar to those described for 10 starting from 2-iodoanisole ( supporting information scheme s1 ) . the syntheses of compounds 27 ( table 2 ) and 30 ( table 3 ) were completed starting from 4-methoxy-1-naphthaldehyde and 1-nitronaphthalene , respectively ( supporting information schemes s2s3 ) . reagents and conditions : ( a ) nis , tfa , reflux , 24 h ; ( b ) hs(ch2)ncooch3 ( n = 1 , 2 ) , pd(oac)2 , xantphos , cs2co3 , lii , zncl2 , thf , 60 c , overnight , or hs(ch2)3ch3 , pd2(dba)3 , dppf , et3n , nmp , 80 c , 2 h , or hcc(ch2)noh ( n = 1 , 2 ) , pd(pph3)2cl2 , cui , et3n / thf , 60 c , 2 h ( 4:1 ) , 60 c , 2 h ; ( c ) fe , acoh , 70 c , 1 h , or pd / c , h2 30 psi , etoh / etoac ( 6:1 ) , rt , overnight ; ( d ) rso2cl , pyridine , ch2cl2 , rt , overnight , or rcocl , et3n , ch2cl2 , rt , overnight , or , ch3cn , 80 c , 15 h ; ( e ) bbr3 , ch2cl2 , 0 c to rt , 1 h , or bbr3 , ch2cl2 , 0 c to rt , 1 h , quench with meoh at 0 c ; ( g ) phenyl boronic acid , pd(pph3)4 , na2co3 , thf / h2o , 60 c , 2 h ; ( h ) nh4oh , rt , 1 h ; ( i ) nan3 , sicl4 , ch3cn , 80 c , 15 h ; ( j ) lioh , thf , rt , 1 h ; ( k ) h3ccocl , et3n , 0 c , rt , 30 min . structure - based design of analogues based on 1 yielded a focused library of compounds leading to clear sar for this series . the binding affinities of our mcl-1 inhibitors were determined by using competitive fluorescence polarization ( fp ) and surface plasmon resonance ( spr ) binding assays , which test the ability of inhibitors to disrupt interaction between mcl-1 and two different bh3 peptides , fluorescently labeled bid and biotin - labeled bim , respectively . concurrently , hsqc nmr experiments were performed to provide structural insights of protein - bound ligand and experimental validation for the modeling studies . the predicted binding model showed that the thiophene ring at r1 of 1 projects into the h2 pocket ( figure 1b ) , which is the biggest and deepest pocket among the four hydrophobic pockets of mcl-1 . to investigate the importance of hydrophobic interaction at this site and increase the binding affinity of 1 , a series of analogues with variation at r1 was synthesized and evaluated ( table 1 ) . when r1 is changed to a methyl group in 2 , the binding affinity is significantly reduced , confirmed by spr ( ic50 > 100 m ) and nmr experiments which showed lack of chemical shift perturbation of backbone residues in the mcl-1 bh3 binding site after adding 2 ( supporting information figure s1 ) . as was expected , isosteric replacement of the thiophene in 1 to a phenyl in 3 maintained binding affinity with ki of 3.56 0.45 m . to probe the hydrophobic interactions in the h2 pocket , analogues with alkyl and halogen substituents of various sizes at the para - position of the phenyl ring were prepared . from this set of compounds , a trend emerged showing improved binding with increasing size and hydrophobicity of substituents . tert - butyl phenyl ( 7 ) exhibited 4.5-fold enhancement in fp assay ( ki = 0.81 0.04 m ) and 1.5-fold in spr assay ( ic50 = 8.73 0.95 m ) , and bromo phenyl ( 10 ) 7-fold in fp assay ( ki = 0.49 0.06 m ) and 5-fold in spr assay ( ic50 = 2.40 0.17 m ) improved binding over 3 . introduction of a more polar methoxy group in 11 was accommodated but resulted in a 3-fold decreased binding ( ki = 9.31 3.1 m ) compared to 3 . bromine substitution at the meta-(12 ) and ortho - positions ( 13 ) of the phenyl ring was also explored , and the obtained binding results from both assays , fp ( ki of 2.35 0.19 m and 4.42 0.86 m , respectively ) and spr ( ic50 = 9.68 0.88 m and ic50 = 6.18 1.24 m , respectively ) , indicated that para - bromo substitution in 10 is the best , exhibiting the highest binding affinity to mcl-1 among these three isomers . because of peak overlap and possibility of ambiguous assignment of the peaks in the presence of a ligand and to improve the quality and accuracy of ligand - induced mcl-1 chemical shift perturbation , resonance assignments were also determined for 10 in complex with mcl-1 using c , n double - labeled mcl-1 protein in the presence of a 2-fold excess of 10 . the hsqc spectrum of 10 showed a similar pattern of chemical shift perturbation as with 1 , which we attribute to the structural similarity of the two analogues . consistent with the 7-fold improved binding of 10 in comparison with 1 , the chemical shift perturbations were larger for 10 ( supporting information figure s2a ) . in particular , significant perturbation of residues ser 247 , arg 248 , and val 253 on -helix 4 , which forms the upper rim of the h2 pocket , and residues val 216 , val 220 , and gln 221 located toward the c - terminus of -helix 2 , which are at the border between the h3 and h4 pockets , were observed for 10 , suggesting that the mcl-1 conformational flexibility accommodates larger r1 substituent into the h2 pocket . chemical shift perturbation plots derived from h , n - hsqc experiments of the other two bromophenyl isomers showed a similar perturbation pattern as 10 with chemical shift changes that correlate well with the binding data in which the strongest perturbation is observed for 10 , followed by 12 and 13 ( supporting information figure s2a c ) . to further extend into the h2 pocket and gain additional interaction , analogues with biphenyl substituents analogues 16 and 17 , with para- and meta - biphenyl substituents , respectively , showed a similar 10-fold improvement in fp - based binding assay compared to 3 with ki values of 0.37 0.10 and 0.38 0.03 m , respectively , further confirmed with spr assay showing 6-fold improvement . on the other hand , the ortho - biphenyl analogue 18 showed almost the same binding affinity as 3 with ki of 2.03 0.41 m and ic50 = 9.90 3.50 m , in fp and spr assays , respectively . the predicted binding models of these compounds showed that the distal phenyl ring of 16 and 17 inserts deeper into the h2 pocket ( parts a and b of figure 2 , respectively ) . for 18 , this phenyl ring is partially solvent exposed ( figure 2c ) , which might explain its lower affinity compared to 16 and 17 . in agreement with their binding affinities , chemical shift perturbation plots of 16 and 17 show stronger perturbation of residues involved in the binding site compared to 18 ( figure 2d f ) . putative binding modes of ( a ) 16 , ( b ) 17 , ( c ) 18 to mcl-1 ( pdb i d : 2nla ) . significant shift ( > 0.09 ppm ) is represented with purple , moderate shift ( 0.03 and 0.09 ppm ) represented with pink . plots of chemical shift changes of mcl-1 amide upon addition of ( d ) 16 ( mcl-1:16 ratio of 1:2 ) , ( e ) 17 ( mcl-1:17 ratio of 1:2 ) , ( f ) , and 18 ( mcl-1:18 ratio of 1:2 ) as a function of mcl-1 residue numbers . analysis of the hsqc spectra of analogues 1618 resulted in an important finding that strongly supports their predicted binding mode . one long - standing question for us was the possibility of a flipped binding mode for this class of analogues where r1 would occupy the h4 pocket instead of h2 . the possibility of a flipped conformation has been previously documented for a different class of dual mcl-1 and bcl - xl inhibitors . the differential chemical shift mapping method was applied , using the three biphenyl analogues ( 16 , 17 , and 18 ) differing only in the orientation of distal phenyl ring . comparison of the spectra of the mcl-1 bound to these three analogues shows distinct changes in chemical shifts exclusively for the resonances of the residues that are in direct contact with the modified structure of the inhibitors , facilitating binding site mapping . careful analysis of the hsqc spectra of these analogues reveals a different direction of the chemical shift perturbations of the residues forming the h2 pocket ( met 250 ) or in its vicinity ( phe 228 ) as well as residues located on -helix 4 ( val 249 , val 253 ) ( figures 3a c ) . on the other hand , residues which are not part of the h2 pocket , such as leu267 , val265 , his224 , and arg263 , show very similar chemical shift perturbations in the presence of the biphenyl analogues ( supporting information figure s6 ) . this finding provides conclusive evidence for biphenyl occupation of the h2 pocket and further supports that -helix 4 of mcl-1 is flexible . overlaid n h hsqc spectra of mcl-1 ( red ) and in the presence of 16 ( mcl-1:16 ratio of 1:2 ) ( purple ) , 17 ( mcl-1:17 ratio of 1:2 ) ( blue ) , 18 ( mcl-1:18 ratio of 1:2 ) ( green ) for ( a ) phe 228 , ( b ) met 250 , and ( c ) val 249 and val 253 . ( d ) overlay of putative binding modes of 16 ( purple ) , 17 ( blue ) , and 18 ( green ) to mcl-1 ( pdb i d : 2nla ) highlighting in red val 249 , met 250 , and val 253 on helix 4 , phe 228 on helix 3 of mcl-1 . because of encouraging binding data with 16 , analogues 19 and 20 with halogenated biphenyl rings at r1 were synthesized to further increase potency . satisfyingly , 19 with para - chlorobiphenyl showed a 21-fold improved binding compared to 3 , becoming the most potent analogue in our series , with a ki = 0.17 0.04 m determined by the fp binding assay using fluorescent labeled bid bh3 peptide and ic50 of 0.88 0.15 m in displacement of biotin labeled bim bh3 peptide in the spr - based assay ( 14-fold improvement ) . the chemical shift perturbation plot of 19 ( supporting information figure s3 ) showed a similar pattern of perturbations to 16 , suggesting a similar binding conformation but with a lower perturbation magnitude , likely due to the lower solubility of 19 in our nmr studies . to improve the physicochemical properties of the biphenyl analogues , we synthesized and evaluated analogues 21 with para - phenoxyphenyl at r1 and its fluorine substituted congener , 22 . analogue 21 showed similar binding affinity as 19 ( ki = 0.18 0.05 m in fp , and ic50 = 1.45 0.29 m in spr ) but stronger chemical shift perturbation ( figure 4a c ) , probably due to its improved solubility . computational docking of 21 predicted a stacking of the distal phenyl of the para - phenoxyphenyl moiety with the phenyl of phe270 of mc-1 ( figure 4d ) , which could account for its improved affinity . compounds 19 and 21 became the most potent analogues of our series and exhibited bies of 13.5 and 14.0 , respectively , maintaining 1 s bie . in comparison with compound 53 ( ki of 0.055 m ) , recently reported mcl-1 selective inhbitor with an indole core structure , discovered and optimized by fragment - based screening strategy,19 and 21 have binding affinity in a similar nanomolar range , being 3-fold less potent than 53 . to further increase the aqueous solubility of this class of analogues , a para - phenylpiperazine group at r1 was introduced in 23 which led to a 6-fold decrease in binding based on fp assay ( ki = 2.22 0.36 m ) and 8-fold decrease based on spr assay ( ic50 = 16.20 5.80 m ) in comparison with the biphenyl substituent in 16 . this can be attributed to a relatively polar substitutent projecting into the hydrophobic h2 pocket and to a less optimal conformational preference of phenylpiperazine versus biphenyl for the h2 pocket . ( a ) surface of the mcl-1 protein ( pdb i d : 2nla ) is colored according to the chemical shift intensity . significant shift ( > 0.09 ppm ) is represented with purple , moderate shift ( 0.03 and 0.09 ppm ) represented with pink . ( b ) plot of chemical shift changes of mcl-1 amide upon addition of 21 ( mcl-1:21 ratio of 1:2 ) as a function of mcl-1 residue numbers . ( c ) overlaid n h hsqc spectra of mcl-1 ( red ) and in the presence of 21 ( mcl-1:21 ratio of 1:2 ) ( black ) , ( mcl-1:21 ratio of 1:1 ) ( purple ) . ( d ) mcl-1 residues shown to be perturbed in hsqc nmr in the presence of 21 ( mcl-1:21 ratio of 1:2 ) ( green ) . to briefly explore the importance of the naphthalene core to the binding potency , which occupies the h3 hydrophobic pocket , analogue 24 with a phenyl core was synthesized . binding studies showed a significant drop in potency ( ki = 283.36 81.50 m in fp and ic50 > 100 m in spr ) compared to the corresponding compound 10 with the naphthalene core , supporting the importance of hydrophobic interactions at h3 to the overall mcl-1 binding . the next focus of our sar studies was the sulfonamide linker for which docking results suggested lack of any specific interactions with mcl-1 . to investigate the importance of the sulfonamide linker , replacement of the sulfonamide with a carboxamide in 25 led to significant 83-fold decrease of the binding affinity with ki value of 40.8 8.50 m compared to 10 , possibly due to the unfavorable orientation of r1 by the carboxamide linker . the decreased binding potency was confirmed by spr and nmr ( supporting information figure s4 ) binding studies . to explore the impact of the flexibility of the linker , a methylene linker distal ( 26 ) or proximal ( 27 ) to the naphthalene core was inserted and both analogues showed decreased binding affinity with ki = 5.23 0.36 m and ki = 64.75 12.61 m , relative to 9 ( 6-fold ) and 3 ( 18-fold ) , respectively . as expected , change of the sulfonamide linker in 26 to a carboxamide in 28 decreased the potency , but only by 2-fold ( ki = 12.53 1.24 m ) , suggesting that the insertion of the methylene linker between the carboxamide and the pendant aryl ring provids a degree of freedom to better orient r1 into h2 pocket of mcl-1 . modeling studies showed that the thioacetic acid moiety at r2 mimics the conserved asp of bh3-only peptides and that the carboxylate is involved in electrostatic interaction with asn 260 and arg 263 of mcl-1 . therefore , to further explore this site , analogues with different substituents at r2 were synthesized ( table 3 ) . removal of the acid side chain ( 30 ) or its replacement with thiobutyl ( 31 ) did not show binding up to 100 m in both fp and spr assays . introducing a methyl ester ( 32 ) or a primary carboxamide ( 33 ) resulted in decreased binding by 10-and 3-fold in fp assay respectively compared to 10 ( 6- and 2-fold decrease in spr assay respectively ) , consistent with each of these functional groups , forming a weaker interaction with arg 263 when the carboxylic acid was changed to an alcohol and the sulfur to methylene ( 34 ) or ethylene ( 35 ) , both analogues showed decreased binding compared to the parent congener 10 with ki values of 3.79 0.62 and 2.18 0.91 m , respectively . as was expected , bioisosteric replacement of the carboxylic acid group with a tetrazole in analogues 36 and 37 maintained the binding affinity in comparison with their parent congeners , 10 and 21 . homologation of the thioacetic acid in 38 had no detrimental effect on binding and showed a similar ki of 1.13 0.34 m in fp assay and ic50 = 5.03 1.88 m in spr assay as 15 , which can be explained with the flexibility of both the thiopropanoic acid moiety and arg 263 side chain . however , changing the point of fusion of the naphthalene ring in 38 substantially affected the binding and 39 with 1-naphthyl substituent showed a significant reduction in binding to mcl-1 . our docking studies suggest that this might be attributed to a clash with the residues in the h2 pocket of mcl-1 . nd : the ic50 was not determined because the compound showed nonspecific binding to the control surface ; * compounds were tested up to 100 m . modeling showed that the phenolic group of the core naphthalene scaffold forms a hydrogen bond with his 224 of mcl-1 , consistent with the reported acidic hotspot in the h3 site of mcl-1 close to his 224 . several analogues were synthesized to probe the contribution of the phenolic group to binding to mcl-1 ( table 4 ) . when the hydroxyl group is changed to a methoxy in analogue 40 , the potency decreased by 170 fold ( ki = 86.98 15.35 m ) compared to the phenolic congener 10 . the analogue 41 , where r2 is methyl thioacetate and r3 is methoxy , did not show binding up to 100 m , and the loss of the binding was confirmed with spr and hsqc experiment ( supporting information figure s5 ) . similar significant loss of binding is also apparent in compounds 42 to 45 compared to their corresponding phenolic analogues , clearly indicating the importance of the phenolic group to the overall binding to mcl-1 . compound 46 , where the hydroxyl group was acetylated exhibited a ki of 33.97 15.96 m , which is a 69-fold decrease in binding compared to 10 . however , 46 showed improved binding relative to 40 , which might be attributed to the formation of a hydrogen bond between the carbonyl of the acetyl group and the protonated his 224 , supported by computational prediction . we determined the selectivity of this class of compounds against four other bcl-2 antiapoptotic proteins ( bcl-2 , bcl - xl , bcl - w , bfl-1/a1 ) . the most potent analogues were tested in competitive fp - based assays that were optimized for each protein , and ki values were calculated using equations developed previously ( table 5 ) . in general , all the analogues inhibit mcl-1 most potently with the following order of selectively : bfl-1/a1 > bcl - w > bcl-2 > bcl - xl . as the bh3 domain binding profile of bfl-1/a1 , as well as its bh3 binding groove , is most similar to that of mcl-1 , it is not surprising that the tested compounds showed less selective inhibition of a1 . the most potent analogues in this series , 19 and 21 , show a profile for selectively inhibiting mcl-1 with 7-and 19-fold versus bfl-1/a1 , 8-and 9-fold versus bcl - w , 36- and 42-fold versus bcl-2 , and 56- and 59-fold versus bcl - xl , respectively . other reported selective mcl-1 inhibitors , mim1 and 53 , also show high selectivity against bcl - xl ( ic50 > 50 m and ki > 15 m , respectively ) , while 19 and 21 show better selectivity against bcl-2 in comparison with 53 which has 16-fold selectivity with ki value of 0.87 m . to verify the specific binding of novel inhibitors to mcl-1 , we employed a pull - down assay using biotin - labeled noxa ( bl - noxa ) and whole cell lysate from the human breast cancer cell line 2lmp . as shown in figure 5 , mcl-1 was pulled down by bl - noxa and , as was expected , the bim bh3 peptide disrupted the interaction between bl - noxa and mcl-1 . preincubation with several mcl-1 inhibitors , 10 , 19 , and 21 , completely blocked the binding of bl - noxa to mcl-1 , similar to bim peptide , demonstrating that these inhibitors can recognize and specifically bind to the bh3 binding groove of endogenous mcl-1 protein . biotin - labeled noxa ( bl - noxa , 0.1 m ) was incubated with whole cell lysates of 2lmp cells with or without tested mcl-1 inhibitors and bim bh3 peptide as a positive control , followed by incubation with precleared streptavidin agarose beads . it is well established that bax and bak are required for the initiation of intrinsic , mitochondrial , apoptotic cell death , and they are maintained in an inactive state through interaction with the antiapoptotic proteins . therefore , cells subjected to inhibitors of antiapoptotic proteins are expected to undergo cell death in a bax / bak - dependent manner . to determine the contribution of bak and bax in the cell death induced by our mcl-1 inhibitors , we employed murine embryonic fibroblasts ( mefs ) wild - type ( wt ) and deficient in both bax and bak ( double knock out , dko ) . exposure of the wt mefs to 21 resulted in a concentration - dependent cell death ( assessed by pi staining ) , while bax / bak deletion significantly rescued cells from 21 induced cell death ( figure 6 ) . although 21 initiates cell death also in dko mefs ( 23% positive pi at 16 m ) , it is clear that 21 is more potent in wt mefs ( 61% positive pi at 16 m ) , indicating that bax and/or bak are involved in cell death induction . taken together , our data suggest that 21 induces cell death in a bax / bak - dependent manner due to its mcl-1 inhibitory function . cell death induced by mcl-1 inhibitor 21 is bax / bak - dependent . mefs deficient in bax and bak ( gray bars ) along with their wild - type counterpart ( black bars ) were exposed for 15 h to different concentrations of 21 , and the cell viability was assessed with pi staining . the significance was calculated using unpaired t test , and the number of data is shown for each tested concentration with corresponding significance : ( * * ) p < 0.01 and ( * * * ) p < 0.001 . to further confirm the specificity of our novel mcl-1 inhibitors and to determine whether different prosurvival bcl-2 proteins could suppress the apoptotic activities of novel mcl-1 inhibitors , we used reported cell lines developed by retroviral transduction of lymphoma cells isolated from e-myc transgenic mice which differ only in their expression of prosurvival bcl-2 family proteins . lymphoma cells overexpressing mcl-1 and bcl-2 were treated with varying concentrations of tested compounds for 1518 h , and then cell viability was determined by flow cytometry using a fluorescent reactive dye ( live / dead fixable violet stain kit ) . a selective inhibitor of bcl-2 , bcl - xl , and bcl - w , was used as a positive control . as predicted , lymphoma cells overexpressing mcl-1 were significantly sensitive to 19 and 21 as assessed by an increased percentage of cell death in a concentration - dependent manner . in contrast , 19 and 21 were ineffective against e-myc / bcl-2 lymphomas ( figure 7 ) . importantly , 41 did not show any activity against both cell lines overexpressing mcl-1 or bcl-2 , consistent with our binding studies which showed that 41 does not bind to mcl-1 . as expected , lymphoma cells overexpressing bcl-2 were sensitive to cell death induced by abt-263 , while cells overexpressing mcl-1 were insensitive to abt-263 , consistent with its binding specificity . collectively , these results , demonstrate that 19 and 21 specifically bind and inhibit mcl-1 and have no effect on bcl-2 , which is consistent with our biochemical data for their selectivity profiles . furthermore , this supports the concept that tumor cells addicted to mcl-1 protein will be the most sensitive target cell population for selective small - molecule mcl-1 inhibitors . sensitivity of e-myc lymphoma cells overexpressing mcl-1 and bcl-2 antiapoptotic proteins to inhibitor - induced cell death . e-myc / mcl-1 and e-myc / bcl-2 lymphomas were treated for 1518 h with increasing concentrations of 19 , 21 , 41 , and abt-263 . dead cells were assessed by live / dead fixable dead cell stain kit ( vivid ) . the significance was calculated using unpaired t test , and the number of data is shown for each tested concentration with corresponding significance : ( * ) is p < 0.05 , ( * * ) p < 0.01 , and ( * * * ) p < 0.001 . we next evaluated our most potent compounds for their ability to inhibit cell growth in the leukemia cell lines hl-60 , mv4,11 , and k-562 ( figure 8) . it has been shown that aml - derived cell lines , hl-60 and mv4,11 , are sensitive to inhibition of the antiapoptotic protein mcl-1 , while cml - derived k-562 cell line is less sensitive to mcl-1 inhibition . tested compounds from our series showed inhibition of the cell growth in a dose - dependent manner with similar potencies , ic50 values ranging from 2.06 to 11.72 m against the hl-60 and mv4,11 cell lines ( figure 8a ) . interestingly , these compounds showed decreased ability to inhibit the cell growth of k-562 , with ic50 values of 11.76 to 29.89 m . compound 41 , which does not bind to mcl-1 , did not show inhibition up to 50 m . it is important to be pointed out that 36 and 37 , where the carboxylic acid was replaced with a bioisostere tetrazole group , show similar cellular activity in all tested cell lines in comparison with their parent compounds , 10 and 21 , respectively . these results demonstrate that the tetrazole group can effectively replace the acid group , achieving not only the same binding affinity to mcl-1 but also comparable cellular activity . furthermore , compound 32 , in which the acid group has been replaced with methyl ester and showed 10-fold less binding to mcl-1 as compared with 10 , has also similar ic50 values in tested cell lines as 10 . this is probably due to enzymatic hydrolysis of the methyl ester group in the cells and releasing the corresponding compound with free acid group , thus functioning as a pro - drug . these data also demonstrate that the compounds with free acidic group in this series are cell permeable , although the mechanism of their cell permeability need to be further studied . compounds 21 and 37 were evaluated for their ability to induce apoptosis in the hl-60 cell line using annexin - v and propidium iodide ( pi ) double staining by flow cytometry ( figure 8b ) . treatment of the hl-60 cells by 2.5 , 5.0 , and 10 m of 21 and 37 for 20 h results in 28.9% and 23.5% , 37.8% and 51.6% , and 78.4% and 74.2% of apoptotic cells ( early + late ) , respectively , as compared to 7.6% and 7.3% of apoptotic cells in the dmso controls . these results further confirmed that these two analogues have similar cellular activity , and tetrazole can effectively replace the acid group . to determine if the induction of apoptosis by compound 37 in the hl-60 cell line depends upon caspases , we treated the cells with compound 37 alone or in the presence of z - ved - fmk , a pan - caspase inhibitor ( figure 8b and supporting information figure s7 ) . the obtained results showed that the induction of apoptosis was significantly inhibited in the presence of z - vad - fmk , clearly indicating that the apoptotic activity of compound 37 is mediated by caspases . of note , z - vad - fmk alone has no effect on cells ( supporting information figure s7 ) . compound 41 at 40 m has no effect on apoptosis induction just like untreated control , which is consistent with its lack of binding to mcl-1 and inhibition of cell growth . we further tested compounds 19 and 21 in hl-60 cell line for their ability to induce caspase-3 activity , one of the important biochemical markers of apoptosis ( figure 8c ) . both compounds induce activation of caspase-3 activity in a dose - dependent manner , with 21 effectively inducing activation over a 24 h period starting at 2.5 m . importantly , these results correlate with the ability of 21 to induce apoposis and inhibit hl-60 cell growth . cell death and apoptosis induction by mcl-1 inhibitors in human leukemic cell lines . ( a ) inhibition of cell growth by designed mcl-1 inhibitors in the hl-60 , mv4,11 , and k-562 leukemia cell lines . cells were treated for 3 days , and cell growth was determined using celltiter glo luminescent cell viability assay . ( b ) analysis of apoptosis induced by 21 and 37 in the hl-60 leukemia cell line . cells were treated with 21 , 37 , and 41 for 20 h using indicated concentrations , and apoptosis was analyzed with annexin - v and propidium iodide ( pi ) double staining by flow cytometry . early apoptotic cells were defined as annexin - v positive / pi - negative , and late apoptotic cells as annexin - v / pi - double positive . induction of the apoptosis by 37 was tested also in the presence of z - vad - fmk . ( c ) induction of caspase-3 by 19 and 21 in the hl-60 cell line . cells were treated for 20 h , and caspase-3 was detected with fluorometric - based assay . applying a hts approach we have identified a novel class of small molecules as selective mcl-1 inhibitors . employing structure - based design supported by nmr studies , we synthesized a focused library of analogues and established a sar for binding to mcl-1 . careful hsqc analysis of analogues 1618 provided strong evidence for the predicted binding model of these compounds and further confirmation that r1 substituent of this class of inhibitors binds to one well - defined pocket of mcl-1 known as h2 pocket . analogues 19 and 21 , with ki values of 170 and 180 nm , respectively , were developed as the most potent compounds in this series with an overall 9-fold increase in binding compared to 1 . binding studies showed that 19 and 21 maintained the selectivity profile of 1 . using wild - type and bax / bak double knockout mefs cells , the contribution of these two multidomain pro - apoptotic proteins in 21-induced cell death was determined and demonstrated that 21 primarily causes cell death in wild - type mefs in a bax / bak - dependent manner . furthermore , 19 and 21 led to sensitization of e-myc lymphomas overexpressing mcl-1 but did not show effect on cells overexpressing bcl-2 antiapoptotic protein , confirming their selective targeting of mcl-1 . most potent developed 3-substituted - n-(4-hydroxynaphthalen-1-yl)arylsulfonamide mcl-1 inhibitors inhibited the cell growth of aml - derived cell lines , hl-60 and mv4,11 . compounds 19 and 21 induced activation of caspase-3 in hl-60 cell line and 21 effectively induced apoptosis starting at 2.5 m . future studies will be directed toward optimization of this class of compounds and in vivo evaluation . silica gel chromatography was performed with silica gel ( 220240 mesh ) obtained from silicycle . purities of final compounds were assessed by analytical reverse - phase hplc performed with one of the two methods . method a : agilent 1100 series with an agilent zorbax eclipse plus c18 ( 4.6 mm 75 mm , 3.5 m particle size ) column with the gradient 10% acn / water ( 1 min ) , 1090% acn / water ( 6 min ) , and 90% acn / water ( 2 min ) flow = 1 ml / min . method b : shimadzu system with a restek ultra c18 ( 4.6 mm 150 mm , 5 m particle size ) column with the gradient 50% acn / water ( 5 min ) , 5070% acn / water ( 2 min ) , and 70%90% acn / water ( 8 min ) flow = 1 ml / min . semipreparative reverse - phase hplc was performed on a shimadzu system with a restek ultra c18 ( 21.2 mm 150 mm , 5 m particle size ) column . all nmr spectra were obtained in dmso - d6 or cdcl3 , and results were recorded at 400 or 500 mhz on a varian 400 or 500 instrument . mass spectra were recorded on a micromass lct time - of - flight instrument utilizing electrospray ionization operating in positive - ion ( esi+ ) or negative - ion ( esi ) modes where indicated . high resolution mass spectrometry ( hrms ) analysis was performed on an agilent q - tof system . analogues 48 , 15 , and 39 were purchased from commercial vendors . synthesized using reported procedure with modification . a mixture of 1-methoxy-4-nitronaphthalene ( 2.1 g , 10.4 mmol ) and n - iodosuccinimide ( 2.7 g , 12 mmol ) in tfa ( 40 ml ) was heated to reflux and stirred for 20 h under nitrogen . the reaction mixture was diluted with etoac ( 40 ml ) , washed with saturated aqueous na2s2o3 solution ( 30 ml ) , saturated aqueous nahco3 ( 30 ml 2 ) , and brine ( 30 ml ) . the organic layer was dried ( mgso4 ) , filtered , and silica was added to filtrate and the solvent was removed under reduced pressure . the adsorbed crude residue was purified by flash column chromatography ( 100% hexane ) on silica gel to give 47 ( 2.4 g , 70% ) as a light - yellow solid . note : rf of starting material and product were very close and a good separation was achieved with a relatively long silica gel column and 100% hexane gradient . h nmr ( 400 mhz , cdcl3 ) 8.59 ( s , 1h ) , 8.58 ( s , 1h ) , 8.21 ( d , j = 8.42 hz , 1h ) , 7.74 ( t , j = 7.53 hz , 1h ) , 7.65 ( t , j = 7.53 hz , 1h ) , 4.03 ( s , 3h ) . c nmr ( 100 mhz , cdcl3 ) 161.69 , 142.85 , 134.07 , 129.99 , 128.55 , 128.08 , 126.55 , 123.92 , 123.02 , 83.35 , 62.20 . esi ms : m / z 330.0 ( m + h ) . synthesized using reported procedures with modification . to a solution of cs2co3 ( 1.5 g , 4.5 mmol ) in dry thf ( 7 ml ) under nitrogen was added methylthioglycolate ( 277 l , 2.9 mmol ) . the mixture was stirred at room temperature for 10 min . at this time , a solution of zncl2 ( 288 mg , 2.1 mmol ) in dry thf ( 3 ml ) was added and the mixture was stirred at room temperature for an additional 10 min . meanwhile , in a separate flask , pd(oac)2 ( 36 mg , 0.16 mmol ) and xantphos ( 90 mg , 0.15 mmol ) were premixed in dry thf ( 5 ml ) under nitrogen and stirred at room temperature for about 20 min . to the solution of thiol , cs2co3 , and zncl2 was added 47 ( 1.0 g , 3.1 mmol ) , lii ( 200 mg , 1.5 mmol ) , and premixed solution of the catalyst and ligand . the mixture was stirred at 60 c under nitrogen for 20 h. the reaction mixture was filtered to remove cs2co3 and silica was added to the mixture and the solvent was removed under reduced pressure . the adsorbed crude residue was purified by column chromatography ( hexane / etoac 4:1 ) on silica gel to give 48a ( 606 mg , 66% ) as a yellow oil which solidified . h nmr ( 400 mhz , cdcl3 ) 8.59 ( d , j = 8.50 hz , 1h ) , 8.37 ( s , 1h ) , 8.19 ( d , j = 8.50 hz , 1h ) , 7.70 ( t , j = 7.57 hz , 1h ) , 7.64 ( t , j = 7.57 hz , 1h ) , 4.07 ( s , 3h ) , 3.77 ( s , 2h ) , 3.70 ( s , 3h ) . c nmr ( 100 mhz , cdcl3 ) 169.48 , 159.95 , 142.50 , 129.73 , 128.88 , 127.85 , 127.22 , 125.94 , 123.71 , 122.93 , 122.68 , 61.92 , 52.72 , 35.10 . esi ms : m / z 308.1 ( m + h ) . synthesized using a similar procedure used to prepare 48a except using methyl 3-mercaptopropionate . the mixture was stirred at 60 c under nitrogen for 5 h. crude was purified using flash column chromatography ( hexane / etoac 4:1 ) on silica gel with dry loading to give 48b ( 194 mg , 66% ) as a yellow oil . h nmr ( 400 mhz , cdcl3 ) 8.56 ( d , j = 8.48 hz , 1h ) , 8.27 ( s , 1h ) , 8.16 ( d , j = 8.48 hz , 1h ) , 7.707.64 ( m , 1h ) , 7.647.58 ( m , 1h ) , 4.03 ( s , 3h ) , 3.65 ( s , 3h ) , 3.28 ( t , j = 7.24 hz , 2h ) , 2.65 ( t , j = 7.24 hz , 2h ) . c nmr ( 100 mhz , cdcl3 ) 171.64 , 159.79 , 142.48 , 129.51 , 128.93 , 127.81 , 126.94 , 125.60 , 123.62 , 123.45 , 122.57 , 61.59 , 51.90 , 34.11 , 28.11 . esi ms : m / z 322.0 ( m + h ) , 343.9 ( m + na ) . synthesized using a reported procedure . a stirred mixture of 47 ( 300 mg , 0.91 mmol ) , pd2(dba)3 ( 42 mg , 0.05 mmol ) , dppf ( 104 mg , 0.18 mmol ) , and et3n ( 0.2 ml ) in dry nmp ( 7 ml ) was flushed with nitrogen for 15 min at room temperature . butanethiol ( 83 l , 0.77 mmol ) was then added , and the reaction mixture was heated to 80 c and stirred for 2 h. the mixture was diluted with etoac ( 10 ml ) and washed with h2o ( 10 ml 4 ) and brine ( 10 ml ) . the organic layer was dried ( mgso4 ) , filtered , and silica added to the filtrate , and the solvent was removed under reduced pressure . the adsorbed crude residue was purified by flash column chromatography ( hexane to hexane / etoac 99:1 ) on silica gel to give 48c ( 189 mg , 71% ) as a yellow oil . h nmr ( 400 mhz , cdcl3 ) 8.58 ( ddd , j = 0.72 , 1.50 , 8.36 hz , 1h ) , 8.26 ( s , 1h ) , 8.16 ( ddd , j = 0.72 , 1.50 , 8.36 hz , 1h ) , 7.687.59 ( m , 2h ) , 4.03 ( s , 3h ) , 3.03 ( t , j = 7.36 hz , 2h ) , 1.67 ( p , j = 7.36 hz , 2h ) , 1.48 ( h , j = 7.36 hz , 2h ) , 0.93 ( t , j = 7.36 hz , 3h ) . c nmr ( 100 mhz , cdcl3 ) 158.48 , 142.53 , 129.05 , 128.81 , 127.69 , 125.69 , 125.57 , 125.03 , 123.62 , 122.34 , 61.31 , 32.23 , 31.10 , 21.92 , 13.60 . esi ms : m / z 292.0 ( m + h ) . synthesized using a reported procedure . a mixture of 47 ( 453 mg , 1.4 mmol ) , pd(pph3)2cl2 ( 48 mg , 0.07 mmol ) , and cui ( 28 mg , 0.15 mmol ) in et3n ( 8 ml ) and dry thf ( 3 ml ) was added dropwise to a solution of 2-propyn-1-ol ( 0.15 ml , 2.6 mmol ) in et3n ( 3 ml ) under nitrogen at room temperature . reaction mixture was heated to 60 c and stirred for 2 h then diluted with etoac ( 10 ml ) and washed with saturated aqueous nh4cl ( 15 ml 2 ) and brine ( 15 ml ) . the organic layer was dried ( mgso4 ) , filtered , and concentrated under reduced pressure . the crude was purified by flash column chromatography ( hexane / etoac 3:2 ) on silica gel to give 48d ( 342 mg , 97% ) as a yellow solid . h nmr ( 400 mhz , cdcl3 ) 8.59 ( d , j = 8.66 hz , 1h ) , 8.29 ( s , 1h ) , 8.26 ( d , j = 8.66 hz , 1h ) , 7.747.66 ( m , 1h ) , 7.647.55 ( m , 1h ) , 4.58 ( s , 2h ) , 4.29 ( s , 3h ) . c nmr ( 100 mhz , cdcl3 ) 162.40 , 141.04 , 130.49 , 129.78 , 128.26 , 127.56 , 126.29 , 123.44 , 123.24 , 107.40 , 93.96 , 80.83 , 61.95 , 51.67 . esi ms : m / z 258.1 ( m + h ) . synthesized using a similar procedure used to prepare 48d except using 3-butyn-1-ol as the alkyne . the crude was purified by flash column chromatography ( hexane / etoac 3:2 ) on silica gel to give 48e ( 338 mg , 83% ) as a yellow solid . h nmr ( 400 mhz , cdcl3 ) 8.53 ( d , j = 8.56 hz , 1h ) , 8.23 ( s , 1h ) , 8.19 ( d , j = 8.56 hz , 1h ) , 7.63 ( t , j = 7.59 hz , 1h ) , 7.53 ( t , j = 7.59 hz , 1h ) , 4.23 ( s , 3h ) , 3.87 ( t , j = 6.14 hz , 2h ) , 2.76 ( t , j = 6.14 hz , 2h ) , 2.45 ( s , 1h ) . c nmr ( 100 mhz , cdcl3 ) 162.29 , 141.11 , 130.19 , 129.91 , 128.36 , 127.48 , 126.03 , 123.43 , 123.10 , 109.99 , 108.73 , 93.90 , 61.77 , 60.96 , 24.07 . synthesized using a reported procedure . to a suspension of iron powder ( 538 mg , 9.6 mmol ) in glacial acetic acid ( 5 ml ) was added 48a ( 195 mg , 0.63 mmol ) dissolved in glacial acetic acid ( 5 ml ) . the mixture was stirred at 70 c under nitrogen for 1 h , when the mixture turned milky . the mixture was then diluted with etoac ( 15 ml ) and washed with saturated aqueous nahco3 ( 20 ml 2 ) and brine ( 20 ml ) . organic layer was dried ( mgso4 ) , filtered , and the solvent removed under reduced pressure to give 49a as a crude as a purple oil . h nmr ( 400 mhz , cdcl3 ) 8.03 ( d , j = 8.29 hz , 1h ) , 7.76 ( d , j = 8.29 hz , 1h ) , 7.50 ( t , j = 7.58 hz , 1h ) , 7.43 ( t , j = 7.58 hz , 1h ) , 6.78 ( s , 1h ) , 3.91 ( s , 3h ) , 3.73 ( s , 2h ) , 3.68 ( s , 3h ) . c nmr ( 100 mhz , cdcl3 ) 170.48 , 147.81 , 138.68 , 128.54 , 126.54 , 125.33 , 124.24 , 123.36 , 122.45 , 121.35 , 111.08 , 61.41 , 52.53 , 35.37 . esi ms : m / z 278.1 ( m + h ) , 300.1 ( m + na ) . synthesized using the procedure for 49a except using 48b as the starting material . h nmr ( 400 mhz , cdcl3 ) 8.02 ( d , j = 8.36 hz , 1h ) , 7.75 ( d , j = 8.36 hz , 1h ) , 7.49 ( t , j = 7.56 hz , 1h ) , 7.42 ( t , j = 7.56 hz , 1h ) , 6.72 ( s , 1h ) , 6.26 ( s , 2h ) , 3.89 ( s , 3h ) , 3.65 ( s , 3h ) , 3.22 ( t , j = 7.46 hz , 2h ) , 2.64 ( t , j = 7.46 hz , 2h);. c nmr ( 100 mhz , cdcl3 ) 172.38 , 147.73 , 138.77 , 128.63 , 126.51 , 125.12 , 123.92 , 123.56 , 122.36 , 121.35 , 110.85 , 61.21 , 51.78 , 34.48 , 27.90 . h nmr ( 400 mhz , cdcl3 ) 8.03 ( d , j = 8.30 hz , 1h ) , 7.75 ( d , j = 8.30 hz , 1h ) , 7.49 ( t , j = 7.42 hz , 1h ) , 7.40 ( t , j = 7.42 hz , 1h ) , 6.72 ( s , 1h ) , 6.30 ( s , 2h ) , 3.92 ( s , 3h ) , 2.96 ( t , j = 7.33 hz , 2h ) , 1.65 ( p , j = 7.33 hz , 2h ) , 1.47 ( h , j = 7.33 hz , 2h ) , 0.92 ( t , j = 7.33 hz , 3h ) . c nmr ( 100 mhz , cdcl3 ) 146.85 , 138.46 , 128.54 , 126.42 , 125.50 , 124.73 , 123.45 , 122.20 , 121.30 , 110.24 , 61.02 , 32.29 , 31.50 , 22.02 , 13.67 . syntheszied using a reported procedure . a stirred solution of 48d ( 325 mg , 1 mmol ) in a mixture of etoh ( 12 ml ) and etoac ( 2 ml ) was hydrogenated in the presence of 10% pd / c ( 80 mg ) at room temperature and under 30 psi of h2 overnight . the suspension was filtered through a pad of celite , and the filtrate was concentrated under reduced pressure . h nmr ( 400 mhz , cdcl3 ) 8.02 ( d , j = 8.35 hz , 1h ) , 7.76 ( d , j = 8.35 hz , 1h ) , 7.517.43 ( m , 1h ) , 7.437.35 ( m , 1h ) , 6.54 ( s , 1h ) , 3.85 ( s , 3h ) , 3.54 ( t , j = 6.08 hz , 2h ) , 2.80 ( t , j = 7.27 hz , 2h ) , 1.85 ( p , j = 6.69 hz , 2h ) . c nmr ( 100 mhz , cdcl3 ) 146.20 , 138.66 , 129.80 , 128.24 , 126.01 , 124.51 , 123.79 , 122.41 , 121.33 , 111.39 , 62.18 , 61.32 , 33.18 , 25.51 . esi ms : m / z 232.1 ( m + h ) . synthesized using the procedure for 49d except using 48e as the starting material . h nmr ( 400 mhz , cdcl3 ) 7.99 ( d , j = 8.39 hz , 1h ) , 7.72 ( d , j = 8.39 hz , 1h ) , 7.43 ( t , j = 7.33 hz , 1h ) , 7.34 ( t , j = 7.33 hz , 1h ) , 6.52 ( s , 1h ) , 3.79 ( s , 3h ) , 3.56 ( t , j = 6.43 hz , 2h ) , 2.67 ( t , j = 6.43 hz , 2h ) , 1.65 ( dt , j = 6.60 , 14.36 hz , 2h ) , 1.56 ( dt , j = 6.60 , 14.36 hz , 2h ) . c nmr ( 100 mhz , cdcl3 ) 145.98 , 138.36 , 130.77 , 128.39 , 125.85 , 124.33 , 123.72 , 122.34 , 121.36 , 111.52 , 62.32 , 61.97 , 32.45 , 29.21 , 26.89 . a solution of the crude amine 49a dissolved in dry ch2cl2 ( 4 ml ) was added to 2-thiophenesulfonyl chloride ( 119 mg , 0.65 mmol ) . addition of pyridine ( 0.08 ml , 0.99 mmol ) was followed , and the mixture was stirred at room temperature under nitrogen overnight . the mixture was diluted with etoac ( 10 ml ) and washed with h2o ( 10 ml 3 ) and brine ( 10 ml ) . the organic layer was dried ( mgso4 ) , filtered , and the solvent removed under reduced pressure . crude was purified by flash column chromatography ( hexane / etoac 7:3 ) on silica gel to give 49f ( 176 mg , 66% over two steps ) as a purple oil which solidified upon standing . h nmr ( 400 mhz , cdcl3 ) 8.01 ( d , j = 8.40 hz , 1h ) , 7.82 ( d , j = 8.40 hz , 1h ) , 7.497.43 ( m , 2h ) , 7.427.39 ( m , 1h ) , 7.397.34 ( m , 2h ) , 6.926.88 ( m , 1h ) , 3.95 ( s , 3h ) , 3.68 ( s , 3h ) , 3.65 ( s , 2h ) . c nmr ( 100 mhz , cdcl3 ) 169.91 , 154.12 , 139.48 , 133.09 , 132.51 , 130.06 , 128.60 , 127.74 , 127.39 , 126.96 , 125.74 , 123.39 , 122.29 , 61.49 , 52.68 , 35.12 . esi ms : m / z 423.9 ( m + h ) , 445.8 ( m + na ) . synthesized using the procedure for 49f except using methanesulfonyl chloride , which afforded the title compound ( 69 mg , 39% over two steps ) as a light - pink solid . h nmr ( 400 mhz , cdcl3 ) 8.138.08 ( m , 1h ) , 8.058.00 ( m , 1h ) , 7.65 ( s , 1h ) , 7.607.54 ( m , 2h ) , 6.80 ( s , 1h ) , 3.99 ( s , 3h ) , 3.75 ( s , 2h ) , 3.69 ( s , 3h ) , 3.04 ( s , 3h ) . c nmr ( 100 mhz , cdcl3 ) 169.98 , 154.06 , 129.61 , 128.85 , 127.84 , 127.36 , 127.19 , 125.15 , 123.67 , 122.67 , 122.07 , 61.50 , 52.64 , 39.91 , 35.00 . esi ms : m / z 355.9 ( m + h ) , 377.9 ( m + na ) . synthesized using the procedure for 49 g except using benzenesulfonyl chloride , which afforded the title compound ( 119 mg , 64% over two steps ) as a purple oil which solidified upon standing . h nmr ( 400 mhz , cdcl3 ) 8.01 ( d , j = 8.40 hz , 1h ) , 7.74 ( t , j = 9.06 hz , 3h ) , 7.47 ( q , j = 8.19 , 8.80 hz , 2h ) , 7.36 ( t , j = 7.61 hz , 3h ) , 7.29 ( s , 1h ) , 6.93 ( s , 1h ) , 3.95 ( s , 3h ) , 3.68 ( s , 3h ) , 3.61 ( s , 2h ) . c nmr ( 100 mhz , cdcl3 ) 169.80 , 154.02 , 139.11 , 132.95 , 129.95 , 128.98 , 128.63 , 127.82 , 127.33 , 126.95 , 126.90 , 125.67 , 123.36 , 122.31 , 122.28 , 61.49 , 52.63 , 35.06 . esi ms : m / z 417.9 ( m + h ) , 439.9 ( m + na ) . synthesized using the procedure for 49f except using 4-chlorobenzenesulfonyl chloride , which afforded the title compound ( 280 mg , 67% over two steps ) as a light - pink solid . h nmr ( 400 mhz , cdcl3 ) 8.02 ( d , j = 8.40 hz , 1h ) , 7.74 ( d , j = 8.40 hz , 1h ) , 7.66 ( t , j = 1.96 hz , 1h ) , 7.65 ( t , j = 1.96 hz , 1h ) , 7.47 ( ddd , j = 1.17 , 6.86 , 8.24 hz , 1h ) , 7.38 ( ddd , j = 1.17 , 6.86 , 8.24 hz , 1h ) , 7.357.33 ( m , 2h ) , 7.32 ( t , j = 1.94 hz , 1h ) , 7.07 ( s , 1h ) , 3.95 ( s , 3h ) , 3.69 ( s , 3h ) , 3.63 ( s , 2h ) . c nmr ( 100 mhz , cdcl3 ) 169.84 , 154.18 , 139.50 , 137.67 , 129.86 , 129.25 , 128.78 , 128.66 , 127.49 , 127.05 , 125.81 , 123.42 , 122.41 , 122.14 , 61.50 , 52.64 , 35.02 . esi ms : m / z 451.9 ( m + h ) , 473.8 ( m + na ) . synthesized using the procedure for 49f except using 4-bromobenzenesulfonyl chloride , which afforded the title compound ( 185 mg , 61% over two steps ) as a pink / purple solid . h nmr ( 400 mhz , cdcl3 ) 8.04 ( d , j = 8.38 hz , 1h ) , 7.72 ( d , j = 8.38 hz , 1h ) , 7.617.59 ( m , 1h ) , 7.58 ( t , j = 2.04 hz , 1h ) , 7.547.47 ( m , 3h ) , 7.447.38 ( m , 1h ) , 7.34 ( s , 1h ) , 6.76 ( s , 1h ) , 3.97 ( s , 3h ) , 3.71 ( s , 3h ) , 3.64 ( s , 2h ) . c nmr ( 100 mhz , cdcl3 ) 169.83 , 154.25 , 138.21 , 132.29 , 129.91 , 128.89 , 128.71 , 128.07 , 127.41 , 127.12 , 125.92 , 123.47 , 122.48 , 122.17 , 122.09 , 61.56 , 52.70 , 35.03 . esi hrms : m / z 493.9724 ( m h ) . synthesized using the procedure for 49f except using 4-methoxybenzenesulfonyl chloride , which afforded the title compound ( 190 mg , 77% over two steps ) as a purple oil which solidified upon standing . h nmr ( 400 mhz , cdcl3 ) 8.02 ( d , j = 8.42 hz , 1h ) , 7.80 ( d , j = 8.42 hz , 1h ) , 7.697.63 ( m , 2h ) , 7.48 ( t , j = 7.64 hz , 1h ) , 7.40 ( t , j = 7.64 hz , 1h ) , 7.29 ( s , 1h ) , 6.866.80 ( m , 3h ) , 3.95 ( s , 3h ) , 3.79 ( s , 3h ) , 3.70 ( s , 3h ) , 3.63 ( s , 2h ) . c nmr ( 100 mhz , cdcl3 ) 169.82 , 163.12 , 153.79 , 130.64 , 129.85 , 129.52 , 128.61 , 128.11 , 126.95 , 126.90 , 125.20 , 123.36 , 122.31 , 114.12 , 61.50 , 55.56 , 52.65 , 35.09 . esi ms : m / z 447.8 ( m + h ) , 469.8 ( m + na ) . synthesized using the procedure for 49f except using 3-bromobenzenesulfonyl chloride , which afforded the title compound ( 331 mg , 74% ) as a purple / pink solid . h nmr ( 400 mhz , cdcl3 ) 8.04 ( d , j = 8.40 hz , 1h ) , 7.88 ( s , 1h ) , 7.73 ( d , j = 8.40 hz , 1h ) , 7.62 ( t , j = 9.41 hz , 2h ) , 7.49 ( t , j = 7.59 hz , 1h ) , 7.40 ( t , j = 7.59 hz , 1h ) , 7.34 ( s , 1h ) , 7.267.21 ( m , 1h ) , 6.82 ( s , 1h ) , 3.98 ( s , 3h ) , 3.70 ( s , 3h ) , 3.65 ( s , 2h ) . c nmr ( 100 mhz , cdcl3 ) 169.72 , 154.55 , 141.06 , 135.89 , 130.41 , 130.24 , 130.03 , 128.75 , 127.31 , 127.04 , 127.02 , 126.33 , 125.89 , 123.45 , 122.90 , 122.48 , 122.09 , 61.48 , 52.55 , 35.16 . esi ms : m / z 495.8 , 497.8 ( m + h ) , 517.8 , 519.8 ( m + na ) . synthesized using the procedure for 49f except using 2-bromobenzenesulfonyl chloride , which afforded the title compound ( 314 mg , 74% over two steps ) as a purple oil . h nmr ( 400 mhz , cdcl3 ) 8.168.11 ( m , 1h ) , 8.058.00 ( m , 1h ) , 7.93 ( d , j = 7.72 hz , 1h ) , 7.78 ( d , j = 7.72 hz , 1h ) , 7.547.48 ( m , 3h ) , 7.427.32 ( m , 2h ) , 7.19 ( s , 1h ) , 7.13 ( s , 1h ) , 3.94 ( s , 3h ) , 3.68 ( s , 3h ) , 3.49 ( s , 2h ) . c nmr ( 100 mhz , cdcl3 ) 169.52 , 154.35 , 138.58 , 135.17 , 133.92 , 132.04 , 130.52 , 128.75 , 127.83 , 127.55 , 127.11 , 127.07 , 125.00 , 123.16 , 122.90 , 122.26 , 119.94 , 61.40 , 52.50 , 35.13 . esi ms : m / z 495.8 , 597.8 ( m + h ) , 517.8 , 519.8 ( m + na ) . synthesized using the procedure for 49f except using 1-benzothiophene-2-sulfonyl chloride , which afforded the title compound ( 132 mg , 57% over two steps ) as a purple oil which solidified upon standing . h nmr ( 400 mhz , cdcl3 ) 8.02 ( d , j = 8.40 hz , 1h ) , 7.90 ( d , j = 8.40 hz , 1h ) , 7.79 ( d , j = 7.99 hz , 1h ) , 7.74 ( d , j = 7.99 hz , 1h ) , 7.69 ( s , 1h ) , 7.487.42 ( m , 2h ) , 7.41 ( s , 1h ) , 7.407.35 ( m , 2h ) , 7.01 ( s , 1h ) , 3.96 ( s , 3h ) , 3.64 ( s , 3h ) , 3.51 ( s , 2h ) . c nmr ( 100 mhz , cdcl3 ) 169.77 , 154.23 , 141.85 , 139.53 , 137.38 , 130.61 , 130.05 , 128.66 , 127.47 , 127.37 , 127.06 , 125.72 , 125.47 , 125.44 , 123.47 , 122.62 , 122.34 , 122.25 , 61.51 , 52.58 , 34.95 . esi ms : m / z 473.9 ( m + h ) , 495.8 ( m + na ) . synthesized using the procedure for 49f except using 4-biphenylsulfonyl chloride , which afforded the title compound ( 152 mg , 71% over two steps ) as a purple oil which solidified upon standing . h nmr ( 400 mhz , cdcl3 ) 8.02 ( d , j = 8.34 hz , 1h ) , 7.837.76 ( m , 3h ) , 7.58 ( d , j = 8.31 hz , 2h ) , 7.51 ( d , j = 7.14 hz , 2h ) , 7.497.42 ( m , 2h ) , 7.427.35 ( m , 3h ) , 7.33 ( s , 1h ) , 6.90 ( s , 1h ) , 3.96 ( s , 3h ) , 3.65 ( s , 3h ) , 3.60 ( s , 2h ) . c nmr ( 100 mhz , cdcl3 ) 169.75 , 153.99 , 145.89 , 139.09 , 137.68 , 129.97 , 128.98 , 128.66 , 128.50 , 127.90 , 127.86 , 127.56 , 127.23 , 126.98 , 126.93 , 125.58 , 123.43 , 122.35 , 122.33 , 61.49 , 52.58 , 35.02 . esi ms : m / z 494.1 ( m + h ) , 516.1 ( m + na ) . a stirred mixture of 49k ( 307 mg , 0.62 mmol ) , phenylboronic acid ( 113 mg , 0.91 mmol ) , 2 m aqueous na2co3 ( 0.93 ml ) , and pd(pph3)4 ( 72 mg , 0.06 mmol ) in thf ( 6 ml ) /h2o ( 1 ml ) was heated at 60 c under nitrogen for 2 h. mixture was diluted with etoac ( 10 ml ) and washed with h2o ( 15 ml 2 ) and brine ( 15 ml ) . organic layer was dried ( mgso4 ) , filtered , and concentrated under reduced pressure . the crude was purified by flash column chromatography on silica to give 49o ( 284 mg , 92% ) as a light - brown oil . h nmr ( 400 mhz , cdcl3/ couple of drops of d2o ) 8.00 ( d , j = 8.39 hz , 1h ) , 7.83 ( s , 1h ) , 7.797.73 ( m , 2h ) , 7.69 ( dd , j = 7.58 , 14.09 hz , 2h ) , 7.477.40 ( m , 2h ) , 7.377.31 ( m , 6h ) , 3.92 ( s , 3h ) , 3.62 ( s , 3h ) , 3.58 ( s , 2h ) . c nmr ( 100 mhz , cdcl3/ couple of drops of d2o ) 169.86 , 154.00 , 142.22 , 139.49 , 138.99 , 133.60 , 131.46 , 131.02 , 130.00 , 129.44 , 128.89 , 128.63 , 128.14 , 127.89 , 127.03 , 126.98 , 126.88 , 125.92 , 125.87 , 125.80 , 123.46 , 122.38 , 122.27 , 61.43 , 52.60 , 34.97 . esi ms : m / z 494.1 ( m + h ) . synthesized using the procedure for 49f except using 2-biphenylsulfonyl chloride which was synthesized as the title compound ( 57 mg , 58% over two steps ) was obtained as a light - orange oil . h nmr ( 400 mhz , cdcl3 ) 8.16 ( d , j = 8.20 hz , 1h ) , 8.00 ( d , j = 8.20 hz , 1h ) , 7.62 ( t , j = 7.54 hz , 1h ) , 7.51 ( q , j = 7.13 , 7.54 hz , 3h ) , 7.447.37 ( m , 2h ) , 7.377.29 ( m , 5h ) , 6.93 ( s , 1h ) , 5.86 ( s , 1h ) , 3.91 ( s , 3h ) , 3.67 ( s , 3h ) , 3.44 ( s , 2h ) . c nmr ( 100 mhz , cdcl3 ) 169.66 , 152.79 , 140.89 , 138.72 , 138.15 , 132.77 , 132.73 , 129.66 , 129.43 , 128.59 , 128.52 , 128.39 , 128.20 , 128.11 , 127.94 , 127.07 , 126.69 , 123.16 , 122.20 , 122.04 , 120.40 , 61.44 , 52.62 , 34.96 . esi ms : m / z 494.0 ( m + h ) . synthesized using the procedure for 49f except using 4-chlorobiphenyl-4-sulfonyl chloride , which afforded the title compound ( 284 mg , 80% over two steps ) as a pink oil which solidified upon standing . h nmr ( 400 mhz , cdcl3 ) 8.02 ( d , j = 7.14 hz , 1h ) , 7.80 ( d , j = 7.14 hz , 1h ) , 7.78 ( d , j = 7.54 hz , 2h ) , 7.54 ( d , j = 7.54 hz , 2h ) , 7.497.44 ( m , 2h ) , 7.447.40 ( m , 3h ) , 7.407.35 ( m , 1h ) , 7.33 ( s , 1h ) , 6.97 ( s , 1h ) , 3.96 ( s , 3h ) , 3.65 ( s , 3h ) , 3.61 ( s , 2h ) . c nmr ( 100 mhz , cdcl3 ) 169.73 , 154.05 , 144.56 , 138.08 , 137.53 , 134.79 , 129.97 , 129.19 , 128.67 , 128.48 , 128.01 , 127.80 , 127.40 , 126.98 , 126.93 , 125.64 , 123.40 , 122.35 , 122.33 , 61.50 , 52.60 , 35.03 . esi ms : m / z 528.1 ( m + h ) , 550.1 ( m + na ) . synthesized using the procedure for 49f except using 2,4-difluoro - biphenyl-4-sulfonyl chloride , which afforded the title compound ( 75 mg , 58% over two steps ) as a purple oil which solidified upon standing . h nmr ( 400 mhz , cdcl3 ) 8.02 ( d , j = 8.41 hz , 1h ) , 7.837.76 ( m , 3h ) , 7.50 ( d , j = 8.16 hz , 2h ) , 7.46 ( d , j = 7.94 hz , 1h ) , 7.40 ( d , j = 7.94 hz , 1h ) , 7.377.29 ( m , 2h ) , 6.98 ( s , 1h ) , 6.966.86 ( m , 2h ) , 3.96 ( s , 3h ) , 3.66 ( s , 3h ) , 3.61 ( s , 2h ) . c nmr ( 100 mhz , cdcl3 ) 169.84 , 154.01 , 139.74 , 138.18 , 131.40 ( dd , j = 4.57 , 9.75 hz ) , 130.03 , 129.43 ( d , j = 3.03 hz ) , 128.65 , 127.76 , 127.61 , 127.02 , 126.96 , 125.70 , 123.44 , 122.37 , 122.32 , 111.98 ( dd , j = 3.71 , 21.44 hz ) , 105.88103.01 ( m ) , 61.51 , 52.63 , 34.95 . esi ms : m / z 530.1 ( m + h ) . synthesized using the procedure for 49f except using 4-phenoxybenzenesulfonyl chloride , which afforded the title compound ( 244 mg , 72% over two steps ) as a light - brown oil . h nmr ( 400 mhz , cdcl3 ) 8.02 ( d , j = 8.43 hz , 1h ) , 7.80 ( d , j = 8.43 hz , 1h ) , 7.66 ( d , j = 8.14 hz , 2h ) , 7.527.46 ( m , 1h ) , 7.427.32 ( m , 4h ) , 7.23 ( s , 1h ) , 7.17 ( t , j = 7.38 hz , 1h ) , 6.94 ( d , j = 8.52 hz , 2h ) , 6.87 ( d , j = 8.52 hz , 2h ) , 3.95 ( s , 3h ) , 3.69 ( s , 3h ) , 3.66 ( s , 2h ) . c nmr ( 100 mhz , cdcl3 ) 169.85 , 161.65 , 155.11 , 153.85 , 132.59 , 130.09 , 129.85 , 129.59 , 128.60 , 128.03 , 126.90 , 126.85 , 125.50 , 124.81 , 123.42 , 122.38 , 122.31 , 120.02 , 117.64 , 61.50 , 52.67 , 35.05 . esi ms : m / z 509.8 ( m + h ) . synthesized using the procedure for 49f except using 4-(4-fluoro - phenoxy)-benzenesulfonyl chloride , which afforded the title compound ( 93 mg , 72% over two steps ) as a yellow oil which solidified upon standing . h nmr ( 400 mhz , cdcl3 ) 8.02 ( d , j = 8.42 hz , 1h ) , 7.80 ( d , j = 8.42 hz , 1h ) , 7.66 ( d , j = 8.72 hz , 2h ) , 7.517.45 ( m , 1h ) , 7.427.36 ( m , 1h ) , 7.34 ( s , 1h ) , 7.077.00 ( m , 2h ) , 6.946.88 ( m , 2h ) , 6.84 ( d , j = 8.72 hz , 2h ) , 3.95 ( s , 3h ) , 3.69 ( s , 3h ) , 3.66 ( s , 2h ) . c nmr ( 100 mhz , cdcl3 ) 169.87 , 161.82 , 160.79 , 158.36 , 153.87 , 150.83 ( d , j = 2.72 hz ) , 132.65 , 129.83 , 129.66 , 128.61 , 127.99 , 126.88 ( d , j = 4.95 hz ) , 125.47 , 123.39 , 122.35 ( d , j = 6.41 hz ) , 121.67 ( d , j = 8.41 hz ) , 117.23 , 116.86 , 116.63 , 61.50 , 52.69 , 35.06 . esi ms : m / z 528.1 ( m + h ) , 550.1 ( m + na ) . as reported previously , to a solution of 1,1-sulfonyldiimidazole ( 1.98 g , 10 mmol ) in ch2cl2 ( 40 ml ) was added methyl trifluoromethanesulfonate ( 1.64 g , 10 mmol ) at 0 c . the solvent was removed after 3 h stirring . to the resulting residue in ch3cn ( 40 ml ) was added 1-phenylpiperazine ( 1.08 g , 6.67 mmol ) . after being stirred at room temperature overnight , the reaction mixture was concentrated under reduced pressure , and the crude was purified using flash column chromatography on silica to give 1-((1h - imidazol-1-yl)sulfonyl)-4-phenylpiperazine ( 1.17 g , 60% yield over 2 steps ) as a white solid . h nmr ( 400 mhz , cdcl3 ) 7.92 ( s , 1h ) , 7.307.23 ( m , 3h ) , 7.16 ( s , 1h ) , 6.93 ( t , j = 7.33 hz , 1h ) , 6.896.84 ( m , 2h ) , 3.373.30 ( m , 4h ) , 3.263.20 ( m , 4h ) . c nmr ( 100 mhz , cdcl3 ) 150.21 , 136.62 , 130.80 , 129.34 , 121.43 , 117.64 , 117.20 , 48.89 , 46.42 . to a solution of 1-((1h - imidazol-1-yl)sulfonyl)-4-phenylpiperazine ( 85 mg , 0.29 mmol ) in ch2cl2 ( 3 ml ) cooled at 0 c was added methyl trifluoromethanesulfonate ( 0.035 ml , 0.32 mmol ) . after being stirred for 2 h at 0 c , the reaction mixture was concentrated under reduced pressure to give 3-methyl-1-((4-phenylpiperazin-1-yl)sulfonyl)-1h - imidazol-3-ium as a beige solid which was used in the next step without further purification . a solution of 3-methyl-1-((4-phenylpiperazin-1-yl)sulfonyl)-1h - imidazol-3-ium ( 0.29 mmol ) and aniline 49a ( 0.29 mmol ) in ch3cn ( 3 ml ) was stirred at 80 c for 15 h. the reaction mixture was diluted with etoac ( 10 ml ) , washed with 1 n hcl , 1 n naoh , h2o , and brine , dried ( mgso4 ) , and filtered . the solvent was removed under reduced pressure , and the crude was purified using flash column chromatography on silica to give 49u ( 36 mg , 25% over three steps ) as a purple solid . h nmr ( 400 mhz , cdcl3 ) 8.128.06 ( m , 1h ) , 8.068.00 ( m , 1h ) , 7.71 ( s , 1h ) , 7.597.52 ( m , 2h ) , 7.22 ( t , j = 7.94 hz , 2h ) , 6.886.81 ( m , 3h ) , 6.75 ( s , 1h ) , 3.98 ( s , 3h ) , 3.74 ( s , 2h ) , 3.68 ( s , 3h ) , 3.433.37 ( m , 4h ) , 3.153.08 ( m , 4h ) . c nmr ( 100 mhz , cdcl3 ) 169.86 , 153.45 , 150.61 , 129.16 , 128.93 , 128.69 , 128.57 , 127.15 , 127.07 , 123.77 , 123.51 , 122.71 , 121.71 , 120.67 , 116.73 , 61.50 , 52.59 , 49.15 , 46.48 , 35.20 . esi ms : m / z 502.1 ( m + h ) , 524.1 ( m + na ) . synthesized using the procedure for 49f except using 4-bromobenzoyl chloride and et3n as the base , which afforded the title compound ( 245 mg , 66% ) as a pink solid . h nmr ( 400 mhz , cdcl3 ) 8.14 ( s , 1h ) , 8.09 ( d , j = 8.38 hz , 1h ) , 7.88 ( s , 1h ) , 7.827.73 ( m , 3h ) , 7.637.56 ( m , 2h ) , 7.567.43 ( m , 2h ) , 3.98 ( s , 3h ) , 3.74 ( s , 2h ) , 3.68 ( s , 3h ) . c nmr ( 100 mhz , cdcl3 ) 170.08 , 165.42 , 153.22 , 133.17 , 132.01 , 131.74 , 131.53 , 128.82 , 128.59 , 128.43 , 126.80 , 126.77 , 123.92 , 123.50 , 122.67 , 121.57 , 61.45 , 52.57 , 35.18 . esi ms : m / z 460.0 , 462.0 ( m + h ) , 482.0 , 484 ( m + na ) . synthesized using the procedure for 49f except using 4-chlorobenzylsulfonyl chloride , which afforded the title compound ( 110 mg , 41% over two steps ) as a light - pink solid . h nmr ( 400 mhz , cdcl3 ) 8.10 ( d , j = 8.31 hz , 1h ) , 7.75 ( d , j = 8.31 hz , 1h ) , 7.65 ( s , 1h ) , 7.57 ( t , j = 7.54 hz , 1h ) , 7.50 ( t , j = 7.54 hz , 1h ) , 7.227.16 ( m , 4h ) , 6.73 ( s , 1h ) , 4.37 ( s , 2h ) , 4.00 ( s , 3h ) , 3.75 ( s , 2h ) , 3.70 ( s , 3h ) . c nmr ( 100 mhz , cdcl3 ) 170.00 , 153.34 , 135.04 , 132.09 , 128.91 , 128.74 , 128.31 , 127.98 , 127.18 , 127.08 , 126.88 , 123.66 , 122.67 , 122.59 , 121.65 , 61.55 , 57.41 , 52.66 , 34.96 . esi ms : m / z 465.8 ( m + h ) , 487.8 ( m + na ) . synthesized using the procedure for 49f except using 4-chlorophenylacetyl chloride and et3n as the base , which afforded the title compound ( 208 mg , 69% over two steps ) as a light - yellow solid . h nmr ( 400 mhz , cdcl3 ) 8.05 ( d , j = 8.34 hz , 1h ) , 7.82 ( s , 1h ) , 7.49 ( m , 1h ) , 7.457.38 ( m , 4h ) , 7.387.32 ( m , 3h ) , 3.94 ( s , 3h ) , 3.82 ( s , 2h ) , 3.73 ( s , 2h ) , 3.67 ( s , 3h ) . c nmr ( 100 mhz , cdcl3 ) 170.16 , 169.79 , 153.07 , 133.95 , 132.72 , 130.93 , 129.52 , 128.47 , 128.24 , 127.90 , 126.80 , 126.72 , 123.43 , 123.32 , 122.67 , 120.80 , 61.41 , 52.59 , 43.65 , 35.14 . esi ms : m / z 429.9 ( m + h ) , 451.8 ( m + na ) . synthesized using the procedure for 49f except using phenylacetyl chloride . crude was triturated with cold methylene chloride to yield the title compound ( 154 mg , 49% over two steps ) as a white solid . h nmr ( 400 mhz , cdcl3 ) 8.06 ( d , j = 8.38 hz , 1h ) , 7.94 ( s , 1h ) , 7.537.44 ( m , 4h ) , 7.40 ( q , j = 7.27 hz , 2h ) , 7.33 ( s , 1h ) , 7.297.24 ( m , 2h ) , 3.96 ( s , 3h ) , 3.89 ( s , 2h ) , 3.78 ( s , 2h ) , 3.71 ( s , 3h ) . c nmr ( 100 mhz , cdcl3 ) 170.10 , 169.71 , 152.55 , 134.54 , 129.69 , 129.46 , 128.70 , 128.40 , 127.92 , 127.64 , 126.59 , 123.48 , 122.57 , 122.48 , 120.78 , 61.40 , 52.61 , 44.64 , 35.16 . esi ms : m / z 396.1 ( m + h ) , 418.1 ( m + na ) . synthesized using the procedure for 49f except using 49c and 4-bromobenzenesulfonyl chloride , which afforded the title compound ( 55 mg , 57% over two steps ) as a light - brown oil which solidified . h nmr ( 400 mhz , cdcl3 ) 8.01 ( d , j = 8.44 hz , 1h ) , 7.77 ( d , j = 8.44 hz , 1h ) , 7.59 ( d , j = 8.36 hz , 2h ) , 7.49 ( d , j = 8.36 hz , 2h ) , 7.46 ( d , j = 7.85 hz , 1h ) , 7.35 ( t , j = 7.85 hz , 1h ) , 7.21 ( s , 1h ) , 7.16 ( s , 1h ) , 3.94 ( s , 3h ) , 2.81 ( t , j = 7.24 hz , 2h ) , 1.58 ( p , j = 7.24 hz , 2h ) , 1.45 ( h , j = 7.24 hz , 2h ) , 0.92 ( t , j = 7.24 hz , 3h ) . c nmr ( 100 mhz , cdcl3 ) 152.71 , 138.26 , 132.23 , 128.92 , 128.88 , 128.65 , 127.99 , 127.25 , 126.93 , 126.37 , 125.79 , 124.49 , 122.19 , 122.04 , 61.01 , 31.92 , 31.20 , 21.95 , 13.67 . esi ms : m / z 479.8 , 481.8 ( m + h ) . synthesized using a reported procedure . compound 41 ( 82 mg , 0.16 mmol ) was added to aqueous nh4oh ( 29% , 3 ml ) . the mixture was stirred at room temperature for 1 h. workup included diluting the mixture with etoac ( 10 ml ) and washing with h2o ( 10 ml 2 ) and brine ( 10 ml ) . organic layer was dried ( mgso4 ) , filtered , and concentrated under reduced pressure to give 42 ( 69 mg , 90% ) as a pink solid . h nmr ( 400 mhz , dmso - d6 ) 10.32 ( s , 1h ) , 7.95 ( d , j = 8.50 hz , 1h ) , 7.85 ( d , j = 8.50 hz , 1h ) , 7.72 ( d , j = 8.20 hz , 2h ) , 7.60 ( d , j = 8.20 hz , 2h ) , 7.567.50 ( m , 2h ) , 7.437.37 ( m , 1h ) , 7.21 ( s , 1h ) , 7.15 ( s , 1h ) , 3.88 ( s , 3h ) , 3.53 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 169.76 , 151.88 , 139.67 , 132.70 , 129.69 , 129.18 , 129.05 , 128.23 , 127.46 , 127.03 , 126.41 , 125.24 , 124.89 , 123.95 , 121.82 , 61.27 , 35.91 . esi ms : m / z 480.7 , 482.7 ( m + h ) , 502.7 , 504.7 ( m + na ) . esi hrms : m / z 478.9742 ( m h ) . synthesized using the procedure for 42 except using 49s as the starting material , which afforded the title compound ( 179 mg , quantitative ) as a light - purple solid . h nmr ( 500 mhz , dmso - d6 ) 10.14 ( s , 1h ) , 7.99 ( d , j = 7.8 hz , 1h ) , 7.87 ( d , j = 8.3 hz , 1h ) , 7.66 ( d , j = 8.3 hz , 2h ) , 7.567.46 ( m , 2h ) , 7.41 ( t , j = 7.0 hz , 2h ) , 7.397.33 ( m , 1h ) , 7.237.14 ( m , 2h ) , 7.11 ( s , 1h ) , 7.046.94 ( m , 4h ) , 3.82 ( s , 3h ) , 3.50 ( s , 2h ) . c nmr ( 125 mhz , dmso - d6 ) 169.84 , 155.74 , 130.72 , 129.77 , 129.55 , 128.23 , 127.07 , 125.57 , 125.09 , 124.94 , 124.55 , 121.45 , 120.02 , 118.17 , 109.99 , 61.17 , 36.13 . esi ms : m / z 495.0 ( m + h ) , 517.0 ( m + na ) . synthesized using the procedure for 49f except using 49d and 4-bromobenzenesulfonyl chloride , which afforded the title compound ( 163 mg , 33% over two steps ) as a white solid . h nmr ( 400 mhz , dmso - d6 ) 10.11 ( s , 1h ) , 7.94 ( t , j = 7.53 hz , 2h ) , 7.70 ( d , j = 8.53 hz , 2h ) , 7.53 ( d , j = 8.53 hz , 2h ) , 7.517.45 ( m , 1h ) , 7.427.35 ( m , 1h ) , 6.86 ( s , 1h ) , 4.47 ( s , 1h ) , 3.79 ( s , 3h ) , 3.37 ( t , j = 5.87 hz , 2h ) , 2.692.60 ( m , 2h ) , 1.591.49 ( m , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 152.33 , 139.44 , 132.62 , 130.38 , 130.34 , 129.28 , 128.55 , 128.23 , 127.00 , 126.88 , 126.78 , 126.01 , 124.09 , 122.21 , 62.33 , 60.67 , 33.79 , 25.69 . esi ms : m / z 449.9 , 451.9 ( m + h ) , 471.9 , 473.9 ( m + na ) . synthesized using the procedure for 49f except using 49e and 4-bromobenzenesulfonyl chloride , which afforded the title compound ( 226 mg , 49% over two steps ) as a light - pink solid . h nmr ( 400 mhz , dmso - d6 ) 10.09 ( s , 1h ) , 7.997.91 ( m , 2h ) , 7.70 ( d , j = 8.43 hz , 2h ) , 7.54 ( d , j = 8.43 hz , 2h ) , 7.49 ( t , j = 7.34 hz , 1h ) , 7.40 ( t , j = 7.34 hz , 1h ) , 6.81 ( s , 1h ) , 4.33 ( t , j = 5.42 hz , 1h ) , 3.78 ( s , 3h ) , 3.37 ( q , j = 5.42 hz , 2h ) , 2.59 ( t , j = 7.01 hz , 2h ) , 1.491.28 ( m , 4h ) . c nmr ( 100 mhz , dmso - d6 ) 152.35 , 139.45 , 132.62 , 130.46 , 130.42 , 129.33 , 128.54 , 128.24 , 126.97 , 126.82 , 126.78 , 126.02 , 124.16 , 122.21 , 62.36 , 60.92 , 32.66 , 28.85 , 26.90 . esi ms : m / z 464.1 , 466.1 ( m + h ) , 486.1 , 488.1 ( m + na ) . esi hrms : m / z 462.0378 ( m h ) . synthesized using a reported procdure . to a suspension of 42 ( 127 mg , 0.26 mmol ) and sodium azide ( 255 mg , 3.9 mmol ) in dry ch3cn ( 5 ml ) in a glass tube was added sicl4 ( 0.15 ml , 1.3 mmol ) via syringe . the tube was sealed , and the stirring reaction mixture was heated to 80 c for 15 h. the reaction was quenched with 2 m na2co3 . the aqueous phase was acidified with 1 n hcl , and the mixture was extracted with etoac ( 15 ml 5 ) . the combined organic extracts were washed with brine ( 20 ml ) , dried ( mgso4 ) , filtered , and concentrated under reduced pressure to give 49cc ( 66 mg , 50% ) as a tan solid . h nmr ( 400 mhz , dmso - d6 ) 10.31 ( s , 1h ) , 7.93 ( d , j = 8.40 hz , 1h ) , 7.83 ( d , j = 8.40 hz , 1h ) , 7.65 ( d , j = 8.04 hz , 2h ) , 7.557.48 ( m , 3h ) , 7.447.38 ( m , 1h ) , 7.14 ( s , 1h ) , 6.53 ( s , 1h ) , 4.42 ( s , 2h ) , 3.78 ( s , 3h ) . c nmr ( 100 mhz , dmso - d6 ) 153.77 , 139.46 , 132.64 , 130.46 , 129.17 , 129.07 , 128.36 , 127.58 , 127.06 , 126.23 , 123.97 , 122.53 , 122.22 , 61.68 , 25.09 . esi ms : m / z 505.8 , 507.8 ( m + h ) , 527.8 , 529.8 ( m + na ) . synthesized using the procedure for 49cc except using 49aa as the starting material which afforded the title compound ( 128 mg , 95% ) as a tan solid . h nmr ( 500 mhz , dmso - d6 ) 10.14 ( s , 1h ) , 7.95 ( d , j = 8.4 hz , 1h ) , 7.87 ( d , j = 8.4 hz , 1h ) , 7.60 ( d , j = 8.0 hz , 2h ) , 7.56 ( t , j = 7.5 hz , 1h ) , 7.487.43 ( m , 1h ) , 7.40 ( t , j = 7.4 hz , 2h ) , 7.20 ( t , j = 7.3 hz , 1h ) , 7.17 ( s , 1h ) , 7.016.94 ( m , 4h ) , 4.44 ( s , 2h ) , 3.79 ( s , 3h ) . c nmr ( 125 mhz , dmso - d6 ) 160.90 , 155.45 , 153.57 , 134.18 , 130.75 , 130.55 , 129.76 , 129.50 , 128.35 , 127.49 , 126.94 , 126.14 , 125.17 , 124.15 , 122.62 , 122.16 , 120.13 , 118.27 , 61.66 , 25.13 . esi ms : m / z 517.9 ( m h ) . synthesized using the procedure for 49f except using 49b and 2-naphthalenesulfonyl chloride , which afforded the title compound ( 180 mg , 59% over two steps ) as a light - pink solid . h nmr ( 400 mhz , cdcl3 ) 8.30 ( s , 1h ) , 7.98 ( d , j = 8.39 hz , 1h ) , 7.907.85 ( m , 2h ) , 7.857.80 ( m , 2h ) , 7.807.76 ( m , 1h ) , 7.59 ( t , j = 7.36 hz , 1h ) , 7.53 ( t , j = 7.36 hz , 1h ) , 7.43 ( t , j = 7.39 hz , 1h ) , 7.34 ( t , j = 7.39 hz , 1h ) , 7.19 ( s , 1h ) , 6.98 ( s , 1h ) , 3.89 ( s , 3h ) , 3.64 ( s , 3h ) , 2.94 ( t , j = 7.23 hz , 2h ) , 2.40 ( t , j = 7.23 hz , 2h ) . c nmr ( 100 mhz , cdcl3 ) 171.84 , 153.85 , 135.99 , 134.83 , 131.94 , 129.66 , 129.43 , 129.20 , 129.02 , 128.91 , 128.75 , 127.82 , 127.78 , 127.54 , 126.95 , 126.72 , 125.21 , 123.73 , 122.39 , 122.19 , 109.99 , 61.20 , 51.79 , 34.05 , 27.65 . esi ms : m / z 481.9 ( m + h ) , 503.9 ( m + na ) . to a solution of 41 ( 425 mg , 0.85 mmol ) in dry thf ( 2 ml ) was added 1n aqueous lioh ( 4 ml ) . the mixture was stirred at room temperature under nitrogen for 1 h. reaction mixture was diluted with water ( 10 ml ) and washed with etoac ( 10 ml 2 ) . aqueous phase was acidified with 1 n hcl and extracted with etoac ( 10 ml 3 ) . combined organic extracts were washed with brine , dried ( mgso4 ) , and filtered . the solvent was removed under reduced pressure . crude was triturated with cold ch2cl2 to give 40 ( 305 mg , 74% ) as a white / tan solid . hplc ( method a , tr = 6.85 min ) , purity > 99% . h nmr ( 400 mhz , dmso - d6 ) 10.32 ( s , 1h ) , 7.92 ( d , j = 8.38 hz , 1h ) , 7.87 ( d , j = 8.38 hz , 1h ) , 7.68 ( d , j = 8.08 hz , 2h ) , 7.57 ( d , j = 8.08 hz , 2h ) , 7.51 ( t , j = 7.56 hz , 1h ) , 7.40 ( t , j = 7.56 hz , 1h ) , 7.09 ( s , 1h ) , 3.84 ( s , 3h ) , 3.65 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 170.59 , 152.07 , 139.60 , 132.69 , 129.83 , 129.18 , 129.13 , 128.28 , 127.54 , 127.09 , 126.57 , 124.60 , 124.56 , 124.01 , 121.86 , 61.30 , 34.63 . esi hrms : m / z 479.9578 ( m h ) . synthesized using the procedure for 40 except 49n was used as the starting material . crude was triturated with cold ch2cl2 to give 43 ( 88 mg , 70% ) as an white solid . h nmr ( 400 mhz , dmso - d6 ) 12.74 ( s , 1h ) , 10.21 ( s , 1h ) , 7.95 ( d , j = 8.44 hz , 1h ) , 7.91 ( d , j = 8.44 hz , 1h ) , 7.797.70 ( m , 4h ) , 7.687.61 ( m , 2h ) , 7.537.48 ( m , 1h ) , 7.45 ( t , j = 7.28 hz , 2h ) , 7.39 ( t , j = 7.28 hz , 2h ) , 7.08 ( s , 1h ) , 3.83 ( s , 3h ) , 3.61 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 170.52 , 151.84 , 144.73 , 139.09 , 138.87 , 129.90 , 129.50 , 128.93 , 128.26 , 127.89 , 127.78 , 127.49 , 126.47 , 124.56 , 124.21 , 124.18 , 121.77 , 61.26 , 34.56 . esi hrms : m / z 478.0787 ( m h ) . synthesized using the procedure for 40 except 49o was used as the starting material . crude was triturated with cold ch2cl2 to give 44 ( 152 mg , 59% ) as a white solid . h nmr ( 400 mhz , dmso - d6 ) 12.69 ( s , 1h ) , 10.20 ( s , 1h ) , 7.90 ( d , j = 8.46 hz , 1h ) , 7.86 ( d , j = 8.46 hz , 1h ) , 7.82 ( d , j = 7.81 hz , 1h ) , 7.72 ( s , 1h ) , 7.63 ( d , j = 7.81 hz , 1h ) , 7.55 ( t , j = 7.74 hz , 1h ) , 7.507.44 ( m , 1h ) , 7.437.38 ( m , 4h ) , 7.387.30 ( m , 2h ) , 7.11 ( s , 1h ) , 3.82 ( s , 3h ) , 3.61 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 170.46 , 151.97 , 141.49 , 140.71 , 138.92 , 131.42 , 130.33 , 129.80 , 129.49 , 129.43 , 128.66 , 128.24 , 127.47 , 127.14 , 126.43 , 125.88 , 125.21 , 124.64 , 124.59 , 124.04 , 121.77 , 61.23 , 34.56 . esi hrms : m / z 478.0788 ( m h ) . to a stirred solution of 49f ( 48 mg , 0.12 mmol ) suspended in dry ch2cl2 ( 1.5 ml ) was added bbr3 ( 1 m in ch2cl2 , 0.5 ml , 0.5 mmol ) dropwise at 0 c under nitrogen . after 1 h of stirring , the starting material was entirely consumed and the product formed as determined by tlc and ms ( esi ) . the mixture was slowly added to a stirred solution of saturated aqueous nh4cl ( 20 ml ) at 0 c . the combined organic extracts were washed with brine ( 15 ml ) , dried ( mgso4 ) , and filtered , and the solvent was removed under reduced pressure . the crude was purified using a c18 reverse phase semipreparative hplc column with solvent a ( 0.1% of tfa in water ) and solvent b ( 0.1% of tfa in ch3cn ) as eluents to give 1 ( 59 ) ( 28 mg , 59% ) as a white / tan solid . h nmr ( 400 mhz , dmso - d6 ) 10.10 ( s , 1h ) , 8.178.14 ( m , 1h ) , 7.897.84 ( m , 2h ) , 7.527.40 ( m , 2h ) , 7.36 ( dd , j = 1.32 , 3.74 hz , 1h ) , 7.09 ( s , 1h ) , 7.07 ( dd , j = 3.74 , 4.97 hz , 1h ) , 3.56 ( s , 2h ) ; c nmr ( 100 mhz , dmso - d6 ) 171.54 , 152.87 , 140.58 , 133.57 , 132.80 , 131.71 , 129.80 , 128.03 , 127.20 , 126.22 , 125.46 , 124.33 , 123.61 , 122.83 , 113.36 , 37.25 . esi hrms : m / z 393.9870 ( m h ) . synthesized using the procedure for 1 except 49 t was used as the starting material . the title compound ( 27 mg , 48% ) as a white solid after hplc purification . h nmr ( 400 mhz , dmso - d6 ) 9.40 ( s , 1h ) , 8.20 ( d , j = 8.09 hz , 1h ) , 8.14 ( d , j = 8.09 hz , 1h ) , 7.56 ( p , j = 6.86 hz , 2h ) , 7.47 ( s , 1h ) , 3.69 ( s , 2h ) , 2.99 ( s , 3h ) . c nmr ( 100 mhz , dmso - d6 ) 171.73 , 152.58 , 131.89 , 129.38 , 127.31 , 126.30 , 125.65 , 125.18 , 124.32 , 122.82 , 113.55 , 39.79 , 37.03 . esi hrms : m / z 326.0164 ( m h ) . synthesized using the procedure for 1 except 49h was used as the starting material . the title compound ( 21 mg , 22% ) was obtained as a white solid after hplc purification . h nmr ( 400 mhz , dmso - d6 ) 12.76 ( s , 1h ) , 9.91 ( s , 1h ) , 9.79 ( s , 1h ) , 8.14 ( d , j = 8.22 hz , 1h ) , 7.88 ( d , j = 8.22 hz , 1h ) , 7.677.61 ( m , 2h ) , 7.617.56 ( m , 1h ) , 7.547.47 ( m , 3h ) , 7.467.39 ( m , 1h ) , 7.01 ( s , 1h ) , 3.51 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.46 , 152.56 , 140.16 , 133.03 , 131.56 , 129.53 , 129.44 , 127.25 , 127.05 , 126.17 , 125.46 , 124.56 , 123.76 , 122.75 , 113.34 , 37.16 . esi hrms : m / z 388.0319 ( m h ) . synthesized using the procedure for 1 except 49i was used as the starting material . the title compound ( 48 mg , 20% ) was obtained as a white solid after trituration with a mixture of ch3cn : h2o 1:1 and cold ch2cl2 and without a need for hplc purification . h nmr ( 400 mhz , dmso - d6 ) 12.77 ( s , 1h ) , 10.03 ( s , 1h ) , 9.84 ( s , 1h ) , 8.15 ( d , j = 8.12 hz , 1h ) , 7.85 ( d , j = 8.12 hz , 1h ) , 7.667.59 ( m , 2h ) , 7.597.52 ( m , 2h ) , 7.46 ( dt , j = 7.28 , 14.85 hz , 2h ) , 7.05 ( s , 1h ) , 3.54 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.48 , 152.72 , 139.06 , 137.95 , 131.47 , 129.66 , 129.60 , 129.19 , 127.15 , 126.25 , 125.48 , 124.24 , 123.63 , 122.84 , 113.38 , 37.11 . esi hrms : m / z 421.9930 ( m h ) . synthesized using the procedure for 1 except 41 was used as the starting material . the title compound ( 134 mg , 45% ) was obtained as a white solid after trituration with a mixture of ch3cn : h2o 1:1 and cold ch2cl2 and without a need for hplc purification . h nmr ( 400 mhz , dmso - d6 ) 12.80 ( s , 1h ) , 10.05 ( s , 1h ) , 9.87 ( s , 1h ) , 8.14 ( d , j = 8.17 hz , 1h ) , 7.84 ( d , j = 8.17 hz , 1h ) , 7.71 ( d , j = 8.48 hz , 2h ) , 7.54 ( d , j = 8.48 hz , 2h ) , 7.48 ( t , j = 7.03 hz , 1h ) , 7.43 ( t , j = 7.03 hz , 1h ) , 7.04 ( s , 1h ) , 3.53 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.52 , 152.74 , 139.45 , 132.56 , 131.45 , 129.66 , 129.28 , 127.18 , 126.92 , 126.27 , 125.48 , 124.21 , 123.63 , 122.86 , 113.38 , 37.10 . esi hrms : m / z 465.9418 ( m h ) . synthesized using the procedure for 1 except 49j was used as the starting material . the title compound ( 25 mg , 29% ) was obtained as a white solid after hplc purification . hplc ( method a , tr = 6.20 min ) , purity 95% . h nmr ( 400 mhz , dmso - d6 ) 9.70 ( s , 1h ) , 8.10 ( d , j = 8.07 hz , 1h ) , 7.89 ( d , j = 7.78 hz , 1h ) , 7.52 ( d , j = 8.63 hz , 2h ) , 7.497.36 ( m , 2h ) , 7.006.93 ( m , 3h ) , 3.75 ( s , 3h ) , 3.47 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.47 , 162.75 , 152.44 , 131.81 , 131.63 , 129.49 , 129.37 , 127.05 , 126.18 , 125.48 , 124.92 , 123.93 , 122.75 , 114.57 , 113.35 , 56.02 , 37.24 . esi hrms : m / z 418.0424 ( m h ) . synthesized using the procedure for 1 except 49k was used as the starting material . the title compound ( 21 mg , 35% ) was obtained as a white / tan solid after trituration with a mixture of ch3cn : h2o 1:1 and cold ch2cl2 and without a need for hplc purification . h nmr ( 400 mhz , dmso - d6 ) 10.08 ( s , 1h ) , 8.15 ( d , j = 7.56 hz , 1h ) , 7.877.78 ( m , 2h ) , 7.75 ( t , j = 1.78 hz , 1h ) , 7.627.56 ( m , 1h ) , 7.527.39 ( m , 3h ) , 7.01 ( s , 1h ) , 3.52 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.52 , 152.92 , 142.07 , 135.93 , 131.78 , 131.49 , 129.77 , 129.59 , 127.18 , 126.34 , 126.28 , 125.53 , 124.02 , 123.58 , 122.89 , 122.35 , 113.43 , 37.24 . esi hrms : m / z 465.9414 ( m h ) . synthesized using the procedure for 1 except 49l was used as the starting material . the title compound ( 34 mg , 40% ) was obtained as a white solid after hplc purification . h nmr ( 400 mhz , dmso - d6 ) 12.80 ( s , 1h ) , 10.23 ( s , 1h ) , 9.88 ( s , 1h ) , 8.168.10 ( m , 1h ) , 8.078.02 ( m , 1h ) , 7.86 ( dd , j = 1.52 , 7.83 hz , 1h ) , 7.78 ( dd , j = 1.52 , 7.83 hz , 1h ) , 7.527.46 ( m , 3h ) , 7.42 ( td , j = 1.33 , 7.60 hz , 1h ) , 7.05 ( s , 1h ) , 3.46 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.42 , 152.60 , 139.23 , 135.82 , 134.64 , 131.92 , 131.77 , 129.49 , 128.52 , 127.18 , 126.32 , 125.44 , 123.98 , 123.78 , 122.78 , 119.92 , 113.54 , 37.03 . esi hrms : m / z 465.9408 ( m h ) . synthesized using the procedure for 1 except 49 the title compound ( 13 mg , 22% ) was obtained as an orange solid after hplc purification . h nmr ( 400 mhz , dmso - d6 ) 10.32 ( s , 1h ) , 8.11 ( d , j = 8.14 hz , 1h ) , 8.02 ( d , j = 8.14 hz , 1h ) , 7.89 ( t , j = 8.09 hz , 2h ) , 7.72 ( s , 1h ) , 7.47 ( t , j = 7.56 hz , 1h ) , 7.41 ( t , j = 7.35 hz , 2h ) , 7.387.32 ( m , 1h ) , 7.14 ( s , 1h ) , 3.40 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.57 , 153.29 , 141.31 , 141.04 , 137.80 , 131.73 , 129.99 , 129.94 , 127.65 , 127.22 , 126.21 , 125.86 , 125.54 , 123.96 , 123.54 , 123.41 , 122.92 , 113.33 , 37.41 . esi hrms : m / z 444.0041 ( m h ) . synthesized using the procedure for 1 except 49n was used as the starting material . the title compound ( 29 mg , 45% ) was obtained as a white / tan solid after hplc purification . h nmr ( 400 mhz , dmso - d6 ) 9.92 ( s , 1h ) , 8.11 ( d , j = 7.93 hz , 1h ) , 7.90 ( d , j = 7.93 hz , 1h ) , 7.76 ( d , j = 7.47 hz , 2h ) , 7.727.62 ( m , 4h ) , 7.507.42 ( m , 3h ) , 7.437.36 ( m , 2h ) , 7.02 ( s , 1h ) , 3.46 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.52 , 152.74 , 144.57 , 139.00 , 138.90 , 131.65 , 129.57 , 129.52 , 128.92 , 128.00 , 127.63 , 127.46 , 127.08 , 126.18 , 125.55 , 124.53 , 123.85 , 122.81 , 113.42 , 37.27 . esi hrms : m / z 464.0630 ( m h ) . synthesized using the procedure for 1 except 49o was used as the starting material . the title compound ( 48 mg , 34% ) was obtained as a white / tan solid after hplc purification . h nmr ( 400 mhz , dmso - d6 ) 12.73 ( s , 1h ) , 9.93 ( s , 1h ) , 9.79 ( s , 1h ) , 8.10 ( d , j = 8.25 hz , 1h ) , 7.887.81 ( m , 2h ) , 7.79 ( s , 1h ) , 7.607.50 ( m , 2h ) , 7.507.41 ( m , 4h ) , 7.417.32 ( m , 3h ) , 7.04 ( s , 1h ) , 3.45 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.44 , 152.66 , 141.46 , 140.82 , 139.01 , 131.50 , 131.30 , 130.18 , 129.60 , 129.55 , 128.66 , 127.17 , 127.07 , 126.23 , 126.07 , 125.51 , 125.26 , 124.59 , 123.73 , 122.81 , 113.44 , 37.16 . esi hrms : m / z 464.0632 ( m h ) . synthesized using the procedure for 1 except 49p was used as the starting material . the title compound ( 20 mg , 43% ) was obtained as a white / tan solid after hplc purification . h nmr ( 400 mhz , dmso - d6 ) 12.77 ( s , 1h ) , 9.76 ( s , 1h ) , 9.70 ( s , 1h ) , 8.12 ( d , j = 8.03 hz , 1h ) , 7.95 ( d , j = 8.03 hz , 1h ) , 7.74 ( d , j = 8.30 hz , 1h ) , 7.58 ( t , j = 7.46 hz , 1h ) , 7.52 ( t , j = 7.46 hz , 1h ) , 7.487.41 ( m , 1h ) , 7.417.34 ( m , 1h ) , 7.23 ( q , j = 4.82 , 5.81 hz , 1h ) , 7.17 ( t , j = 7.46 hz , 3h ) , 7.016.95 ( m , 1h ) , 6.93 ( s , 1h ) , 6.91 ( s , 1h ) , 3.43 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.57 , 152.24 , 141.36 , 139.92 , 138.75 , 133.21 , 132.68 , 131.42 , 129.62 , 129.37 , 128.85 , 128.36 , 127.55 , 127.48 , 126.97 , 126.21 , 125.49 , 124.57 , 123.79 , 122.69 , 113.65 , 37.07 . esi hrms : m / z 464.0636 ( m h ) . synthesized using the procedure for 1 except 49q was used as the starting material . the title compound ( 36 mg , 25% ) was obtained as a white solid after hplc purification . hplc ( method b , tr = 10.23 min ) , purity > 99% . h nmr ( 400 mhz , dmso - d6 ) 12.75 ( s , 1h ) , 9.96 ( s , 1h ) , 9.81 ( s , 1h ) , 8.10 ( d , j = 8.09 hz , 1h ) , 7.88 ( d , j = 8.09 hz , 1h ) , 7.77 ( d , j = 8.24 hz , 2h ) , 7.727.64 ( m , 4h ) , 7.52 ( d , j = 8.24 hz , 2h ) , 7.477.36 ( m , 2h ) , 6.99 ( s , 1h ) , 3.45 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.45 , 152.51 , 143.18 , 139.25 , 137.67 , 133.88 , 131.56 , 129.50 , 129.39 , 129.27 , 128.05 , 127.65 , 127.09 , 126.24 , 125.50 , 124.54 , 123.84 , 122.78 , 113.44 , 37.04 . esi hrms : m / z 498.0247 ( m h ) . synthesized using the procedure for 1 except 49r was used as the starting material . the title compound ( 31 mg , 48% ) as a white solid after hplc purification . h nmr ( 400 mhz , dmso - d6 ) 12.71 ( s , 1h ) , 9.97 ( s , 1h ) , 9.80 ( s , 1h ) , 8.10 ( d , j = 8.16 hz , 1h ) , 7.86 ( d , j = 8.16 hz , 1h ) , 7.69 ( d , j = 7.72 hz , 2h ) , 7.62 ( d , j = 7.72 hz , 2h ) , 7.597.53 ( m , 1h ) , 7.477.34 ( m , 3h ) , 7.19 ( t , j = 8.31 hz , 1h ) , 7.00 ( s , 1h ) , 3.46 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.46 , 152.55 , 139.44 , 138.77 , 132.51 ( dd , j = 4.88 , 9.80 hz ) , 131.58 , 129.82 ( d , j = 2.86 hz ) , 129.50 , 127.66 , 127.06 , 126.23 , 125.50 , 124.50 , 123.81 , 122.78 , 113.47 , 112.73 ( dd , j = 3.78 , 21.26 hz ) , 105.15 ( t , j = 26.72 hz ) , 37.04 . esi hrms : m / z 500.0441 ( m h ) . synthesized using the procedure for 1 except 49s was used as the starting material . the title compound ( 18 mg , 13% ) was obtained as a white solid after hplc purification . hplc ( method b , tr = 10.24 min ) , purity 99% . h nmr ( 400 mhz , dmso - d6 ) 12.81 ( s , 1h ) , 9.87 ( s , 1h ) , 9.83 ( s , 1h ) , 8.14 ( d , j = 8.31 hz , 1h ) , 7.86 ( d , j = 8.31 hz , 1h ) , 7.59 ( d , j = 8.75 hz , 2h ) , 7.50 ( t , j = 7.09 hz , 1h ) , 7.44 ( t , j = 7.81 hz , 3h ) , 7.23 ( t , j = 7.09 hz , 1h ) , 7.056.98 ( m , 5h ) , 3.55 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.53 , 160.76 , 155.60 , 152.58 , 134.17 , 131.57 , 130.75 , 129.84 , 129.76 , 127.06 , 126.16 , 125.48 , 125.07 , 124.60 , 123.78 , 122.80 , 120.03 , 118.33 , 113.38 , 37.15 . esi hrms : m / z 480.0579 ( m h ) . synthesized using the procedure for 1 except 49 t was used as the starting material . the title compound ( 36 mg , 44% ) as a white solid after hplc purification . h nmr ( 400 mhz , dmso - d6 ) 12.78 ( s , 1h ) , 9.83 ( s , 1h ) , 9.79 ( s , 1h ) , 8.11 ( d , j = 8.21 hz , 1h ) , 7.83 ( d , j = 8.21 hz , 1h ) , 7.55 ( d , j = 8.46 hz , 2h ) , 7.44 ( dt , j = 6.92 , 21.86 hz , 2h ) , 7.25 ( t , j = 8.52 hz , 2h ) , 7.117.02 ( m , 2h ) , 6.996.96 ( m , 2h ) , 6.95 ( s , 1h ) , 3.51 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.53 , 161.09 , 152.57 , 151.54 ( d , j = 2.57 hz ) , 134.14 , 131.59 , 129.86 , 129.68 , 127.08 , 126.19 , 125.48 , 124.61 , 123.80 , 122.80 , 122.17 ( d , j = 8.70 hz ) , 117.90 , 117.45 , 117.22 , 113.38 , 37.17 . esi hrms : m / z 498.0485 ( m h ) . synthesized using the procedure for 1 except 49u was used as the starting material . the title compound ( 14.5 mg , 48% ) was obtained as a white solid after hplc purification . h nmr ( 400 mhz , dmso - d6 ) 9.77 ( s , 1h ) , 9.59 ( s , 1h ) , 8.15 ( t , j = 7.91 hz , 2h ) , 7.597.53 ( m , 1h ) , 7.537.48 ( m , 2h ) , 7.207.14 ( m , 2h ) , 6.89 ( d , j = 7.91 hz , 2h ) , 6.77 ( t , j = 7.26 hz , 1h ) , 3.64 ( s , 2h ) , 3.243.16 ( m , 4h ) , 3.143.04 ( m , 4h ) . c nmr ( 100 mhz , dmso - d6 ) 171.57 , 152.02 , 150.84 , 131.08 , 129.40 , 128.56 , 127.32 , 126.28 , 125.52 , 125.43 , 123.76 , 122.95 , 120.04 , 116.48 , 113.63 , 48.54 , 46.36 , 37.06 . esi hrms : m / z 472.1002 ( m h ) . synthesized using the procedure for 1 except 49v was used as the starting material . the title compound was obtained ( 8.5 mg , 30% ) as a white solid after hplc purification . h nmr ( 400 mhz , dmso - d6 ) 10.36 ( s , 1h ) , 8.23 ( d , j = 5.36 hz , 1h ) , 8.02 ( d , j = 8.05 hz , 2h ) , 7.83 ( d , j = 5.36 hz , 1h ) , 7.78 ( d , j = 8.05 hz , 2h ) , 7.577.50 ( m , 3h ) , 3.72 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.74 , 165.71 , 151.94 , 133.90 , 131.89 , 130.66 , 130.30 , 128.64 , 127.11 , 126.17 , 126.13 , 125.80 , 125.47 , 123.80 , 122.97 , 113.65 , 37.15 . esi hrms : m / z 429.9747 ( m h ) . synthesized using the procedure for 1 except 49w was used as the starting material . the title compound ( 15.5 mg , 20% ) was obtained as a white solid after hplc purification . h nmr ( 400 mhz , dmso - d6 ) 12.86 ( s , 1h ) , 9.84 ( s , 1h ) , 9.55 ( s , 1h ) , 8.20 ( d , j = 8.36 hz , 1h ) , 8.07 ( d , j = 8.36 hz , 1h ) , 7.607.51 ( m , 2h ) , 7.44 ( s , 1h ) , 7.437.38 ( m , 4h ) , 4.49 ( s , 2h ) , 3.71 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.81 , 152.35 , 133.45 , 133.18 , 131.79 , 129.37 , 128.93 , 128.72 , 127.23 , 126.30 , 125.63 , 124.99 , 124.28 , 122.79 , 113.67 , 57.16 , 36.96 . esi hrms : m / z 436.0086 ( m h ) . synthesized using the procedure for 1 except 49x was used as the starting material . the title compound ( 19 mg , 10% ) was obtained as a white solid after trituration with a mixture of ch3cn : h2o 1:1 and cold ch2cl2 and without a need for hplc purification . h nmr ( 400 mhz , dmso - d6 ) 9.96 ( s , 1h ) , 8.238.17 ( m , 1h ) , 7.897.83 ( m , 1h ) , 7.577.50 ( m , 3h ) , 7.477.39 ( m , 4h ) , 3.78 ( s , 2h ) , 3.66 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.72 , 169.89 , 151.36 , 135.73 , 131.66 , 131.49 , 129.70 , 128.69 , 127.15 , 127.00 , 126.11 , 126.07 , 125.40 , 123.21 , 123.02 , 113.51 , 42.24 , 37.21 . esi hrms : m / z 400.0417 ( m h ) . synthesized using the procedure for 1 except 49y was used as the starting material . the title compound ( 50 mg , 61% ) was obtained as a white solid after trituration with a mixture of ch3cn : h2o 1:1 and cold ch2cl2 and without a need for hplc purification . h nmr ( 400 mhz , dmso - d6 ) 12.78 ( s , 1h ) , 9.97 ( s , 1h ) , 9.66 ( s , 1h ) , 8.268.12 ( m , 1h ) , 7.927.77 ( m , 1h ) , 7.597.47 ( m , 3h ) , 7.42 ( d , j = 7.64 hz , 2h ) , 7.37 ( t , j = 7.40 hz , 2h ) , 7.317.24 ( m , 1h ) , 3.77 ( s , 2h ) , 3.67 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 171.73 , 170.25 , 151.26 , 136.74 , 129.70 , 129.58 , 128.76 , 127.10 , 126.97 , 126.92 , 126.19 , 126.06 , 125.38 , 123.22 , 122.99 , 113.49 , 43.09 , 37.13 . esi hrms : m / z 366.0802 ( m h ) . synthesized using the procedure for 1 except 49z was used as the starting material . the title compound ( 10 mg , 8% ) was obtained as a white solid after hplc purification . h nmr ( 400 mhz , cdcl3 ) 8.23 ( d , j = 7.40 hz , 1h ) , 7.79 ( d , j = 7.40 hz , 1h ) , 7.597.50 ( m , 4h ) , 7.507.45 ( m , 1h ) , 7.37 ( s , 1h ) , 7.26 ( s , 1h ) , 6.60 ( s , 1h ) , 3.49 ( s , 1h ) , 2.65 ( t , j = 7.24 hz , 2h ) , 1.521.45 ( m , 2h ) , 1.451.35 ( m , 2h ) , 0.90 ( t , j = 7.24 hz , 3h ) . c nmr ( 100 mhz , cdcl3 ) 154.04 , 138.33 , 132.18 , 131.86 , 131.23 , 128.98 , 128.09 , 127.90 , 126.29 , 123.80 , 123.47 , 122.92 , 122.16 , 111.25 , 36.61 , 31.70 , 21.74 , 13.60 . esi hrms : m / z 463.9998 ( m h ) . to a suspension of 41 ( 75 mg , 0.15 mmol ) in dry ch2cl2 ( 1.5 ml ) was added bbr3 ( 1 m in ch2cl2 , 0.4 ml , 0.4 mmol ) dropwise at 0 c . the mixture was allowed to warm up to room temperature and stirred under nitrogen . the starting material was entirely consumed as determined by tlc and analytical hplc after 30 min . the mixture was again cooled down to 0 c and meoh ( 2 ml ) was added . after addition , the mixture was allowed to warm up to room temperature and stirred for 1 h when a new spot formed as monitored by tlc . the mixture was slowly added to a stirring solution of saturated aqueous nh4cl ( 15 ml ) at 0 c . the combined organic extracts were washed with brine ( 15 ml ) , dried ( mgso4 ) , and filtered . the crude was purified using a c18 reverse phase semipreparative hplc column with solvent a ( 0.1% of tfa in water ) and solvent b ( 0.1% of tfa in ch3cn ) as eluents to give 32 ( 31 mg , 43% ) as a white solid . h nmr ( 400 mhz , dmso - d6 ) 10.08 ( s , 1h ) , 9.90 ( s , 1h ) , 8.15 ( d , j = 8.22 hz , 1h ) , 7.85 ( d , j = 8.22 hz , 1h ) , 7.73 ( d , j = 8.58 hz , 2h ) , 7.55 ( d , j = 8.58 hz , 2h ) , 7.527.41 ( m , 2h ) , 6.99 ( s , 1h ) , 3.60 ( s , 5h ) . c nmr ( 100 mhz , dmso - d6 ) 170.21 , 152.67 , 139.44 , 132.58 , 131.45 , 129.39 , 129.23 , 127.20 , 126.93 , 126.30 , 125.51 , 124.25 , 123.67 , 122.85 , 113.07 , 52.64 , 36.18 . esi hrms : m / z 479.9586 ( m h ) . synthesized using the procedure for 1 except 41 was used as the starting material . the title compound ( 24 mg , 42% ) as a yellow solid after trituration with a mixture of ch3cn : h2o 1:1 and cold ch2cl2 and without a need for hplc purification . h nmr ( 400 mhz , dmso - d6 ) 11.14 ( s , 1h ) , 9.98 ( s , 1h ) , 8.14 ( d , j = 8.15 hz , 1h ) , 7.877.78 ( m , 2h ) , 7.69 ( d , j = 8.16 hz , 2h ) , 7.567.48 ( m , 3h ) , 7.44 ( p , j = 6.86 hz , 2h ) , 6.98 ( s , 1h ) , 3.47 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 173.00 , 154.82 , 139.41 , 132.55 , 132.15 , 131.51 , 129.34 , 127.54 , 126.92 , 126.20 , 125.68 , 123.58 , 123.23 , 112.74 . esi hrms : m / z 464.9591 ( m h ) . synthesized using the procedure for 1 except 49aa was used as the starting material . the title compound ( 16 mg , 32% ) was obtained as a white solid after hplc purification . h nmr ( 400 mhz , dmso - d6 ) 9.87 ( s , 1h ) , 9.17 ( s , 1h ) , 8.11 ( d , j = 8.34 hz , 1h ) , 7.82 ( d , j = 8.34 hz , 1h ) , 7.68 ( d , j = 8.50 hz , 2h ) , 7.49 ( d , j = 8.50 hz , 2h ) , 7.38 ( t , j = 7.23 hz , 1h ) , 7.31 ( t , j = 7.23 hz , 1h ) , 6.73 ( s , 1h ) , 3.34 ( t , j = 6.35 hz , 2h ) , 2.61 ( t , j = 7.48 hz , 2h ) , 1.54 ( p , j = 6.68 hz , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 149.29 , 139.59 , 132.49 , 130.41 , 129.31 , 128.21 , 126.80 , 126.16 , 125.66 , 125.40 , 123.69 , 123.54 , 122.46 , 122.39 , 60.51 , 33.15 , 26.18 . esi hrms : m / z 434.0063 ( m h ) . synthesized using the procedure for 1 except 45 was used as the starting material . the title compound ( 49 mg , 34% ) was obtained as a white solid after hplc purification . h nmr ( 400 mhz , dmso - d6 ) 9.84 ( s , 1h ) , 9.13 ( s , 1h ) , 8.12 ( d , j = 8.17 hz , 1h ) , 7.87 ( d , j = 8.17 hz , 1h ) , 7.69 ( d , j = 8.49 hz , 2h ) , 7.51 ( d , j = 8.49 hz , 2h ) , 7.39 ( t , j = 7.31 hz , 1h ) , 7.33 ( t , j = 7.31 hz , 1h ) , 6.67 ( s , 1h ) , 4.36 ( s , 1h ) , 3.423.33 ( m , 2h ) , 2.58 ( t , j = 5.70 hz , 2h ) , 1.451.26 ( m , 4h ) . c nmr ( 100 mhz , dmso - d6 ) 149.19 , 139.58 , 132.50 , 130.54 , 129.39 , 128.16 , 126.78 , 126.20 , 125.66 , 125.39 , 123.64 , 123.61 , 122.58 , 122.46 , 61.16 , 32.47 , 29.39 , 26.67 . esi hrms : m / z 448.0223 ( m h ) . synthesized using the procedure for 1 except 49cc was used as the starting material . the title compound ( 20 mg , 42% ) was obtained as a white solid after hplc purification . h nmr ( 400 mhz , dmso - d6 ) 10.00 ( s , 1h ) , 8.13 ( d , j = 8.04 hz , 1h ) , 7.75 ( d , j = 8.04 hz , 1h ) , 7.64 ( d , j = 8.62 hz , 2h ) , 7.497.43 ( m , 3h ) , 7.427.36 ( m , 1h ) , 6.91 ( s , 1h ) , 4.25 ( s , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 153.78 , 139.47 , 132.51 , 131.79 , 130.43 , 129.21 , 127.45 , 126.87 , 126.27 , 125.59 , 124.10 , 123.57 , 123.08 , 26.54 . esi hrms : m / z 489.9647 ( m h ) . synthesized using the procedure for 1 except 49dd was used as the starting material . the title compound ( 32 mg , 53% ) as a white solid after hplc purification . h nmr ( 500 mhz , dmso - d6 ) 9.84 ( s , 1h ) , 8.15 ( d , j = 8.4 hz , 1h ) , 7.78 ( d , j = 8.4 hz , 1h ) , 7.52 ( d , j = 8.7 hz , 2h ) , 7.517.46 ( m , 1h ) , 7.447.38 ( m , 3h ) , 7.20 ( t , j = 7.4 hz , 1h ) , 6.996.94 ( m , 5h ) , 4.28 ( s , 2h ) . c nmr ( 125 mhz , dmso - d6 ) 160.70 , 155.63 , 153.61 , 134.27 , 131.88 , 130.73 , 130.54 , 129.77 , 127.33 , 126.16 , 125.60 , 125.06 , 124.51 , 123.71 , 123.04 , 119.98 , 118.35 , 111.64 , 26.59 . esi ms : m / z 503.9 ( m h ) . synthesized using the procedure for 1 except 49ee ( 23 mg , 15% ) was obtained as a white solid after hplc purification . hplc ( method b , tr = 7.08 min ) , purity > 99% . h nmr ( 400 mhz , dmso - d6 ) 12.31 ( s , 1h ) , 10.01 ( s , 1h ) , 9.60 ( s , 1h ) , 8.23 ( s , 1h ) , 8.148.06 ( m , 2h ) , 8.067.97 ( m , 3h ) , 7.80 ( d , j = 8.52 hz , 1h ) , 7.68 ( t , j = 7.36 hz , 1h ) , 7.60 ( t , j = 7.36 hz , 1h ) , 7.43 ( p , j = 6.84 hz , 2h ) , 6.87 ( s , 1h ) , 2.62 ( t , j = 6.83 hz , 2h ) , 2.17 ( t , j = 6.83 hz , 2h ) . c nmr ( 100 mhz , dmso - d6 ) 172.97 , 152.44 , 137.07 , 134.56 , 131.90 , 131.48 , 129.68 , 129.51 , 129.23 , 129.20 , 128.33 , 128.20 , 128.00 , 126.94 , 126.22 , 125.51 , 124.61 , 123.96 , 122.93 , 122.70 , 113.33 , 34.12 , 29.59 . esi hrms : m / z 452.0630 ( m h ) . synthesized using a reported procedure . a stirred solution of 10 ( 50 mg , 0.11 mmol ) in dry thf ( 1.5 ml ) et3n ( 31 l , 0.22 mmol ) was added , and the mixture was stirred for 5 min before acetyl chloride ( 10 l , 0.14 mmol ) was added at 0 c . the mixture was stirred under nitrogen for an additional 20 min then diluted with etoac ( 10 ml ) and washed with h2o ( 10 ml 3 ) . the organic layer was dried ( mgso4 ) , filtered , and concentrated under reduced pressure . the crude was subjected to flash column chromatography ( hexane / etoac 4:1 ) on silica gel to afford 46 ( 34 mg , 61% ) as a white solid . h nmr ( 400 mhz , cdcl3 ) 8.29 ( d , j = 8.10 hz , 1h ) , 7.96 ( d , j = 8.48 hz , 2h ) , 7.76 ( d , j = 8.10 hz , 1h ) , 7.71 ( d , j = 8.48 hz , 2h ) , 7.697.59 ( m , 2h ) , 7.37 ( s , 1h ) , 3.63 ( s , 2h ) , 1.79 ( s , 3h ) . c nmr ( 100 mhz , cdcl3 ) 170.13 , 161.35 , 147.04 , 137.50 , 132.14 , 131.33 , 131.18 , 129.73 , 129.46 , 128.87 , 128.46 , 126.94 , 125.55 , 122.76 , 121.64 , 114.58 , 28.48 , 24.29 . sensitive and quantitative fp - based binding assays were developed and optimized to determine the binding affinities of small - molecule inhibitors to the recombinant mcl-1 , a1/bfl-1 , bcl - w , bcl-2 , and bcl - xl proteins . protocols for expression and purification of used antiapoptotic proteins and determination of kd values of fluorescent probes to proteins are provided in the supporting information . on the basis of the kd values , the concentrations of the proteins used in the competitive binding experiments were 90 nm for mcl-1 , 40 nm for bcl - w , 50 nm for bcl - xl , 60 nm for bcl-2 , and 4 nm for a1/bfl-1 . the fluorescent probes , flu - bid and fam - bid , were fixed at 2 nm for all assays except for a1/bfl-1 , where fam - bid was used at 1 nm . then 5 l of the tested compound in dmso and 120 l of protein / probe complex in the assay buffer ( 100 mm potassium phosphate , ph 7.5 ; 100 g / ml bovine gamma globulin ; 0.02% sodium azide , purchased from invitrogen , life technologies , supplemented with 0.01% triton x-100 ) were added to assay plates ( microfluor 2black , thermo scientific ) , incubated at room temperature for 3 h , and the polarization values ( mp ) were measured at an excitation wavelength at 485 nm and an emission wavelength at 530 nm using the plate reader synergy h1 hybrid , biotek . ic50 values were determined by nonlinear regression fitting of the competition curves ( graphpad prism 6.0 software ) . the ki values were calculated as described previously . the solution competitive spr - based assay was performed on biacore 2000 . n terminal biotin - labeled bim bh3 peptide ( 141166 amino acids ) was immobilized on streptavidin ( sa ) chip giving density of 1400 ru ( response units ) . the preincubated mcl-1 protein ( 20 nm ) with tested small - molecule inhibitors for at least 30 min was injected over the surfaces of the chip . response units were measured at 15 s in the dissociation phase , and the specific binding was calculated by subtracting the control surface ( fc1 ) signal from the surfaces with immobilized biotin - labeled bim bh3 . ic50 values were determined by nonlinear least - squares analysis using graphpad prism 6.0 software . crystal structure of mcl-1 in complex with mnoxa bh3 peptide ( pdb entry 2nla ) and in silico schrdinger s ifd were used to model the binding poses of our designed compounds with mcl-1 . ifd is allowing incorporation of the protein and ligand flexibility in the docking protocol , which consists of the following steps : ( i ) constrained minimization of the protein with an rmsd cutoff of 0.18 , ( ii ) initial glide docking of the ligand using a softened potential ( van der waals radii scaling ) , ( iii ) one round of prime side chain prediction for each protein / ligand complex , on residues within defined distance of any ligand pose , ( iv ) prime minimization of the same set of residues and the ligand for each protein / ligand complex pose , ( v ) glide redocking of each protein / ligand complex structure within a specified energy of the lowest energy structure , ( vi ) estimation of the binding energy ( ifdscore ) for each output pose . all docking calculations were run in the extra precision ( xp ) mode of glide . the center of the grid box of the mcl-1 was defined by the val 249 ( in h1 ) , phe 270 ( in h2 ) , val 220 ( in h3/h4 ) , and val 216 ( in h4 ) . mc / sd dynamic simulation performs constant temperature calculations that take advantage of the strengths of monte carlo methods for quickly introducing large changes in a few degree of freedom and stochastic dynamics for its effective local sampling of collective motions . the mc / sd dynamic simulation time in our study was set to 100 ps by allowing movement of the docked ligand and the residues , which is less than 6 to the ligand . n - labeled or n , c - labeled mcl-1 proteins for nmr studies were prepared and purified using the same protocol as for unlabeled protein with the exception that the bacteria were grown on m9 minimal media supported with 3 g / l of c - glucose and/or 1 protein samples were prepared in a 20 mm sodium phosphate , 150 mm nacl , and 1 mm dtt solution at ph 7 in 7% d2o . the binding mode of the compounds has been characterized by recording h , n -hsqc experiments with a 138 l solution of uniformly n - labeled mcl-1 ( 75 m ) in the absence and presence of added compounds with the indicated molar ratio concentrations . all spectra were acquired at 30 c on a bruker 600 mhz nmr spectrometer equipped with a cryogenic probe , processed using bruker topspin and rnmr , and were analyzed with sparky . plots of chemical shift changes were calculated as ( ( h ppm ) + ( 0.2(n ppm ) ) ) of mcl-1 amide upon addition of compound . the absence of a bar in a chemical shift plot indicates no chemical shift difference or the presence of a proline or residue that is overlapped or not assigned . human breast cancer 2lmp cells , a subclone of the mda - mb-231 cell line , were lysed in chaps buffer ( 10 mm hepes ( ph 7.4 ) , 2.5 mm edta , 150 mm nacl , 1.0% chap ) . precleared cell lysates were incubated with different concentrations of compounds followed by incubation with biotinylated noxa bh3 peptide ( 1843 ) and streptavidin agarose beads to pull - down mcl-1 protein bound to noxa peptide . beads were washed with chaps buffer , and mcl-1 protein was eluted by boiling in sds - page sample buffer and analyzed by western blotting using mcl-1 antibody ( santa cruz ) . mefs cells , wild - type and bax / bak double knockout , were gifts from shaomeng wang at the university of michigan and were cultured in dmem ( life technologies ) , supplemented with 10% fetal bovine serum ( fbs ) ( thermo scientific hyclone ) . the retroviral transduced lymphoma cells isolated from e-myc transgenic mice were gifts from ricky w. johnstone at university of melbourne , melbourne , australia , and cultured as previously described . human leukemia cell lines hl-60 , k-562 , and mv-411 were obtained from american type culture collection ( atcc ) . the cells were cultured in rpmi 1640 medium ( life technologies ) , all supplemented with 10% fbs . mefs cells were seeded in 12-well plates at 0.5 10 cells / well , left to adhere , and then treated for 15 h with increasing concentrations of the compounds . the cells were harvested , washed with phosphate - buffered saline ( pbs ) , and stained with 0.025 mg / ml propidium iodide ( mp biomedicals ) . the percentage of propodium iodide positive population was determined by flow cytometry and calculated using winlist 3.0 . the mcl-1 and bcl-2 retroviral transduced lymphoma e-myc cells were seeded in 24-well plates at 0.5 10 cells / well . they were treated with different concentrations of tested compounds for 1518 h. the cells were harvested and stained with violet live / dead fixable dead cell stain kit ( invitrogen ) according to manufacturer s protocol . the percentage of fluorescent positive cells was determined by flow cytometry and calculated using winlist 3.0 . the effect of the compounds on tested leukemia cells viability was evaluated by celltiter glo luminescent cell viability assay ( promega ) . cells were plated in 12-well plates at 0.5 10 cells / well and treated with various concentrations of the compounds and incubated for 3 days . cell viability was determined by measuring intracellular atp levels with the celltiter glo reagent and reading the luminescence with the synergy h1 hybrid biotek plate reader . percent cell growth was calculated relative to dmso treated cells , and ic50 values were calculated by nonlinear regression analysis using graphpad prism 6.0 software . hl-60 cells were seeded in 12-well plates at 0.5 10 cells / well . after 20 h treatment with different concentrations of the compounds , caspase 3 activity was determined using the fluorometric substrates devd - afc following the protocol of the caspase-3 fluorometric assay kit ( biovision ) . caspase 3 activity is reported as the fold change relative to dmso treated cells . to determine the induction of apoptosis , hl-60 cells were plated in 12-well plates at 0.5 10 cells / well and treated with various concentrations of tested compounds . after 20 h , cells were harvested , washed with pbs , and treated with annexin - v fitc and propidium iodide using bd annexin v fitc assay kit ( bd biosciences pharmingen ) . the percentage of cells undergoing apoptosis was assessed by flow cytometry within 1 h and analyzed using winlist 3.0 . apoptosis was also determined in the presence of z - vad - fmk ( bachem ) , pan caspase - inhibitor . for this purpose sells were pretreated with 100 m z - vad - fmk for about 1 h before adding tested compounds .

mcl-1 , an antiapoptotic member of the bcl-2 family of proteins , is a validated and attractive target for cancer therapy . overexpression of mcl-1 in many cancers results in disease progression and resistance to current chemotherapeutics . utilizing high - throughput screening , compound 1 was identified as a selective mcl-1 inhibitor and its binding to the bh3 binding groove of mcl-1 was confirmed by several different , but complementary , biochemical and biophysical assays . guided by structure - based drug design and supported by nmr experiments , comprehensive sar studies were undertaken and a potent and selective inhibitor , compound 21 , was designed which binds to mcl-1 with a ki of 180 nm . biological characterization of 21 showed that it disrupts the interaction of endogenous mcl-1 and biotinylated noxa - bh3 peptide , causes cell death through a bak / bax - dependent mechanism , and selectively sensitizes e-myc lymphomas overexpressing mcl-1 , but not e-myc lymphoma cells overexpressing bcl-2 . treatment of human leukemic cell lines with compound 21 resulted in cell death through activation of caspase-3 and induction of apoptosis .

Introduction Results and Discussion Conclusions Experimental Section

evasion of apoptosis or programmed cell death , a key regulator of physiological growth control and regulation of tissue homeostasis , is a hallmark of cancer and a contributor to the emergence of resistance to current therapies . the b - cell lymphoma-2 ( bcl-2 ) family of proteins regulate the intrinsic ( mitochondrial ) pathway of apoptosis through a network of protein protein interactions between pro- and antiapoptotic members . the bcl-2 antiapoptotic proteins , consisting of bcl-2 , bcl - xl , bcl - b , bcl - w , mcl-1 , and a1 , share up to four bh domains which form the hydrophobic bh3-binding groove for binding their cognate partners . the bh3 domain possesses four conserved hydrophobic residues that are involved in the interaction with bh3-binding groove of the pro - survival bcl-2 family members , which results in the sequestering and blocking of the function of pro - death members . overexpression of bcl-2 survival members is observed in different types of human tumor samples and cancer cell lines , and much effort has been focused on developing therapeutics against this family of proteins for the treatment of cancers . several studies have shown that resistance to abt-263 and its analogue abt-737 is linked to high expression levels of mcl-1 , and in many instances this resistance can be overcome by treatment with agents that downregulate , destabilize , or inactivate mcl-1 . these data suggest that therapies specifically targeting mcl-1 , either as a single agent or in combination , can be effective in the treatment of different human cancers . herein we disclose a novel series of small - molecule mcl-1 inhibitors discovered through high - throughput screening ( hts ) followed by the utilization of structure - based design to develop structure activity relationships ( sar ) around lead compound 1 ( figure 1a ) . docking studies and two - dimensional h n heteronuclear single quantum coherence spectroscopy ( hsqc ) nmr studies were employed to provide information about its binding mode which was used for the design and synthesis of additional analogues . hts of a 53.3k small - molecule library was performed using a fluorescence polarization ( fp ) binding assay based on the interaction between recombinant human mcl-1 and fluorescently labeled bid bh3 peptide ( flu - bid ) . compound 1 ( figure 1a ) was one of the validated hits , which was also previously identified as a proteasome inhibitor . compound 1 was resynthesized and its binding to mcl-1 was confirmed with a ki of 1.55 0.18 m . compound 1 exhibits similar potency as mim1 ( ic50 = 4.72 m ) , mcl-1 small - molecule inhibitor with a thiazolyl substituted core , which was also discovered with high - throughput competitive fp screen approach . the bei of compound 1 , calculated as a ratio between pki and molecular weight , is 14.7 , which encouraged us to further pursue with modifications of this scaffold . to explore the binding mode of 1 with mcl-1 , in silico induced fit docking ( ifd ) studies were performed using the crystal structure of mcl-1 in complex with mnoxa bh3 peptide ( pdb i d : 2nla ) . the importance of the two conserved hydrophobic residues , leu and ile , in the bh3 binding motif for the high affinity interaction and selective binding to mcl-1 has been demonstrated with structural studies of bims2a , a highly selective peptide derived from bim bh3-only protein , as well as from recently reported selective small - molecule inhibitor in complex with mcl-1 . a recent report on the conformational flexibility of mcl-1 and its binding hotspots identified his 224 as an acidic hotspot in the h3 site of mcl-1 , supporting the predicted hydrogen bonding in this region of mcl-1 . to validate the computationally predicted binding site and confirm the binding of 1 to the bh3 groove of mcl-1 protein , hsqc nmr spectroscopy studies were performed . overall analysis of the chemical shift changes of the compound 1 in complex with mcl-1 showed that 1 affects the residues forming the bh3-binding groove and provided conclusive evidence that 1 binds mcl-1 protein at the same site that the conserved bh3 peptides interact with mcl-1 protein . a structure - based design approach was undertaken , and a focused library of analogues of 1 was designed and synthesized to improve the potency of this series . when r1 is changed to a methyl group in 2 , the binding affinity is significantly reduced , confirmed by spr ( ic50 > 100 m ) and nmr experiments which showed lack of chemical shift perturbation of backbone residues in the mcl-1 bh3 binding site after adding 2 ( supporting information figure s1 ) . bromine substitution at the meta-(12 ) and ortho - positions ( 13 ) of the phenyl ring was also explored , and the obtained binding results from both assays , fp ( ki of 2.35 0.19 m and 4.42 0.86 m , respectively ) and spr ( ic50 = 9.68 0.88 m and ic50 = 6.18 1.24 m , respectively ) , indicated that para - bromo substitution in 10 is the best , exhibiting the highest binding affinity to mcl-1 among these three isomers . plots of chemical shift changes of mcl-1 amide upon addition of ( d ) 16 ( mcl-1:16 ratio of 1:2 ) , ( e ) 17 ( mcl-1:17 ratio of 1:2 ) , ( f ) , and 18 ( mcl-1:18 ratio of 1:2 ) as a function of mcl-1 residue numbers . ( d ) overlay of putative binding modes of 16 ( purple ) , 17 ( blue ) , and 18 ( green ) to mcl-1 ( pdb i d : 2nla ) highlighting in red val 249 , met 250 , and val 253 on helix 4 , phe 228 on helix 3 of mcl-1 . satisfyingly , 19 with para - chlorobiphenyl showed a 21-fold improved binding compared to 3 , becoming the most potent analogue in our series , with a ki = 0.17 0.04 m determined by the fp binding assay using fluorescent labeled bid bh3 peptide and ic50 of 0.88 0.15 m in displacement of biotin labeled bim bh3 peptide in the spr - based assay ( 14-fold improvement ) . in comparison with compound 53 ( ki of 0.055 m ) , recently reported mcl-1 selective inhbitor with an indole core structure , discovered and optimized by fragment - based screening strategy,19 and 21 have binding affinity in a similar nanomolar range , being 3-fold less potent than 53 . ( b ) plot of chemical shift changes of mcl-1 amide upon addition of 21 ( mcl-1:21 ratio of 1:2 ) as a function of mcl-1 residue numbers . to briefly explore the importance of the naphthalene core to the binding potency , which occupies the h3 hydrophobic pocket , analogue 24 with a phenyl core was synthesized . to investigate the importance of the sulfonamide linker , replacement of the sulfonamide with a carboxamide in 25 led to significant 83-fold decrease of the binding affinity with ki value of 40.8 8.50 m compared to 10 , possibly due to the unfavorable orientation of r1 by the carboxamide linker . as expected , change of the sulfonamide linker in 26 to a carboxamide in 28 decreased the potency , but only by 2-fold ( ki = 12.53 1.24 m ) , suggesting that the insertion of the methylene linker between the carboxamide and the pendant aryl ring provids a degree of freedom to better orient r1 into h2 pocket of mcl-1 . however , changing the point of fusion of the naphthalene ring in 38 substantially affected the binding and 39 with 1-naphthyl substituent showed a significant reduction in binding to mcl-1 . modeling showed that the phenolic group of the core naphthalene scaffold forms a hydrogen bond with his 224 of mcl-1 , consistent with the reported acidic hotspot in the h3 site of mcl-1 close to his 224 . several analogues were synthesized to probe the contribution of the phenolic group to binding to mcl-1 ( table 4 ) . the analogue 41 , where r2 is methyl thioacetate and r3 is methoxy , did not show binding up to 100 m , and the loss of the binding was confirmed with spr and hsqc experiment ( supporting information figure s5 ) . similar significant loss of binding is also apparent in compounds 42 to 45 compared to their corresponding phenolic analogues , clearly indicating the importance of the phenolic group to the overall binding to mcl-1 . compound 46 , where the hydroxyl group was acetylated exhibited a ki of 33.97 15.96 m , which is a 69-fold decrease in binding compared to 10 . however , 46 showed improved binding relative to 40 , which might be attributed to the formation of a hydrogen bond between the carbonyl of the acetyl group and the protonated his 224 , supported by computational prediction . as the bh3 domain binding profile of bfl-1/a1 , as well as its bh3 binding groove , is most similar to that of mcl-1 , it is not surprising that the tested compounds showed less selective inhibition of a1 . the most potent analogues in this series , 19 and 21 , show a profile for selectively inhibiting mcl-1 with 7-and 19-fold versus bfl-1/a1 , 8-and 9-fold versus bcl - w , 36- and 42-fold versus bcl-2 , and 56- and 59-fold versus bcl - xl , respectively . as shown in figure 5 , mcl-1 was pulled down by bl - noxa and , as was expected , the bim bh3 peptide disrupted the interaction between bl - noxa and mcl-1 . preincubation with several mcl-1 inhibitors , 10 , 19 , and 21 , completely blocked the binding of bl - noxa to mcl-1 , similar to bim peptide , demonstrating that these inhibitors can recognize and specifically bind to the bh3 binding groove of endogenous mcl-1 protein . exposure of the wt mefs to 21 resulted in a concentration - dependent cell death ( assessed by pi staining ) , while bax / bak deletion significantly rescued cells from 21 induced cell death ( figure 6 ) . cell death induced by mcl-1 inhibitor 21 is bax / bak - dependent . mefs deficient in bax and bak ( gray bars ) along with their wild - type counterpart ( black bars ) were exposed for 15 h to different concentrations of 21 , and the cell viability was assessed with pi staining . to further confirm the specificity of our novel mcl-1 inhibitors and to determine whether different prosurvival bcl-2 proteins could suppress the apoptotic activities of novel mcl-1 inhibitors , we used reported cell lines developed by retroviral transduction of lymphoma cells isolated from e-myc transgenic mice which differ only in their expression of prosurvival bcl-2 family proteins . lymphoma cells overexpressing mcl-1 and bcl-2 were treated with varying concentrations of tested compounds for 1518 h , and then cell viability was determined by flow cytometry using a fluorescent reactive dye ( live / dead fixable violet stain kit ) . a selective inhibitor of bcl-2 , bcl - xl , and bcl - w , was used as a positive control . as predicted , lymphoma cells overexpressing mcl-1 were significantly sensitive to 19 and 21 as assessed by an increased percentage of cell death in a concentration - dependent manner . importantly , 41 did not show any activity against both cell lines overexpressing mcl-1 or bcl-2 , consistent with our binding studies which showed that 41 does not bind to mcl-1 . as expected , lymphoma cells overexpressing bcl-2 were sensitive to cell death induced by abt-263 , while cells overexpressing mcl-1 were insensitive to abt-263 , consistent with its binding specificity . sensitivity of e-myc lymphoma cells overexpressing mcl-1 and bcl-2 antiapoptotic proteins to inhibitor - induced cell death . e-myc / mcl-1 and e-myc / bcl-2 lymphomas were treated for 1518 h with increasing concentrations of 19 , 21 , 41 , and abt-263 . it has been shown that aml - derived cell lines , hl-60 and mv4,11 , are sensitive to inhibition of the antiapoptotic protein mcl-1 , while cml - derived k-562 cell line is less sensitive to mcl-1 inhibition . tested compounds from our series showed inhibition of the cell growth in a dose - dependent manner with similar potencies , ic50 values ranging from 2.06 to 11.72 m against the hl-60 and mv4,11 cell lines ( figure 8a ) . it is important to be pointed out that 36 and 37 , where the carboxylic acid was replaced with a bioisostere tetrazole group , show similar cellular activity in all tested cell lines in comparison with their parent compounds , 10 and 21 , respectively . furthermore , compound 32 , in which the acid group has been replaced with methyl ester and showed 10-fold less binding to mcl-1 as compared with 10 , has also similar ic50 values in tested cell lines as 10 . treatment of the hl-60 cells by 2.5 , 5.0 , and 10 m of 21 and 37 for 20 h results in 28.9% and 23.5% , 37.8% and 51.6% , and 78.4% and 74.2% of apoptotic cells ( early + late ) , respectively , as compared to 7.6% and 7.3% of apoptotic cells in the dmso controls . to determine if the induction of apoptosis by compound 37 in the hl-60 cell line depends upon caspases , we treated the cells with compound 37 alone or in the presence of z - ved - fmk , a pan - caspase inhibitor ( figure 8b and supporting information figure s7 ) . the obtained results showed that the induction of apoptosis was significantly inhibited in the presence of z - vad - fmk , clearly indicating that the apoptotic activity of compound 37 is mediated by caspases . compound 41 at 40 m has no effect on apoptosis induction just like untreated control , which is consistent with its lack of binding to mcl-1 and inhibition of cell growth . both compounds induce activation of caspase-3 activity in a dose - dependent manner , with 21 effectively inducing activation over a 24 h period starting at 2.5 m . cell death and apoptosis induction by mcl-1 inhibitors in human leukemic cell lines . employing structure - based design supported by nmr studies , we synthesized a focused library of analogues and established a sar for binding to mcl-1 . using wild - type and bax / bak double knockout mefs cells , the contribution of these two multidomain pro - apoptotic proteins in 21-induced cell death was determined and demonstrated that 21 primarily causes cell death in wild - type mefs in a bax / bak - dependent manner . furthermore , 19 and 21 led to sensitization of e-myc lymphomas overexpressing mcl-1 but did not show effect on cells overexpressing bcl-2 antiapoptotic protein , confirming their selective targeting of mcl-1 . to the solution of thiol , cs2co3 , and zncl2 was added 47 ( 1.0 g , 3.1 mmol ) , lii ( 200 mg , 1.5 mmol ) , and premixed solution of the catalyst and ligand . sensitive and quantitative fp - based binding assays were developed and optimized to determine the binding affinities of small - molecule inhibitors to the recombinant mcl-1 , a1/bfl-1 , bcl - w , bcl-2 , and bcl - xl proteins . on the basis of the kd values , the concentrations of the proteins used in the competitive binding experiments were 90 nm for mcl-1 , 40 nm for bcl - w , 50 nm for bcl - xl , 60 nm for bcl-2 , and 4 nm for a1/bfl-1 . crystal structure of mcl-1 in complex with mnoxa bh3 peptide ( pdb entry 2nla ) and in silico schrdinger s ifd were used to model the binding poses of our designed compounds with mcl-1 . the center of the grid box of the mcl-1 was defined by the val 249 ( in h1 ) , phe 270 ( in h2 ) , val 220 ( in h3/h4 ) , and val 216 ( in h4 ) .

cancer is diagnosed in more than 12,000 children in the united states every year . over the past 25 years , the 5- and 10-year survival rates for childhood cancers have substantially improved , from less than 50% in the 1970s to almost 80% today [ 2 , 3 ] . some of this improvement comes from the use of anthracycline chemotherapeutic agents , which are widely used to treat childhood leukemias and lymphomas , as well as other malignancies . their use has helped create a growing population of long - term childhood cancer survivors of more than 325,000 in the united states alone . however , this growing population of survivors is at a substantial risk for treatment - related complications that can markedly affect their quality of life . increasingly , survivors and their clinicians are realizing the importance of continuous monitoring long after their cancer treatment has been completed . within the first 30 years after diagnosis recent 5-year estimates indicate that the leading non - cancer - related cause of morbidity and mortality in long - term survivors of childhood cancer is cardiovascular - related disease [ 510 ] . survivors are 8 times more likely than the general population to die from cardiovascular - related disease , and compared to sibling controls , they are 15 times as likely to suffer from heart failure ( hf ) , more than 10 times as likely to have coronary artery disease , and more than 9 times as likely to have had a cerebrovascular accident during the first 30 years after cancer diagnosis . anthracyclines , such as doxorubicin , are among the leading causes of these cardiovascular events . the cardiotoxic nature of anthracyclines , coupled with their widespread use , may explain the large impact they have made on survivor morbidity and mortality . childhood cancer survivors exposed to anthracyclines are at a significantly increased risk of hf , and this risk increases as the cumulative dose increases ; 30 years after diagnosis , more than 7.5% will have experienced hf . the risks of cardiac events persist up to 45 years beyond treatment and are the second most common cause of death , after secondary malignancy . in addition to clinical outcomes , detailed cardiac evaluations have shown that more than half of anthracycline - exposed childhood cancer survivors have subclinical cardiac abnormalities , including decreased left ventricular ( lv ) mass and wall thickness , increased lv afterload , and decreased lv contractility . the severity of the cardiotoxic effects of anthracyclines vary and are categorized by time of onset as acute , occurring during or immediately after treatment , early , occurring within 1 year of exposure , and late , occurring 1 or more years after initial exposure ( table 1 ) [ 11 , 15 ] . severe cardiotoxicity , during or shortly after treatment , is strongly associated with hf later in life , despite an initial asymptomatic interval . in a followup study of long - term survivors of childhood cancer who were treated with anthracyclines and who experienced acute hf , all had a temporary recovery though nearly half of these patients later had recurrent hf . understanding the early- and late - onset cardiovascular complications of long - term survivors of childhood cancer is important for oncologists , cardiologists , and other health care providers involved in the care of such patients , not only after cancer treatment but also when selecting treatments at the time of diagnosis . here , we review the cardiovascular complications associated with the use of anthracyclines in treating malignancies in children and discuss methods for preventing , screening , and treating such complications in childhood cancer survivors . anthracyclines are commonly used to treat a variety of solid and hematologic tumors in both adults and children . however , because they have clinically relevant dose - dependent cardiotoxicity , lower doses are used in treating childhood cancer [ 18 , 19 ] . anthracyclines express their anticancer effects through two major mechanisms : ( 1 ) their intercalation between base pairs of dna prevents cancer cells from replicating and ( 2 ) their inhibition of topoisomerase - ii activity prevents the uncoiling process of dna that is necessary for replication and transcription . however , the specific mechanisms of anthracycline cardiotoxicity are complex and , despite decades of research , remain unclear . the oxidative stress hypothesis is among the most widely studied and accepted cellular mechanism thought to cause cardiotoxicity [ 2023 ] . once administered , anthracyclines are believed to enter cells through passive diffusion , where they can reach intracellular concentrations several hundred times higher than that in extracellular compartments . once inside the cell , anthracyclines may form complexes with intracellular iron , leading to the production of free radicals , which can cause dna damage and lipid peroxidation . the damage caused by free radicals , and by reactive oxygen species ( ros ) , can eventually lead to cell death and larger - scale organ damage . why the heart is particularly vulnerable to oxidative stress caused by anthracyclines is not clear , but the abundance of mitochondria found in cardiomyocytes may be involved . increasingly , the importance of mitochondria as key mediators of anthracycline - induced cardiotoxicity has been reported . cardiolipin , a phospholipid found in high concentrations on the inner cell membrane of cardiac mitochondria , has a high affinity for anthracyclines . this affinity allows increased concentrations to enter cardiac mitochondria [ 25 , 26 ] , which may impair membrane stability or lead to mitochondrial dna damage by intercalation . anthracycline effects on the mitochondria may also impair the cell 's ability to produce energy and to handle the added oxidative stress of cancer and its treatments [ 21 , 2729 ] . in addition , one of the heart 's key antioxidants , glutathione peroxidase , is depleted in the presence of anthracyclines . several other mechanisms have been suggested for anthracycline cardiotoxicity , including the induction of apoptosis , abnormal creatine kinase activity , the production of vasoactive amines , the formation of toxic metabolites , upregulation of nitric oxide synthetase , and the inhibition of transcription and translation [ 3133 ] . anthracyclines cause uncoupling of the electron transport chain , which creates highly reactive oxygen species , and can impair oxidative phosphorylation and adenosine triphosphate ( atp ) synthesis . anthracyclines can also impair mitochondrial calcium homeostasis , leading to the loss of stability of the mitochondrial membrane , decreased atp , and cell death . in addition , several other changes have been observed in anthracycline - exposed cardiomyocytes , for which the underlying mechanisms have yet to be fully explained . these changes include depleted cardiac stem cells , impaired dna synthesis , impaired cell signaling that triggers cell death , altered gene expression , inhibited calcium release from the sarcoplasmic reticulum , impaired formation of the protein titin in sarcomeres , and impaired mitochondrial creatine kinase activity and function . none of these findings , however , are necessarily completely dependent or independent of an oxidative stress pathway . the fact that many of these subcellular consequences continue to progress for weeks after anthracycline exposure may provide insight into the mechanisms of chronic cardiomyopathy . finally , understanding cardiovascular cell signaling proteins may prove useful by clarifying both the pathway of toxicity and the early detection of anthracycline - induced cardiotoxicity . because of the synergistic cardiotoxic potential of anthracyclines and the erbb2 antibody , trastuzumab , interest in the role of neuregulin ( nrg-1 ) is growing . neuregulin is a growth factor that is an endogenous ligand for the protein erbb2 and is possibly involved in a signaling pathway that regulates sarcomere functioning . however , this decrease has not been clearly associated with serum and echocardiographic markers of cardiac dysfunction . not all children exposed to anthracyclines experience cardiac abnormalities , and the clinical severity of the abnormalities that do occur varies greatly between individuals . hence , determining the factors that may increase susceptibility to the cardiotoxic effects of anthracyclines is of great importance . first , ascertaining which patients are most likely to experience cardiotoxicity can guide treatment , especially as long - term health becomes a more prominent treatment focus . second , identifying risk factors may help to determine the mechanisms directly responsible for cardiac damage and potentially lead to novel strategies for cardioprotection and treatment . one of the main risk factors for anthracycline cardiotoxicity is high cumulative dose . since the 1970s , studies have shown that higher cumulative doses of anthracyclines and higher infusion rates are risk factors for anthracycline cardiotoxicity [ 10 , 19 ] . the strong association between cumulative anthracycline dose and cardiotoxicity appears to be more important with increasing time from treatment [ 44 , 45 ] . this relationship was revealed in a study of nearly 15,000 long - term survivors of childhood cancer treated with anthracyclines who described their cardiac health at up to 30 years after cancer treatment ( figure 1 ) . other known risk factors for anthracycline cardiotoxicity include younger age at treatment , female sex , the use of concomitant cardiotoxic therapies ( such as mediastinal radiation ) , increasing time since treatment , cardiac injury , as indicated by elevations of serum cardiac troponin - t ( ctnt ) , neurohormonal activation of cardiac myocytes in response to pressure and stress , as indicated by elevations of n - terminal probrain natriuretic peptide ( nt - probnp ) , during anthracycline therapy , and hf during anthracycline therapy [ 5 , 10 , 14 , 19 , 28 , 45 ] . although not completely understood , genetic predisposition may also be an important factor in determining the risk of anthracycline cardiotoxicity [ 4648 ] . genetic polymorphisms may alter membrane permeability , antioxidant capacity , or metabolism that favors the creation of cardiac damage . hereditary hemochromatosis , a genetic disorder prevalent in individuals of european descent that can lead to iron overload , is of particular interest because doxorubicin 's cardiotoxic effects depend , at least in part , on its interaction with iron . in mice , hfe deficiency , the defective gene in hereditary hemochromatosis , increases the susceptibility to doxorubicin - induced cardiotoxicity , including mitochondrial degradation and increased mortality , when compared to that found in wild - type mice . this fact suggests that genetic mutations related to defects in iron metabolism may contribute to cardiotoxicity in humans although there is currently no literature on the effect in humans . the concept of genetic predisposition as a risk factor for cardiotoxicity is further supported by findings of greater cardiac susceptibility in patients with trisomy 21 and black race [ 50 , 51 ] . despite the identification of these population - based risk factors , determining therefore , it is important that all children who receive anthracycline therapy be followed closely , both during and after treatment for cardiotoxicity . as a guide , the long - term follow up program resource guide , developed by children 's oncology group 's nursing discipline committee in collaboration with the late effects committee , provides recommendations for screening and management of late effects of therapeutic exposures used during treatment for pediatric malignancies . although identifying multiple risk factors has helped to characterize groups at high risk for cardiac injury , both tolerance of chemotherapy and predisposition to cardiac damage still vary substantially between patients . this difference has led to an increasing interest in the use of monitoring serum biomarkers as a means of evaluating cardiotoxicity during and after treatment with anthracyclines . elevations in cardiac troponin - t and i ( ctnt and ctni ) accurately indicate cardiac damage , and their presence in serum in any detectable amount may indicate irreversible cellular necrosis [ 45 , 54 , 55 ] . cardiac troponin - t is a cardiac - specific isoform that is present in cardiac myocytes in both the contractile unit and the cytoplasm . serum cardiac troponins are widely used in diagnosing and managing ischemic heart disease in adults and also provide valuable clinical information in many other types of cardiac damage in children . initial investigations in animal models found dose - dependent ctnt elevations in response to doxorubicin administration , elevations that were related to the severity of histopathologic findings in cardiac tissue . this relationship is consistent with the hypothesis that anthracyclines damage cardiomyocytes , leading to the release of intracellular ctnt into the circulation . the dana - farber cancer institute childhood acute lymphoblastic leukemia ( all ) consortium protocol 95 - 001 study found that elevated serum ctnt levels during the first 90 days of anthracycline therapy were significantly associated with reduced lv mass and lv end - diastolic posterior wall thickness as measured by echocardiography 4 years later [ 58 , 59 ] . the cardiac contractile protein , ctni , when present in the serum , is related to lv dysfunction and increased cardiovascular risk in adults receiving high - dose chemotherapy [ 54 , 55 ] . these findings provide evidence of the value of serum troponin measurements in childhood cancer patients receiving anthracyclines . n - terminal probrain natriuretic peptide is produced during the cleavage of probnp , a prohormone released from the cardiac ventricles in response to pressure overload and stretching [ 60 , 61 ] . chronic elevations in nt - probnp indicate increased ventricular wall stress , in association with pressure overload and elevated diastolic pressure [ 62 , 63 ] . the aforementioned dana - farber cancer institute protocol 95 - 001 study also assessed serum nt - probnp measurements during and after therapy as possible predictors of cardiac damage . elevations in nt - probnp during the first 90 days of therapy were associated with an abnormal lv thickness - to - dimension ratio 4 years later , suggesting pathologic ventricular remodeling . additionally , a higher percentage of children had elevated levels of nt - probnp than had elevated levels of ctnt before , during , and after treatment . this difference suggests that nt - probnp may detect cardiac stress before any irreversible cell damage and death occurs , which may help identify children early in therapy who are at increased risk of eventual anthracycline - related cardiac abnormalities . as a global inflammatory marker and a critical component of the immune system , high - sensitivity c - reactive protein ( hscrp ) systemic inflammation is associated with increased rates of cardiovascular disease in adults and may also be involved with the mechanisms underlying anthracycline - related cardiotoxicity and pediatric cardiomyopathy . as such , elevations in hscrp may be a strong indicator of cardiac stress . despite a small sample size , one study of 19 children with hf divided into three groups based on symptom severity found that serum hscrp levels were associated with decreased lv function and discriminated between the different groups of symptom severity . another study of 156 survivors of childhood all showed that survivors had significantly elevated levels of hscrp when compared to levels in sibling controls . these findings have encouraged further research on the use of serum hscrp measurements collected during therapy as potential predictors of late cardiac effects . serum hscrp may prove to be a valuable screening tool for identifying long - term survivors at increased risk of subsequent cardiac disease . the ability to identify heart - derived tissue proteins associated with myocardial injury that can be detected before marked elevations in ctnt could have profound implications on earlier detection and clinical monitoring for doxorubicin cardiotoxicity . one pilot study using nanoparticle - mass spectrometry identified several candidate protein biomarkers previously implicated in cardiac dysfunction , remodeling , fibrosis , and hypertrophy . identification of these candidate biomarkers have the potential to increase the predictive value of more routinely used markers , such as ctnt , for earlier detection of cardiac damage , before irreparable damage and loss of cardiomyocytes occur . however , due to the small limited sample size , further studies evaluating the efficacy of nanoparticle - mass spectrometry may advance this novel diagnostic approach . several detailed echocardiographic studies in 115 long - term childhood all survivors treated with doxorubicin have been conducted to better determine the long - term cardiac status of these patients [ 14 , 69 , 70 ] . these studies , ranging from 6 to 12 years of followup after treatment , have documented a persistent and progressive restrictive - like cardiomyopathy in long - term childhood cancer survivors treated with anthracyclines [ 14 , 69 , 70 ] . the restrictive cardiomyopathic - like nature of anthracycline cardiotoxicity may be of great clinical importance in that it suggests theories and treatments derived from studies of dilated cardiomyopathy may be of limited value in understanding anthracycline cardiotoxicity throughout life . the findings of abnormal lv structure and function , as well as a restrictive - like cardiomyopathic pattern , are consistent with those of other long - term followup studies conducted in other groups of anthracycline - treated childhood cancer survivors [ 7274 ] . treatment protocols using lower doses of anthracyclines have reduced the incidence of acute cardiac complications to less than 1% although chronic lv dysfunction is still a major clinical concern [ 50 , 53 ] . in the 1970s , before the association between higher cumulative doses of anthracyclines and the greater risk of cardiotoxicity was known , clinical trials administered cumulative doxorubicin doses greater than 400 mg / m to children with all . lipshultz et al . found that patients who received such high cumulative doses experienced clinically important , progressive lv effects that continued even decades after the completion of doxorubicin treatment [ 14 , 69 , 70 ] . on the basis of this experience , cumulative doses of doxorubicin administered to children and adolescents with all were reduced in the 1980s such that high - risk children received a cumulative dose of no more than 360 mg / m , and standard - risk children received a relatively low cumulative dose of no more than 60 mg / m [ 58 , 75 ] . followup of the high - risk patients suggested that some risk of delayed lv abnormalities remained , although the frequency of overt hf was much lower [ 14 , 69 , 70 ] . in the 1990s , analysis of the combined results of the dana - farber cancer institute 's long - term survivors and those from patients treated in denmark revealed that after a median followup of 8.1 years , the risk of lv abnormalities was lower in patients who received no more than 300 mg / m than it was in those who received more than 300 mg / m of doxorubicin . on the basis of those results , the cumulative doxorubicin dose for high - risk all patients on the dana - farber cancer institute protocol 95 - 001 were further reduced to 300 mg / m . structural modifications of anthracyclines have been considered as possible cardioprotection strategies . of the structural modifications of anthracyclines , liposome - encapsulated anthracyclines escape the leaky capillary system of tumor sites and as such remain concentrated there , in the interstitial fluid . this ability to reduce the plasma levels of free doxorubicin is thought to be the source of the reduced cardiotoxicity provided by liposomal formulations of anthracyclines . although studies of liposomal - encapsulated anthracyclines are limited in children , biopsies have confirmed that these anthracyclines have a lower early cardiotoxicity than conventional anthracyclines , and studies by marina et al . because the cellular mechanisms underlying anthracycline cardiotoxicity are still not completely understood , it has been theorized that continuous infusion might provide some cardioprotective benefit by lowering peak serum levels of the drug . this theory was supported by findings from observational studies in children that found higher dose rates to be associated with cardiotoxicity , independently of the effect of total dose . in addition , in adults receiving anthracyclines , acute cardiotoxicity was diminished in protocols using continuous infusion . on the basis of these findings , continuous infusion was incorporated into many pediatric protocols , despite a lack of evidence on its long - term cardioprotective efficacy [ 44 , 82 ] . a randomized controlled trial of continuous ( over 48 hours ) doxorubicin infusion versus bolus doxorubicin infusion in 121 high - risk pediatric all cases found no cardiac - related benefit to using the continuous infusion after a median of 1.5 years after diagnosis . similar results were found after continued followup of this cohort at a median followup of 8 years . in addition , several retrospective reviews reported no statistically significant differences in the echocardiographic characteristics of children with cancer 5 to 7 years after treatment with either continuous infusion ( over 6 to 24 hours ) or bolus infusion of anthracyclines [ 79 , 80 ] . as mentioned above , anthracycline - related cardiotoxicity is likely related , at least in part , to the generation of ros , which may be exacerbated by iron - dependent mechanisms . this relationship has led to the investigation of several iron - chelating agents , such as dexrazoxane , for preventing anthracycline cardiotoxicity . dexrazoxane is an iron - chelating agent that inhibits the formation of anthracycline - iron complexes that generate toxic and highly charged ros . however , other mechanisms independent of oxidative stress have been proposed as mediators in the cardioprotective effect of iron chelation , such as the mitigation of dna damage caused by dexrazoxane . this proposal has led to great interest in the use of dexrazoxane in children . dexrazoxane is currently recommended by the american society of clinical oncology for preventing cardiotoxicity in specific adult cancer treatment protocols . fearing that the protection dexrazoxane provides to cardiomyocytes might extend to cancer cells as well and perhaps lead to increased rates of second malignancies , some investigators have been reluctant to use dexrazoxane . although such a relationship was previously reported , but questioned [ 85 , 86 ] , in a pediatric hodgkin lymphoma trial , no association between dexrazoxane and second malignancies has been found . a randomized controlled trial of dexrazoxane in children with all treated with doxorubicin used serial ctnt samples taken during therapy to measure cardiac damage . of the 82 children who received doxorubicin plus dexrazoxane , significantly fewer had ctnt elevations when compared with the 76 children who received doxorubicin alone . after 6 to 8 months , almost 50% of those treated with doxorubicin alone had an elevated ctnt level , as opposed to less than 10% of those treated with doxorubicin plus dexrazoxane . a recent followup report of this group at a median of 8.7 years found that event - free survival did not differ significantly between the two groups and that there was no increased risk of recurrence or second malignant neoplasms [ 53 , 87 ] . these findings show that dexrazoxane can provide long - term cardioprotection without hindering the efficacy of doxorubicin . children treated with doxorubicin plus dexrazoxane had progressively less decreased lv fractional shortening and greater lv mass and lv wall thickness over time . most recently , the protective effects of dexrazoxane reported in this trial appear to be sex related , which is consistent with recent findings from animal studies . females showed the greatest protective effect from dexrazoxane therapy , while males receive did not receive such benefit ( figure 2 ) . these findings further highlight that there is still much to learn about the mechanisms underlying such differences [ 53 , 89 ] although sex - related differences in the transport and clearance of doxorubicin have been reported [ 90 , 91 ] , as has dexrazoxane cardioprotection against the sex - related hormone , testosterone . the protective effect of dexrazoxane against anthracycline cardiotoxicity is further supported by studies of childhood cancers other than leukemia [ 81 , 83 ] . however , research is needed to fully understand the subtle risks associated with the use of dexrazoxane , what methods of dexrazoxane administration are most efficient , and what doses are necessary to achieve adequate protection . with more extended followup , the true incidence of chronic hf in long - term childhood cancer survivors treated with anthracyclines may exceed the reported 1% to 16% range [ 44 , 93 ] . chronic anthracycline cardiotoxicity is not only evident as symptomatic lv dysfunction , it also frequently manifests as subclinical abnormalities in lv structure and function . in some cases such effects may also leave long - term childhood cancer survivors more vulnerable to future non - anthracycline - related cardiovascular insults . long - term childhood cancer survivors , like the general population , may have one or more of the traditional risk factors for atherosclerosis , which could provide an additive risk of future cardiovascular complications beyond that directly related to cancer therapies . obesity , physical inactivity , tobacco use , and diabetes mellitus are among the most commonly examined traditional modifiable atherosclerotic risk factors . an improved understanding of the lifetime cardiovascular risk associated with these factors in long - term childhood survivors may help guide treatment and predict any potential additional cardiovascular risk of specific cancer therapies , such as the use of anthracyclines . in the united states , childhood obesity rates have increased to where it has now become epidemic [ 95 , 96 ] . less than 5% of 12-to-19-year - olds were overweight in the late 1960s , while almost 17% were overweight by the year 2008 . this report also shows that almost 32% of children and adolescents between 2 to 19 years old are at risk of overweight . one study showed that almost 80% of obese 10-to-14-year - olds who had an obese parent were obese as adults . this relationship raises two main concerns : ( 1 ) childhood overweight and obesity are associated with poor health outcomes , such as coronary artery disease , hypertension , and diabetes and ( 2 ) childhood obesity is strongly associated with adult obesity , which is associated with an increased risk of atherosclerotic disease and death . obesity contributes to a significant burden in terms of chronic diseases , rising healthcare costs , and , most importantly , disability and premature death . studies of obesity in long - term survivors of childhood cancer have found similarly troubling trends suggesting that survivors may be at increased risk for obesity as a result of their cancer history . this study found that more than one - third of survivors met the criteria for overweight or obese according to the body mass index ( bmi ) , but this was no different than siblings . however , male survivors were found to have greater body fat and trunk fat compared to siblings , while no differences were found between female survivors and siblings . the largest report to date of nearly 8000 long - term survivors found that about 41% were either obese or overweight . this study and others also found that despite a high prevalence of obesity , long - term survivors were not more likely to be obese than the general population although certain groups , such as survivors of all , were at increased risk of obesity [ 101 , 102 ] . the idea that certain subgroups of long - term childhood survivors are at increased risk of obesity is supported by studies of those survivors exposed to cranial radiation . many of these studies reveal treatment - related damage to the hypothalamic - pituitary axis , with subsequent growth hormone deficiency and eventual obesity . however , it should be noted that not all studies have found such a relationship and that other factors , such as obesity before diagnosis , may be more powerful predictors of obesity after treatment . it is clear , though , that obesity is highly prevalent in survivors of all types of childhood cancer and may predispose this group to future health problems , especially atherosclerotic disease , which may be especially problematic for these patients who are less able to compensate for ischemic cardiac insults . physical inactivity is associated with higher risk of cardiovascular disease as well as with other traditional atherosclerotic disease risk factors , such as insulin resistance and obesity . physical activity is not only recommended by several medical associations , including the american academy of pediatrics , but guidelines have been created by several organizations , including the us department of health and human services [ 104 , 105 ] . physical inactivity appears to be more common in long - term childhood survivors than in the general population . miller et al . reported that male survivors watched significantly more hours of television compare to siblings . they also found that increased television viewing hours was associated with higher bmi and percent body fat in male and female survivors of childhood cancer . a survey of nearly 10,000 long - term survivors of childhood cancer and 3,000 of their siblings revealed that survivors were more likely than their healthy siblings and the general population to report being physically inactive and being less likely to meet recommended physical activity guidelines . this survey and others have found that some long - term childhood survivors may be unable to comply with these guidelines as a result of the physical limitations incurred by their cancer - related surgery or treatment - related cardiac damage . hopefully , interventions that lead to appropriate and safe increases in the physical activity of long - term survivors may decrease risk of atherosclerotic disease and its associated negative health outcomes . cigarette smoking is a well - known major preventable risk factor for cardiovascular disease and all - cause mortality among individuals in the general population . the increased rate of cardiovascular disease in smokers has been convincingly evident since the 1960s . one systematic review of the health behaviors of long - term survivors found that most studies have reported lower rates of smoking in survivors relative to the general population although these rates are still high enough to be a concern and to warrant intervention . up to 17% of long - term childhood survivors in the united states are active smokers , compared to more than 20% of the general adult population [ 110 , 111 ] . the increased cardiovascular disease risk associated with smoking may be magnified in long - term survivors who may already have underlying cardiac abnormalities as a result of their malignancy and its treatment . therefore , efforts aimed at smoking prevention and cessation in long - term childhood survivors are essential to improve long - term outcomes . the prevalence of insulin resistance and overt diabetes has risen steadily and parallels the rise in the prevalence of obesity and the decline in physical activity . in the united states today , almost 13% of adults aged 20 years or older have either diabetes or prediabetes , a prevalence that rose from 5.1% between 1988 and 1994 and to 7.7% between 2005 and 2006 . current recommendations for adults state that the added cardiovascular disease risk associated with diabetes is equivalent to that of a previous myocardial infarction . a recent report of more than 8000 long - term survivors found that these survivors are nearly twice as likely to report having diabetes as were their siblings . another longitudinal study of more than 200 long - term survivors found that 4% had diabetes , another 7% had impaired glucose tolerance , and another 4% had hyperinsulinemia . these findings are especially worrisome given that the average age of adult long - term childhood survivors in the study was 25 years and that all were less than 40 years old , age cohorts that would otherwise would be considered at low - risk for impaired glucose metabolism . cancer treatment - related cardiotoxicity may have left many childhood cancer survivors more vulnerable to cardiovascular disease . hypertension is among the leading causes of cardiovascular disease . in 2008 , 29% percent of us adults were hypertensive , an increase from almost 24% in 19881994 . survivors of childhood cancer have a higher risk of developing hypertension compared to the general population . a study of 5,599 childhood cancer survivors and 2,936 siblings reported that survivors were more likely to report taking medication for hypertension than their siblings ( or 1.9 , 95% ci 1.6202 ) . in addition , a preexisting diagnosis of hypertension further increases the risk of clinically significant anthracycline cardiotoxicity . studies in older adults have shown that hypertension may work synergistically with doxorubicin increasing the risk of chf . hershman et al . found that hypertension intensified the effect of doxorubicin on risk of chf in older adults . further , studies in rats have shown a similar trend where hypertensive rats were more sensitive than normotensive rats to the cardiotoxic effects of doxorubicin [ 88 , 118 ] . therefore , monitoring cardiac status during and after anthracycline therapy is important , particularly in the long term , as risks for cardiovascular disease naturally increase with age . effective anti - neoplastic therapies for childhood cancer are one of the great successes of modern medicine and have helped to create a large population of childhood cancer survivors . however , even years after successfully battling cancer , many survivors are burdened by the cardiotoxicity that can result from cancer therapy . there remains a growing need in both basic and clinical research to better understand the mechanisms of anthracycline cardiotoxicity , to develop effective and safe cardioprotection strategies , and to identify the risk factors for cardiac damage . table 2 summarizes a number of the areas where further research could help to fill the current gaps in knowledge . most importantly , cardiologists and oncologists should collaborate to find a balance between the risks of cardiotoxicity and the benefits of oncologic therapy to maximize the quality of life and survival for long - term childhood cancer survivors .

anthracyclines are commonly used to treat childhood leukemias and lymphomas , as well as other malignancies , leading to a growing population of long - term childhood cancer survivors . however , their use is limited by cardiotoxicity , increasing survivors ' vulnerability to treatment - related complications that can markedly affect their quality of life . survivors are more likely to suffer from heart failure , coronary artery disease , and cerebrovascular accidents compared to the general population . the specific mechanisms of anthracycline cardiotoxicity are complex and remain unclear . hence , determining the factors that may increase susceptibility to cardiotoxicity is of great importance , as is monitoring patients during and after treatment . additionally , treatment and prevention options , such as limiting cumulative dosage , liposomal anthracyclines , and dexrazoxane , continue to be explored . here , we review the cardiovascular complications associated with the use of anthracyclines in treating malignancies in children and discuss methods for preventing , screening , and treating such complications in childhood cancer survivors .

1. Introduction 2. Anthracyclines and Mechanisms of Cardiotoxicity 3. Risk Factors for Anthracycline-Related Cardiac Abnormalities 4. Monitoring Long-Term Cancer Survivors 5. Preventing Anthracycline-Induced Cardiotoxicity 6. Conclusions and Directions for Research

some of this improvement comes from the use of anthracycline chemotherapeutic agents , which are widely used to treat childhood leukemias and lymphomas , as well as other malignancies . their use has helped create a growing population of long - term childhood cancer survivors of more than 325,000 in the united states alone . however , this growing population of survivors is at a substantial risk for treatment - related complications that can markedly affect their quality of life . within the first 30 years after diagnosis recent 5-year estimates indicate that the leading non - cancer - related cause of morbidity and mortality in long - term survivors of childhood cancer is cardiovascular - related disease [ 510 ] . survivors are 8 times more likely than the general population to die from cardiovascular - related disease , and compared to sibling controls , they are 15 times as likely to suffer from heart failure ( hf ) , more than 10 times as likely to have coronary artery disease , and more than 9 times as likely to have had a cerebrovascular accident during the first 30 years after cancer diagnosis . anthracyclines , such as doxorubicin , are among the leading causes of these cardiovascular events . the cardiotoxic nature of anthracyclines , coupled with their widespread use , may explain the large impact they have made on survivor morbidity and mortality . childhood cancer survivors exposed to anthracyclines are at a significantly increased risk of hf , and this risk increases as the cumulative dose increases ; 30 years after diagnosis , more than 7.5% will have experienced hf . in addition to clinical outcomes , detailed cardiac evaluations have shown that more than half of anthracycline - exposed childhood cancer survivors have subclinical cardiac abnormalities , including decreased left ventricular ( lv ) mass and wall thickness , increased lv afterload , and decreased lv contractility . the severity of the cardiotoxic effects of anthracyclines vary and are categorized by time of onset as acute , occurring during or immediately after treatment , early , occurring within 1 year of exposure , and late , occurring 1 or more years after initial exposure ( table 1 ) [ 11 , 15 ] . severe cardiotoxicity , during or shortly after treatment , is strongly associated with hf later in life , despite an initial asymptomatic interval . in a followup study of long - term survivors of childhood cancer who were treated with anthracyclines and who experienced acute hf , all had a temporary recovery though nearly half of these patients later had recurrent hf . understanding the early- and late - onset cardiovascular complications of long - term survivors of childhood cancer is important for oncologists , cardiologists , and other health care providers involved in the care of such patients , not only after cancer treatment but also when selecting treatments at the time of diagnosis . here , we review the cardiovascular complications associated with the use of anthracyclines in treating malignancies in children and discuss methods for preventing , screening , and treating such complications in childhood cancer survivors . anthracyclines are commonly used to treat a variety of solid and hematologic tumors in both adults and children . however , because they have clinically relevant dose - dependent cardiotoxicity , lower doses are used in treating childhood cancer [ 18 , 19 ] . however , the specific mechanisms of anthracycline cardiotoxicity are complex and , despite decades of research , remain unclear . once inside the cell , anthracyclines may form complexes with intracellular iron , leading to the production of free radicals , which can cause dna damage and lipid peroxidation . several other mechanisms have been suggested for anthracycline cardiotoxicity , including the induction of apoptosis , abnormal creatine kinase activity , the production of vasoactive amines , the formation of toxic metabolites , upregulation of nitric oxide synthetase , and the inhibition of transcription and translation [ 3133 ] . anthracyclines can also impair mitochondrial calcium homeostasis , leading to the loss of stability of the mitochondrial membrane , decreased atp , and cell death . the fact that many of these subcellular consequences continue to progress for weeks after anthracycline exposure may provide insight into the mechanisms of chronic cardiomyopathy . hence , determining the factors that may increase susceptibility to the cardiotoxic effects of anthracyclines is of great importance . first , ascertaining which patients are most likely to experience cardiotoxicity can guide treatment , especially as long - term health becomes a more prominent treatment focus . one of the main risk factors for anthracycline cardiotoxicity is high cumulative dose . since the 1970s , studies have shown that higher cumulative doses of anthracyclines and higher infusion rates are risk factors for anthracycline cardiotoxicity [ 10 , 19 ] . this relationship was revealed in a study of nearly 15,000 long - term survivors of childhood cancer treated with anthracyclines who described their cardiac health at up to 30 years after cancer treatment ( figure 1 ) . other known risk factors for anthracycline cardiotoxicity include younger age at treatment , female sex , the use of concomitant cardiotoxic therapies ( such as mediastinal radiation ) , increasing time since treatment , cardiac injury , as indicated by elevations of serum cardiac troponin - t ( ctnt ) , neurohormonal activation of cardiac myocytes in response to pressure and stress , as indicated by elevations of n - terminal probrain natriuretic peptide ( nt - probnp ) , during anthracycline therapy , and hf during anthracycline therapy [ 5 , 10 , 14 , 19 , 28 , 45 ] . although not completely understood , genetic predisposition may also be an important factor in determining the risk of anthracycline cardiotoxicity [ 4648 ] . in mice , hfe deficiency , the defective gene in hereditary hemochromatosis , increases the susceptibility to doxorubicin - induced cardiotoxicity , including mitochondrial degradation and increased mortality , when compared to that found in wild - type mice . despite the identification of these population - based risk factors , determining therefore , it is important that all children who receive anthracycline therapy be followed closely , both during and after treatment for cardiotoxicity . as a guide , the long - term follow up program resource guide , developed by children 's oncology group 's nursing discipline committee in collaboration with the late effects committee , provides recommendations for screening and management of late effects of therapeutic exposures used during treatment for pediatric malignancies . this difference has led to an increasing interest in the use of monitoring serum biomarkers as a means of evaluating cardiotoxicity during and after treatment with anthracyclines . this relationship is consistent with the hypothesis that anthracyclines damage cardiomyocytes , leading to the release of intracellular ctnt into the circulation . the dana - farber cancer institute childhood acute lymphoblastic leukemia ( all ) consortium protocol 95 - 001 study found that elevated serum ctnt levels during the first 90 days of anthracycline therapy were significantly associated with reduced lv mass and lv end - diastolic posterior wall thickness as measured by echocardiography 4 years later [ 58 , 59 ] . the aforementioned dana - farber cancer institute protocol 95 - 001 study also assessed serum nt - probnp measurements during and after therapy as possible predictors of cardiac damage . additionally , a higher percentage of children had elevated levels of nt - probnp than had elevated levels of ctnt before , during , and after treatment . as a global inflammatory marker and a critical component of the immune system , high - sensitivity c - reactive protein ( hscrp ) systemic inflammation is associated with increased rates of cardiovascular disease in adults and may also be involved with the mechanisms underlying anthracycline - related cardiotoxicity and pediatric cardiomyopathy . these findings have encouraged further research on the use of serum hscrp measurements collected during therapy as potential predictors of late cardiac effects . serum hscrp may prove to be a valuable screening tool for identifying long - term survivors at increased risk of subsequent cardiac disease . the ability to identify heart - derived tissue proteins associated with myocardial injury that can be detected before marked elevations in ctnt could have profound implications on earlier detection and clinical monitoring for doxorubicin cardiotoxicity . however , due to the small limited sample size , further studies evaluating the efficacy of nanoparticle - mass spectrometry may advance this novel diagnostic approach . several detailed echocardiographic studies in 115 long - term childhood all survivors treated with doxorubicin have been conducted to better determine the long - term cardiac status of these patients [ 14 , 69 , 70 ] . these studies , ranging from 6 to 12 years of followup after treatment , have documented a persistent and progressive restrictive - like cardiomyopathy in long - term childhood cancer survivors treated with anthracyclines [ 14 , 69 , 70 ] . the restrictive cardiomyopathic - like nature of anthracycline cardiotoxicity may be of great clinical importance in that it suggests theories and treatments derived from studies of dilated cardiomyopathy may be of limited value in understanding anthracycline cardiotoxicity throughout life . the findings of abnormal lv structure and function , as well as a restrictive - like cardiomyopathic pattern , are consistent with those of other long - term followup studies conducted in other groups of anthracycline - treated childhood cancer survivors [ 7274 ] . on the basis of this experience , cumulative doses of doxorubicin administered to children and adolescents with all were reduced in the 1980s such that high - risk children received a cumulative dose of no more than 360 mg / m , and standard - risk children received a relatively low cumulative dose of no more than 60 mg / m [ 58 , 75 ] . in the 1990s , analysis of the combined results of the dana - farber cancer institute 's long - term survivors and those from patients treated in denmark revealed that after a median followup of 8.1 years , the risk of lv abnormalities was lower in patients who received no more than 300 mg / m than it was in those who received more than 300 mg / m of doxorubicin . this ability to reduce the plasma levels of free doxorubicin is thought to be the source of the reduced cardiotoxicity provided by liposomal formulations of anthracyclines . although studies of liposomal - encapsulated anthracyclines are limited in children , biopsies have confirmed that these anthracyclines have a lower early cardiotoxicity than conventional anthracyclines , and studies by marina et al . because the cellular mechanisms underlying anthracycline cardiotoxicity are still not completely understood , it has been theorized that continuous infusion might provide some cardioprotective benefit by lowering peak serum levels of the drug . this theory was supported by findings from observational studies in children that found higher dose rates to be associated with cardiotoxicity , independently of the effect of total dose . on the basis of these findings , continuous infusion was incorporated into many pediatric protocols , despite a lack of evidence on its long - term cardioprotective efficacy [ 44 , 82 ] . in addition , several retrospective reviews reported no statistically significant differences in the echocardiographic characteristics of children with cancer 5 to 7 years after treatment with either continuous infusion ( over 6 to 24 hours ) or bolus infusion of anthracyclines [ 79 , 80 ] . as mentioned above , anthracycline - related cardiotoxicity is likely related , at least in part , to the generation of ros , which may be exacerbated by iron - dependent mechanisms . this relationship has led to the investigation of several iron - chelating agents , such as dexrazoxane , for preventing anthracycline cardiotoxicity . however , other mechanisms independent of oxidative stress have been proposed as mediators in the cardioprotective effect of iron chelation , such as the mitigation of dna damage caused by dexrazoxane . this proposal has led to great interest in the use of dexrazoxane in children . fearing that the protection dexrazoxane provides to cardiomyocytes might extend to cancer cells as well and perhaps lead to increased rates of second malignancies , some investigators have been reluctant to use dexrazoxane . of the 82 children who received doxorubicin plus dexrazoxane , significantly fewer had ctnt elevations when compared with the 76 children who received doxorubicin alone . these findings show that dexrazoxane can provide long - term cardioprotection without hindering the efficacy of doxorubicin . these findings further highlight that there is still much to learn about the mechanisms underlying such differences [ 53 , 89 ] although sex - related differences in the transport and clearance of doxorubicin have been reported [ 90 , 91 ] , as has dexrazoxane cardioprotection against the sex - related hormone , testosterone . the protective effect of dexrazoxane against anthracycline cardiotoxicity is further supported by studies of childhood cancers other than leukemia [ 81 , 83 ] . however , research is needed to fully understand the subtle risks associated with the use of dexrazoxane , what methods of dexrazoxane administration are most efficient , and what doses are necessary to achieve adequate protection . with more extended followup , the true incidence of chronic hf in long - term childhood cancer survivors treated with anthracyclines may exceed the reported 1% to 16% range [ 44 , 93 ] . chronic anthracycline cardiotoxicity is not only evident as symptomatic lv dysfunction , it also frequently manifests as subclinical abnormalities in lv structure and function . in some cases such effects may also leave long - term childhood cancer survivors more vulnerable to future non - anthracycline - related cardiovascular insults . long - term childhood cancer survivors , like the general population , may have one or more of the traditional risk factors for atherosclerosis , which could provide an additive risk of future cardiovascular complications beyond that directly related to cancer therapies . an improved understanding of the lifetime cardiovascular risk associated with these factors in long - term childhood survivors may help guide treatment and predict any potential additional cardiovascular risk of specific cancer therapies , such as the use of anthracyclines . this relationship raises two main concerns : ( 1 ) childhood overweight and obesity are associated with poor health outcomes , such as coronary artery disease , hypertension , and diabetes and ( 2 ) childhood obesity is strongly associated with adult obesity , which is associated with an increased risk of atherosclerotic disease and death . obesity contributes to a significant burden in terms of chronic diseases , rising healthcare costs , and , most importantly , disability and premature death . studies of obesity in long - term survivors of childhood cancer have found similarly troubling trends suggesting that survivors may be at increased risk for obesity as a result of their cancer history . however , male survivors were found to have greater body fat and trunk fat compared to siblings , while no differences were found between female survivors and siblings . this study and others also found that despite a high prevalence of obesity , long - term survivors were not more likely to be obese than the general population although certain groups , such as survivors of all , were at increased risk of obesity [ 101 , 102 ] . the idea that certain subgroups of long - term childhood survivors are at increased risk of obesity is supported by studies of those survivors exposed to cranial radiation . many of these studies reveal treatment - related damage to the hypothalamic - pituitary axis , with subsequent growth hormone deficiency and eventual obesity . however , it should be noted that not all studies have found such a relationship and that other factors , such as obesity before diagnosis , may be more powerful predictors of obesity after treatment . it is clear , though , that obesity is highly prevalent in survivors of all types of childhood cancer and may predispose this group to future health problems , especially atherosclerotic disease , which may be especially problematic for these patients who are less able to compensate for ischemic cardiac insults . physical inactivity is associated with higher risk of cardiovascular disease as well as with other traditional atherosclerotic disease risk factors , such as insulin resistance and obesity . physical inactivity appears to be more common in long - term childhood survivors than in the general population . a survey of nearly 10,000 long - term survivors of childhood cancer and 3,000 of their siblings revealed that survivors were more likely than their healthy siblings and the general population to report being physically inactive and being less likely to meet recommended physical activity guidelines . this survey and others have found that some long - term childhood survivors may be unable to comply with these guidelines as a result of the physical limitations incurred by their cancer - related surgery or treatment - related cardiac damage . hopefully , interventions that lead to appropriate and safe increases in the physical activity of long - term survivors may decrease risk of atherosclerotic disease and its associated negative health outcomes . cigarette smoking is a well - known major preventable risk factor for cardiovascular disease and all - cause mortality among individuals in the general population . one systematic review of the health behaviors of long - term survivors found that most studies have reported lower rates of smoking in survivors relative to the general population although these rates are still high enough to be a concern and to warrant intervention . up to 17% of long - term childhood survivors in the united states are active smokers , compared to more than 20% of the general adult population [ 110 , 111 ] . the increased cardiovascular disease risk associated with smoking may be magnified in long - term survivors who may already have underlying cardiac abnormalities as a result of their malignancy and its treatment . therefore , efforts aimed at smoking prevention and cessation in long - term childhood survivors are essential to improve long - term outcomes . a recent report of more than 8000 long - term survivors found that these survivors are nearly twice as likely to report having diabetes as were their siblings . another longitudinal study of more than 200 long - term survivors found that 4% had diabetes , another 7% had impaired glucose tolerance , and another 4% had hyperinsulinemia . these findings are especially worrisome given that the average age of adult long - term childhood survivors in the study was 25 years and that all were less than 40 years old , age cohorts that would otherwise would be considered at low - risk for impaired glucose metabolism . cancer treatment - related cardiotoxicity may have left many childhood cancer survivors more vulnerable to cardiovascular disease . survivors of childhood cancer have a higher risk of developing hypertension compared to the general population . a study of 5,599 childhood cancer survivors and 2,936 siblings reported that survivors were more likely to report taking medication for hypertension than their siblings ( or 1.9 , 95% ci 1.6202 ) . therefore , monitoring cardiac status during and after anthracycline therapy is important , particularly in the long term , as risks for cardiovascular disease naturally increase with age . effective anti - neoplastic therapies for childhood cancer are one of the great successes of modern medicine and have helped to create a large population of childhood cancer survivors . however , even years after successfully battling cancer , many survivors are burdened by the cardiotoxicity that can result from cancer therapy . there remains a growing need in both basic and clinical research to better understand the mechanisms of anthracycline cardiotoxicity , to develop effective and safe cardioprotection strategies , and to identify the risk factors for cardiac damage . most importantly , cardiologists and oncologists should collaborate to find a balance between the risks of cardiotoxicity and the benefits of oncologic therapy to maximize the quality of life and survival for long - term childhood cancer survivors .

blood pressure ( bp ) measured in the clinic does not provide comprehensive information about an individual s bp profile and may be of limited prognostic value , thus requiring out - of - office bp monitoring.1,2 home bp can be measured repeatedly in the comfort of the patient s own home in a relaxing environment , which provides information about day - to - day bp variability ( bpv ) and enables a more accurate understanding of a subject s bp profile . home bp has been widely accepted as a useful tool for clinical management of patients with hypertension and cardiovascular disease . bp is characterized by marked temporal fluctuations showing beat - to - beat variability , 24-hour variability , day - to - day variability , and visit - to - visit variability . clinically , bpv is a well - known risk factor for cardiovascular events and is associated with target organ damage and all - cause mortality that is independent of office bp levels.38 beat - to - beat variability or 24-hour variability reflects increases in central sympathetic drive , decreases in arterial or cardiopulmonary reflex , and increases in arterial stiffness.9,10 humoral , rheological , and emotional factors and behavioral influences also can be involved in bpv . by contrast , day - to - day variability or visit - to - visit variability is proposed to be due to increased arterial stiffness , improper dosing or titration of antihypertensive medication , poor medication compliance , and seasonal variations in bp.11,12 day - to - day bpv is independent of beat - to - beat bpv and may have significantly different effects on hypertension prognosis . recent studies show that increased day - to - day home bpv is associated with cardiovascular risk , severity of target organ damage in patients with hypertension13 or diabetes mellitus,14 and cardiovascular mortality in a community - dwelling population.15 in the anglo - scandinavian cardiac outcomes trial - blood pressure - lowering arm trial , visit - to - visit systolic bpv during treatment was a strong predictor of stroke and coronary events independent of mean levels of clinical or ambulatory systolic bp ( sbp ) in patients with hypertension.16 therefore , reducing both bpv and mean bp has been recognized as a potential target for improved management of hypertension to prevent cardiovascular events , particularly stroke.6,16 several small - scale and heterogeneous human studies on the effects of antihypertensive drugs report conflicting results . calcium channel blockers such as amlodipine , but not beta - blockers , confer favorable effects to decrease home bpv.17 other studies reported increased home bpv after treatment with beta - blockers.18,19 inconsistent results also were observed even within the same class of antihypertensive medication , such as angiotensin ii receptor blockers ( arbs).2022 fimasartan is an arb that effectively and safely reduced high bp in the safe - kanarb study.23 however , its effect on reducing bpv has not yet been determined . the objective of the present study was to determine whether fimasartan reduced bpv in the clinic and at home after 3 months of treatment in hypertensive patients with low - to - moderate cardiovascular risk . this investigation is part of the k - mets study , which is a prospective , multicenter , single - arm , observational study . the study design , socioeconomic and demographic characteristics of the study , and project details have been described in our previous article.24 this study was approved by the institutional review board committee at the cheil general hospital , on behalf of 582 primary care clinics . another ten university hospitals in south korea approved this study through their own institutional review board committees . a total of 10,601 hypertensive patients from 582 primary clinics and eleven university hospitals were enrolled between october 17 , 2011 and october 31 , 2012 . patients were required to meet the following inclusion criteria : 1 ) diagnosed with hypertension , at least 20 years old , and intend to use fimasartan ; 2 ) agree to participate in the study and sign the informed consent form ; and 3 ) maintain a fasting state at each visit . patients who were treated with fimasartan at baseline were excluded.24 finally , 1,396 patients were enrolled who were treated with fimasartan at a daily dose of 30120 mg for 3 months , completed the 3-month follow - up visits , and scheduled bp measurements . patients were enrolled in three different groups : nave , switch , and add - on . in nave group , patients had no previous antihypertensive medication and received fimasartan . in switch group , patients were switched from other antihypertensive drug in the baseline to fimasartan and in an add - on group , patients who received antihypertensive drug also received fimasartan as an add - on therapy . the omron hem-7220 and the omron hem-7200 ( both omron , tokyo , japan ) were used to measure bp in the clinic and at home , respectively.25 these are upper - arm cuff devices based on the cuff automated - oscillometric principle . clinical bp measurements were performed under standardized conditions ( in the same arm by the same physician or nurse ) . an average of two or more bp readings at 2-minute intervals from the same arm was recorded in the morning and evening for 7 consecutive days . morning bp was measured within 1 hour of waking , after urination , in the sitting position , after resting for 5 minutes , and before taking medications or eating . in the evening , bp was measured before going to bed , after resting for 5 minutes , and in the sitting position . an average of 6 days of recordings from the 2nd to the 7th day was used for the analysis . baseline assessment included a health questionnaire and bp measurements and was conducted before and after the 3-month treatment with fimasartan.24 clinical bpv , which measures beat - to - beat variability , was defined as the standard deviation ( sd ) of clinical systolic bp ( c - sbp ) measured three times at 2-minute intervals . home bpv , which represents day - to - day bpv , was defined as the sd of morning home systolic bp ( m - sbp ) . the baseline characteristics of the study subjects were compared between sexes using the test for dichotomous variables or the independent t - test for continuous variables . differences between measured variables ( eg , bp ) were examined using the paired t - test between baseline and after 3 months of follow - up . we analyzed males and females separately because sex may affect bpv.26 to evaluate the potential factors associated with clinical or home sbp variability , both simple and multiple linear regression analyses were conducted . multiple linear regression models included potential factors such as clinical or home average sbp and diastolic bp ( dbp ) , sex , age , history of cardiovascular disease , and smoking . this was done because most of these factors have a positive relationship with bpv , in part due to the stiffening influence on large- and medium - sized arteries , with an increase in the pressure excursions within the arterial compartment . all analyses were performed using sas 9.3 ( sas institute inc . , cary , nc , usa ) . a total of 10,601 hypertensive patients from 582 primary clinics and eleven university hospitals were enrolled between october 17 , 2011 and october 31 , 2012 . patients were required to meet the following inclusion criteria : 1 ) diagnosed with hypertension , at least 20 years old , and intend to use fimasartan ; 2 ) agree to participate in the study and sign the informed consent form ; and 3 ) maintain a fasting state at each visit . patients who were treated with fimasartan at baseline were excluded.24 finally , 1,396 patients were enrolled who were treated with fimasartan at a daily dose of 30120 mg for 3 months , completed the 3-month follow - up visits , and scheduled bp measurements . patients were enrolled in three different groups : nave , switch , and add - on . in nave group , patients had no previous antihypertensive medication and received fimasartan . in switch group , patients were switched from other antihypertensive drug in the baseline to fimasartan and in an add - on group , patients who received antihypertensive drug also received fimasartan as an add - on therapy . the omron hem-7220 and the omron hem-7200 ( both omron , tokyo , japan ) were used to measure bp in the clinic and at home , respectively.25 these are upper - arm cuff devices based on the cuff automated - oscillometric principle . clinical bp measurements were performed under standardized conditions ( in the same arm by the same physician or nurse ) . an average of two or more bp readings at 2-minute intervals from the same arm was recorded in the morning and evening for 7 consecutive days . morning bp was measured within 1 hour of waking , after urination , in the sitting position , after resting for 5 minutes , and before taking medications or eating . in the evening , bp was measured before going to bed , after resting for 5 minutes , and in the sitting position . an average of 6 days of recordings from the 2nd to the 7th day was used for the analysis . baseline assessment included a health questionnaire and bp measurements and was conducted before and after the 3-month treatment with fimasartan.24 clinical bpv , which measures beat - to - beat variability , was defined as the standard deviation ( sd ) of clinical systolic bp ( c - sbp ) measured three times at 2-minute intervals . home bpv , which represents day - to - day bpv , was defined as the sd of morning home systolic bp ( m - sbp ) . the baseline characteristics of the study subjects were compared between sexes using the test for dichotomous variables or the independent t - test for continuous variables . differences between measured variables ( eg , bp ) were examined using the paired t - test between baseline and after 3 months of follow - up . we analyzed males and females separately because sex may affect bpv.26 to evaluate the potential factors associated with clinical or home sbp variability , both simple and multiple linear regression analyses were conducted . multiple linear regression models included potential factors such as clinical or home average sbp and diastolic bp ( dbp ) , sex , age , history of cardiovascular disease , and smoking . this was done because most of these factors have a positive relationship with bpv , in part due to the stiffening influence on large- and medium - sized arteries , with an increase in the pressure excursions within the arterial compartment . all analyses were performed using sas 9.3 ( sas institute inc . , cary , nc , usa ) . a total of 1,396 participants were enrolled ; the mean age was 56.1710.0 years ; 17.55% had diabetes , 6.88% had ischemic heart disease , and 0.93% had stroke . at baseline , the mean c - sbp and clinical dbp levels were 142.3917.00 and 88.0311.35 mmhg , respectively . these values were significantly reduced to 127.3213.14 and 79.379.40 mmhg ( p<0.0001 for both ) , respectively , after 3 months of fimasartan treatment ( table 2 ) . m - sbp and morning home dbp levels were 138.8019.81 and 83.2613.16 mmhg , respectively ; these were significantly reduced after 3 months of fimasartan treatment to 127.3216.72 and 76.6211.32 mmhg ( p<0.0001 for both ) , respectively . the same pattern was observed for evening home sbp ( e - sbp ) and evening home dbp after 3 months of fimasartan treatment ; e - sbp was 137.5720.53 mmhg and declined to 126.3317.06 mmhg , and evening home dbp was 81.3513.29 mmhg and declined to 75.0711.44 mmhg ( p<0.0001 for both ) . at baseline , the sds of c - sbp and clinical dbp were 4.563.22 and 3.192.45 mmhg , respectively ( table 3 ) . after 3 months of fimasartan treatment , the sds of c - sbp and dbp were significantly reduced to 4.243.11 mmhg ( p=0.0026 ) and 2.942.20 mmhg ( p=0.0024 ) , respectively . the sds of m - sbp and morning home dbp also showed significant reduction from 7.926.74 and 5.303.98 mmhg to 6.954.97 and 4.833.23 mmhg ( p<0.0001 and p=0.0002 ) , respectively . the same trend was observed for the sds of evening home sbp and dbp ; sd of e - sbp declined from 8.396.94 to 7.165.38 mmhg ( p<0.0001 ) , and sd of evening home dbp declined from 5.804.13 to 5.163.32 mmhg ( p<0.0001 ) . figure 1 shows that the correlation between the sd of c - sbp and the sd of m - sbp was weak at baseline and then became stronger after 3 months of fimasartan treatment . the same pattern was observed for the sd of c - sbp and e - sbp . figure 2 summarizes the observed bp changes in c - sbp and home sbp after 3 months of follow - up . at baseline , simple and multiple regression analyses indicated that the sd of c - sbp was independently associated with c - sbp ( p<0.0001 for both ) , and the sd of m - sbp was independently associated with m - sbp ( p<0.0001 for both ) . both simple and multiple regression analyses showed that the sd of m - sbp was independently associated with age , female sex , and m - sbp ( p=0.0067 and p=0.0005 , p=0.0032 and p<0.0001 , p<0.0001 and p<0.0001 , respectively ) , but only the simple regression analysis showed an association between the sd of m - sbp and body mass index ( p=0.0246 and p=0.0553 , respectively ) ( table 4 ) . simple and multiple regression analyses ( adjusted for age , sex , body mass index , and change in mean arterial pressure ) were performed to elucidate any factors associated with reduced bpv in the clinic and at home after 3 months of fimasartan treatment . the change in the sd of c - sbp was independently associated with the change in c - sbp ( p<0.0001 and p=0.0268 , respectively ) , and the change in the sd of m - sbp was independently associated with the change in m - sbp ( p<0.0001 and p=0.0258 , respectively ) ( table 5 ) . a total of 1,396 participants were enrolled ; the mean age was 56.1710.0 years ; 17.55% had diabetes , 6.88% had ischemic heart disease , and 0.93% had stroke . at baseline , the mean c - sbp and clinical dbp levels were 142.3917.00 and 88.0311.35 mmhg , respectively . these values were significantly reduced to 127.3213.14 and 79.379.40 mmhg ( p<0.0001 for both ) , respectively , after 3 months of fimasartan treatment ( table 2 ) . m - sbp and morning home dbp levels were 138.8019.81 and 83.2613.16 mmhg , respectively ; these were significantly reduced after 3 months of fimasartan treatment to 127.3216.72 and 76.6211.32 mmhg ( p<0.0001 for both ) , respectively . the same pattern was observed for evening home sbp ( e - sbp ) and evening home dbp after 3 months of fimasartan treatment ; e - sbp was 137.5720.53 mmhg and declined to 126.3317.06 mmhg , and evening home dbp was 81.3513.29 mmhg and declined to 75.0711.44 mmhg ( p<0.0001 for both ) . at baseline , the sds of c - sbp and clinical dbp were 4.563.22 and 3.192.45 mmhg , respectively ( table 3 ) . after 3 months of fimasartan treatment , the sds of c - sbp and dbp were significantly reduced to 4.243.11 mmhg ( p=0.0026 ) and 2.942.20 mmhg ( p=0.0024 ) , respectively . the sds of m - sbp and morning home dbp also showed significant reduction from 7.926.74 and 5.303.98 mmhg to 6.954.97 and 4.833.23 mmhg ( p<0.0001 and p=0.0002 ) , respectively . the same trend was observed for the sds of evening home sbp and dbp ; sd of e - sbp declined from 8.396.94 to 7.165.38 mmhg ( p<0.0001 ) , and sd of evening home dbp declined from 5.804.13 to 5.163.32 mmhg ( p<0.0001 ) . figure 1 shows that the correlation between the sd of c - sbp and the sd of m - sbp was weak at baseline and then became stronger after 3 months of fimasartan treatment . the same pattern was observed for the sd of c - sbp and e - sbp . figure 2 summarizes the observed bp changes in c - sbp and home sbp after 3 months of follow - up . at baseline , simple and multiple regression analyses indicated that the sd of c - sbp was independently associated with c - sbp ( p<0.0001 for both ) , and the sd of m - sbp was independently associated with m - sbp ( p<0.0001 for both ) . both simple and multiple regression analyses showed that the sd of m - sbp was independently associated with age , female sex , and m - sbp ( p=0.0067 and p=0.0005 , p=0.0032 and p<0.0001 , p<0.0001 and p<0.0001 , respectively ) , but only the simple regression analysis showed an association between the sd of m - sbp and body mass index ( p=0.0246 and p=0.0553 , respectively ) ( table 4 ) . simple and multiple regression analyses ( adjusted for age , sex , body mass index , and change in mean arterial pressure ) were performed to elucidate any factors associated with reduced bpv in the clinic and at home after 3 months of fimasartan treatment . the change in the sd of c - sbp was independently associated with the change in c - sbp ( p<0.0001 and p=0.0268 , respectively ) , and the change in the sd of m - sbp was independently associated with the change in m - sbp ( p<0.0001 and p=0.0258 , respectively ) ( table 5 ) . the major finding of this study was that after 3 months of fimasartan treatment , clinical and home bpvs were significantly reduced independent of strong bp reduction . these results were derived primarily from the general practitioners and participants with relatively low - to - moderate hypertension risk , and thus represent unbiased bp data in a real clinical setting . in our study , clinical bpv is calculated as the sd of three consecutive readings on the same patient in the clinic , and thus represents beat - to - beat bpv . by contrast , home bpv represents day - to - day variability of bp measured by the patients themselves at home for 7 consecutive days . bpv is thought to result from various intrinsic and extrinsic factors , although it is poorly understood . beat - to - beat variability is known to be more strongly influenced by increased central sympathetic drive , decreased arterial and cardiopulmonary reflex , and humoral , rheological , behavioral , and emotional factors.27 day - to - day variability has been reported to depend more strongly on arterial stiffness , improper dosing or titration of medication , poor medication compliance , and irregularity of self - measurements performed at home.4,27 clinical bp has been shown to be strongly correlated with home bp , but the correlation between clinical bpv and home bpv has not been elucidated . home bp measurement is reported to reflect the patient s true bp because measurements are recorded multiple times throughout the day in a comfortable environment . the results are supported by demonstrating a relationship of day - to - day bpv with cardiovascular outcomes , which is independent of mean bp.15,16 the finn - home study confirmed this by showing that morning day - to - day bpv is a predictor of cardiovascular events after adjusting for age and mean home bp.28 other studies also demonstrated this association15,28 and the association with increased risk of cardiac , vascular , and renal organ damage.13 the reduction of bpv is recognized as a potential target for improved management of hypertension to prevent cardiovascular events . there have been conflicting results regarding the capacity of antihypertensive drugs to reduce bpv . in general , calcium channel blockers have been reported to reduce bpv , which was determined by the sd of 24-hour ambulatory bp,29,30 and were superior to enalapril in the prevention of coronary events in patients with angiographically proven coronary artery disease and controlled bp.31 some arbs were shown to reduce bpv , whereas others did not show any effects . in the x - cellent study , 3 months of candesartan treatment did not show any effect on bpv evaluated by 24-hour bp monitoring.30 valsartan did not significantly change bpv in patients with hypertension after 12 months of treatment , despite reducing bp.29 in a recent study with home monitoring of bp , valsartan increased individual sd of morning sbp , but telmisartan did not affect bp after its use as an add - on agent in patients on amlodipine monotherapy.32 in an animal study , the superiority of telmisartan over valsartan in sustained bp control and reduction of bpv was attributed to further suppression of sympathetic activity and improvement of the baroreceptor reflex.33 based on this evidence , the arb - mediated reduction of bpv might not be due to its classic effects , but instead may be due to the effects of its own drug characteristics . in our study , 3 months of fimasartan treatment significantly reduced bp and bpv in the clinic and at home . although the methods of measuring bpv differ , the reductions in sd after fimasartan treatment were similar to the results of the x - cellent study with amlopidine.30 a reduction in beat - to - beat variability probably indicates a positive influence on its physiological mechanism , which may be attributable to a fimasartan - mediated reduction in central sympathetic drive . factors that result in day - to - day variability , including increased arterial stiffness , improper dosing or titration of antihypertensive medication , and poor medication compliance , can not be readily changed with 3 months of fimasartan treatment . however , we clearly observed a reduction in bp after fimasartan treatment , indicating better bp control , which in turn might have led to better bpv control . further research is needed to identify the pharmacological mechanisms behind these effects . to the best of our knowledge , the present study provides the first prospective evidence that fimasartan stabilizes day - to - day home systolic bpv . this conclusion was derived from data on measurement of bp in real clinical practice and self - measurement at home , not at a highly selected tertiary center . although this study did not identify the exact mechanism of fimasartan - mediated bpv reduction , the efficacy of fimasartan to reduce bpv was observed in home bpv independent of clinical bpv . the effect of fimasartan on reducing home systolic bpv may be related to its positive effects on hypertension prognosis shown by recent population - based data , which suggest that higher bpv is associated with increased all - cause mortality . therefore , we can presume that hypertensive patients with elevated day - to - day bpv may benefit from treatment with fimasartan . second , the quality of the measurement procedure could have affected the bpv data , although the participants were instructed to measure bp under relatively controlled conditions . the details of home bp data were blinded until final analysis ; therefore , information bias was probably not a significant contributing factor to the measurement and evaluation of home bpv . third , potential confounding factors of bpv , including those related to diet , psychological factors , and drug compliance , were not investigated in the present study . potential confounding factors of bpv , such as diet , psychological factors , and drug compliance , should be investigated in future studies . further studies on the change in clinical bp , morning bp , and bpv between diabetic and nondiabetic patients may be necessary as the findings may provide some insight into the mechanism of fimasartan on day - to - day bpv ( eg , arterial stiffness related to diabetes ) . studies on the effects of arbs in different populations ( eg , uncontrolled or resistant hypertension ) will be necessary . whether the change in bpv is dependent on the fimasartan dosage remains to be analyzed . fimasartan may have additional beneficial effects on cardiovascular protection by reducing bpv in addition to significantly reducing the mean levels of home bp . whether reduction in bpv translates into long - term clinical benefits , such as reduction of all - cause and cardiovascular mortality and target organ damage , needs to be investigated in future research . the observed superiority of fimasartan over other antihypertensive drugs in reducing bpv and preventing cardiovascular events warrants further investigation within clinical settings . the angiotensin receptor antagonist fimasartan is known to have a strong antihypertensive effect . however , the effects on targets other than bp are unknown . this study evaluated whether fimasartan treatment affected clinical and home bp variability in addition to reducing bp . three months of fimasartan treatment reduced day - to - day bp variability independent of bp reduction in patients with mild - to - moderate hypertension . fimasartan treatment also significantly reduced day - to - day bp variability at home independent of bp reduction , which may provide an additional benefit for prevention of cardiovascular events .

backgroundthe angiotensin receptor antagonist fimasartan lowered blood pressure ( bp ) in a previous large population study . the purpose of this study was to evaluate whether fimasartan treatment for 3 months affects clinical and home bp variability in addition to reducing bp.methodsthe study enrolled 1,396 patients ( mean age 56.210.0 years ; males 53.6% ) with mild - to - moderate hypertension who had a complete set of home bp measurements ( morning and evening ) and metabolic risk evaluation . during the 3 months of study , fimasartan alone was used to control bp at a daily dose of 30120 mg . clinical and home bp measurements were performed before and after the 3-month treatment . bp variability included beat - to - beat variability ( clinical ) and day - to - day variability ( home).resultsfimasartan reduced bp after 3 months of treatment . the average reduction of clinical systolic bp ( c - sbp ) was 15.0818.36 mmhg ( p<0.0001 ) , and the average reduction of morning home sbp ( m - sbp ) was 11.4919.33 mmhg ( p<0.0001 ) . beat - to - beat variability as standard deviation ( sd ) of c - sbp was reduced from 4.563.22 to 4.243.11 mmhg ( p=0.0026 ) . day - to - day variability as sd of m - sbp was reduced from 7.926.74 to 6.954.97 mmhg ( p<0.0001 ) . multiple regression analysis revealed an independent association between the change in the sd of c - sbp and the change in c - sbp ( p=0.0268 ) and , similarly , between the change in the sd of m - sbp and the change in m - sbp ( p=0.0258 ) , after adjusting for age , sex , body mass index , and change in mean bp.conclusionthis study indicated that 3 months of fimasartan treatment reduced day - to - day bp variability independent of bp reduction in patients with hypertension .

Introduction Methods Study population BP measurement Data analysis Results Baseline characteristics Changes in BP during the study Changes in BPV during the study Regression analyses of BPV at baseline and after fimasartan treatment can identify factors associated with the change in BPV (SD) in the clinic and at home Discussion Study limitations Perspectives Conclusion

blood pressure ( bp ) measured in the clinic does not provide comprehensive information about an individual s bp profile and may be of limited prognostic value , thus requiring out - of - office bp monitoring.1,2 home bp can be measured repeatedly in the comfort of the patient s own home in a relaxing environment , which provides information about day - to - day bp variability ( bpv ) and enables a more accurate understanding of a subject s bp profile . bp is characterized by marked temporal fluctuations showing beat - to - beat variability , 24-hour variability , day - to - day variability , and visit - to - visit variability . clinically , bpv is a well - known risk factor for cardiovascular events and is associated with target organ damage and all - cause mortality that is independent of office bp levels.38 beat - to - beat variability or 24-hour variability reflects increases in central sympathetic drive , decreases in arterial or cardiopulmonary reflex , and increases in arterial stiffness.9,10 humoral , rheological , and emotional factors and behavioral influences also can be involved in bpv . by contrast , day - to - day variability or visit - to - visit variability is proposed to be due to increased arterial stiffness , improper dosing or titration of antihypertensive medication , poor medication compliance , and seasonal variations in bp.11,12 day - to - day bpv is independent of beat - to - beat bpv and may have significantly different effects on hypertension prognosis . recent studies show that increased day - to - day home bpv is associated with cardiovascular risk , severity of target organ damage in patients with hypertension13 or diabetes mellitus,14 and cardiovascular mortality in a community - dwelling population.15 in the anglo - scandinavian cardiac outcomes trial - blood pressure - lowering arm trial , visit - to - visit systolic bpv during treatment was a strong predictor of stroke and coronary events independent of mean levels of clinical or ambulatory systolic bp ( sbp ) in patients with hypertension.16 therefore , reducing both bpv and mean bp has been recognized as a potential target for improved management of hypertension to prevent cardiovascular events , particularly stroke.6,16 several small - scale and heterogeneous human studies on the effects of antihypertensive drugs report conflicting results . the objective of the present study was to determine whether fimasartan reduced bpv in the clinic and at home after 3 months of treatment in hypertensive patients with low - to - moderate cardiovascular risk . the study design , socioeconomic and demographic characteristics of the study , and project details have been described in our previous article.24 this study was approved by the institutional review board committee at the cheil general hospital , on behalf of 582 primary care clinics . patients who were treated with fimasartan at baseline were excluded.24 finally , 1,396 patients were enrolled who were treated with fimasartan at a daily dose of 30120 mg for 3 months , completed the 3-month follow - up visits , and scheduled bp measurements . the omron hem-7220 and the omron hem-7200 ( both omron , tokyo , japan ) were used to measure bp in the clinic and at home , respectively.25 these are upper - arm cuff devices based on the cuff automated - oscillometric principle . clinical bp measurements were performed under standardized conditions ( in the same arm by the same physician or nurse ) . morning bp was measured within 1 hour of waking , after urination , in the sitting position , after resting for 5 minutes , and before taking medications or eating . baseline assessment included a health questionnaire and bp measurements and was conducted before and after the 3-month treatment with fimasartan.24 clinical bpv , which measures beat - to - beat variability , was defined as the standard deviation ( sd ) of clinical systolic bp ( c - sbp ) measured three times at 2-minute intervals . home bpv , which represents day - to - day bpv , was defined as the sd of morning home systolic bp ( m - sbp ) . differences between measured variables ( eg , bp ) were examined using the paired t - test between baseline and after 3 months of follow - up . multiple linear regression models included potential factors such as clinical or home average sbp and diastolic bp ( dbp ) , sex , age , history of cardiovascular disease , and smoking . patients who were treated with fimasartan at baseline were excluded.24 finally , 1,396 patients were enrolled who were treated with fimasartan at a daily dose of 30120 mg for 3 months , completed the 3-month follow - up visits , and scheduled bp measurements . clinical bp measurements were performed under standardized conditions ( in the same arm by the same physician or nurse ) . baseline assessment included a health questionnaire and bp measurements and was conducted before and after the 3-month treatment with fimasartan.24 clinical bpv , which measures beat - to - beat variability , was defined as the standard deviation ( sd ) of clinical systolic bp ( c - sbp ) measured three times at 2-minute intervals . home bpv , which represents day - to - day bpv , was defined as the sd of morning home systolic bp ( m - sbp ) . differences between measured variables ( eg , bp ) were examined using the paired t - test between baseline and after 3 months of follow - up . multiple linear regression models included potential factors such as clinical or home average sbp and diastolic bp ( dbp ) , sex , age , history of cardiovascular disease , and smoking . a total of 1,396 participants were enrolled ; the mean age was 56.1710.0 years ; 17.55% had diabetes , 6.88% had ischemic heart disease , and 0.93% had stroke . these values were significantly reduced to 127.3213.14 and 79.379.40 mmhg ( p<0.0001 for both ) , respectively , after 3 months of fimasartan treatment ( table 2 ) . m - sbp and morning home dbp levels were 138.8019.81 and 83.2613.16 mmhg , respectively ; these were significantly reduced after 3 months of fimasartan treatment to 127.3216.72 and 76.6211.32 mmhg ( p<0.0001 for both ) , respectively . the same pattern was observed for evening home sbp ( e - sbp ) and evening home dbp after 3 months of fimasartan treatment ; e - sbp was 137.5720.53 mmhg and declined to 126.3317.06 mmhg , and evening home dbp was 81.3513.29 mmhg and declined to 75.0711.44 mmhg ( p<0.0001 for both ) . at baseline , the sds of c - sbp and clinical dbp were 4.563.22 and 3.192.45 mmhg , respectively ( table 3 ) . after 3 months of fimasartan treatment , the sds of c - sbp and dbp were significantly reduced to 4.243.11 mmhg ( p=0.0026 ) and 2.942.20 mmhg ( p=0.0024 ) , respectively . the sds of m - sbp and morning home dbp also showed significant reduction from 7.926.74 and 5.303.98 mmhg to 6.954.97 and 4.833.23 mmhg ( p<0.0001 and p=0.0002 ) , respectively . the same trend was observed for the sds of evening home sbp and dbp ; sd of e - sbp declined from 8.396.94 to 7.165.38 mmhg ( p<0.0001 ) , and sd of evening home dbp declined from 5.804.13 to 5.163.32 mmhg ( p<0.0001 ) . figure 1 shows that the correlation between the sd of c - sbp and the sd of m - sbp was weak at baseline and then became stronger after 3 months of fimasartan treatment . the same pattern was observed for the sd of c - sbp and e - sbp . figure 2 summarizes the observed bp changes in c - sbp and home sbp after 3 months of follow - up . at baseline , simple and multiple regression analyses indicated that the sd of c - sbp was independently associated with c - sbp ( p<0.0001 for both ) , and the sd of m - sbp was independently associated with m - sbp ( p<0.0001 for both ) . both simple and multiple regression analyses showed that the sd of m - sbp was independently associated with age , female sex , and m - sbp ( p=0.0067 and p=0.0005 , p=0.0032 and p<0.0001 , p<0.0001 and p<0.0001 , respectively ) , but only the simple regression analysis showed an association between the sd of m - sbp and body mass index ( p=0.0246 and p=0.0553 , respectively ) ( table 4 ) . simple and multiple regression analyses ( adjusted for age , sex , body mass index , and change in mean arterial pressure ) were performed to elucidate any factors associated with reduced bpv in the clinic and at home after 3 months of fimasartan treatment . the change in the sd of c - sbp was independently associated with the change in c - sbp ( p<0.0001 and p=0.0268 , respectively ) , and the change in the sd of m - sbp was independently associated with the change in m - sbp ( p<0.0001 and p=0.0258 , respectively ) ( table 5 ) . a total of 1,396 participants were enrolled ; the mean age was 56.1710.0 years ; 17.55% had diabetes , 6.88% had ischemic heart disease , and 0.93% had stroke . these values were significantly reduced to 127.3213.14 and 79.379.40 mmhg ( p<0.0001 for both ) , respectively , after 3 months of fimasartan treatment ( table 2 ) . m - sbp and morning home dbp levels were 138.8019.81 and 83.2613.16 mmhg , respectively ; these were significantly reduced after 3 months of fimasartan treatment to 127.3216.72 and 76.6211.32 mmhg ( p<0.0001 for both ) , respectively . the same pattern was observed for evening home sbp ( e - sbp ) and evening home dbp after 3 months of fimasartan treatment ; e - sbp was 137.5720.53 mmhg and declined to 126.3317.06 mmhg , and evening home dbp was 81.3513.29 mmhg and declined to 75.0711.44 mmhg ( p<0.0001 for both ) . at baseline , the sds of c - sbp and clinical dbp were 4.563.22 and 3.192.45 mmhg , respectively ( table 3 ) . after 3 months of fimasartan treatment , the sds of c - sbp and dbp were significantly reduced to 4.243.11 mmhg ( p=0.0026 ) and 2.942.20 mmhg ( p=0.0024 ) , respectively . the sds of m - sbp and morning home dbp also showed significant reduction from 7.926.74 and 5.303.98 mmhg to 6.954.97 and 4.833.23 mmhg ( p<0.0001 and p=0.0002 ) , respectively . the same trend was observed for the sds of evening home sbp and dbp ; sd of e - sbp declined from 8.396.94 to 7.165.38 mmhg ( p<0.0001 ) , and sd of evening home dbp declined from 5.804.13 to 5.163.32 mmhg ( p<0.0001 ) . figure 1 shows that the correlation between the sd of c - sbp and the sd of m - sbp was weak at baseline and then became stronger after 3 months of fimasartan treatment . the same pattern was observed for the sd of c - sbp and e - sbp . figure 2 summarizes the observed bp changes in c - sbp and home sbp after 3 months of follow - up . at baseline , simple and multiple regression analyses indicated that the sd of c - sbp was independently associated with c - sbp ( p<0.0001 for both ) , and the sd of m - sbp was independently associated with m - sbp ( p<0.0001 for both ) . both simple and multiple regression analyses showed that the sd of m - sbp was independently associated with age , female sex , and m - sbp ( p=0.0067 and p=0.0005 , p=0.0032 and p<0.0001 , p<0.0001 and p<0.0001 , respectively ) , but only the simple regression analysis showed an association between the sd of m - sbp and body mass index ( p=0.0246 and p=0.0553 , respectively ) ( table 4 ) . simple and multiple regression analyses ( adjusted for age , sex , body mass index , and change in mean arterial pressure ) were performed to elucidate any factors associated with reduced bpv in the clinic and at home after 3 months of fimasartan treatment . the change in the sd of c - sbp was independently associated with the change in c - sbp ( p<0.0001 and p=0.0268 , respectively ) , and the change in the sd of m - sbp was independently associated with the change in m - sbp ( p<0.0001 and p=0.0258 , respectively ) ( table 5 ) . the major finding of this study was that after 3 months of fimasartan treatment , clinical and home bpvs were significantly reduced independent of strong bp reduction . these results were derived primarily from the general practitioners and participants with relatively low - to - moderate hypertension risk , and thus represent unbiased bp data in a real clinical setting . in our study , clinical bpv is calculated as the sd of three consecutive readings on the same patient in the clinic , and thus represents beat - to - beat bpv . by contrast , home bpv represents day - to - day variability of bp measured by the patients themselves at home for 7 consecutive days . beat - to - beat variability is known to be more strongly influenced by increased central sympathetic drive , decreased arterial and cardiopulmonary reflex , and humoral , rheological , behavioral , and emotional factors.27 day - to - day variability has been reported to depend more strongly on arterial stiffness , improper dosing or titration of medication , poor medication compliance , and irregularity of self - measurements performed at home.4,27 clinical bp has been shown to be strongly correlated with home bp , but the correlation between clinical bpv and home bpv has not been elucidated . the results are supported by demonstrating a relationship of day - to - day bpv with cardiovascular outcomes , which is independent of mean bp.15,16 the finn - home study confirmed this by showing that morning day - to - day bpv is a predictor of cardiovascular events after adjusting for age and mean home bp.28 other studies also demonstrated this association15,28 and the association with increased risk of cardiac , vascular , and renal organ damage.13 the reduction of bpv is recognized as a potential target for improved management of hypertension to prevent cardiovascular events . in general , calcium channel blockers have been reported to reduce bpv , which was determined by the sd of 24-hour ambulatory bp,29,30 and were superior to enalapril in the prevention of coronary events in patients with angiographically proven coronary artery disease and controlled bp.31 some arbs were shown to reduce bpv , whereas others did not show any effects . in the x - cellent study , 3 months of candesartan treatment did not show any effect on bpv evaluated by 24-hour bp monitoring.30 valsartan did not significantly change bpv in patients with hypertension after 12 months of treatment , despite reducing bp.29 in a recent study with home monitoring of bp , valsartan increased individual sd of morning sbp , but telmisartan did not affect bp after its use as an add - on agent in patients on amlodipine monotherapy.32 in an animal study , the superiority of telmisartan over valsartan in sustained bp control and reduction of bpv was attributed to further suppression of sympathetic activity and improvement of the baroreceptor reflex.33 based on this evidence , the arb - mediated reduction of bpv might not be due to its classic effects , but instead may be due to the effects of its own drug characteristics . in our study , 3 months of fimasartan treatment significantly reduced bp and bpv in the clinic and at home . although the methods of measuring bpv differ , the reductions in sd after fimasartan treatment were similar to the results of the x - cellent study with amlopidine.30 a reduction in beat - to - beat variability probably indicates a positive influence on its physiological mechanism , which may be attributable to a fimasartan - mediated reduction in central sympathetic drive . factors that result in day - to - day variability , including increased arterial stiffness , improper dosing or titration of antihypertensive medication , and poor medication compliance , can not be readily changed with 3 months of fimasartan treatment . to the best of our knowledge , the present study provides the first prospective evidence that fimasartan stabilizes day - to - day home systolic bpv . although this study did not identify the exact mechanism of fimasartan - mediated bpv reduction , the efficacy of fimasartan to reduce bpv was observed in home bpv independent of clinical bpv . therefore , we can presume that hypertensive patients with elevated day - to - day bpv may benefit from treatment with fimasartan . further studies on the change in clinical bp , morning bp , and bpv between diabetic and nondiabetic patients may be necessary as the findings may provide some insight into the mechanism of fimasartan on day - to - day bpv ( eg , arterial stiffness related to diabetes ) . fimasartan may have additional beneficial effects on cardiovascular protection by reducing bpv in addition to significantly reducing the mean levels of home bp . this study evaluated whether fimasartan treatment affected clinical and home bp variability in addition to reducing bp . three months of fimasartan treatment reduced day - to - day bp variability independent of bp reduction in patients with mild - to - moderate hypertension . fimasartan treatment also significantly reduced day - to - day bp variability at home independent of bp reduction , which may provide an additional benefit for prevention of cardiovascular events .

pleural effusion is a common clinical problem with diverse etiologies accounting for around 4% patients in pulmonary practice . pleural effusion results from an abnormal collection of fluid due to excessive production or decreased pleural fluid absorption and can be transudative or exudative depending on the fluid composition . for a definitive etiological diagnosis in pleural effusions which remain undiagnosed after diagnostic thoracentesis medical thoracoscopy / pleuroscopy is a minimally invasive procedure that allows complete visualization of the pleural space using a combination of viewing and working instruments enabling the diagnostic and the therapeutic procedures , like pleural biopsy and talc insufflation for pleurodesis to be performed safely . the procedure which can be performed using either rigid or semi - rigid instruments provides minimally invasive access to the pleural space , mediastinum and the lungs . development of the technique of semi - rigid thoracoscopy provided an alternate method to access the pleura using a scope which essentially resembles the flexible bronchoscope . over the past decade , the use of semi - rigid thoracoscope for pleuroscopy has been increasingly evaluated . the aim of our study was to evaluate the diagnostic utility and safety of semi - rigid thoracoscopy in patients with undiagnosed exudative pleural effusions presenting to our center . the study was conducted in the department of pulmonary medicine and sleep disorders at the all india institute of medical sciences ( aiims ) , new delhi , india . medical thoracoscopy ( semi - rigid thoracoscopy ) procedures were performed as part of the routine clinical protocol . patients were asked to report fasting ( for solids and liquids ) for at least 8 hours prior to the thoracoscopy procedure . history of systemic hypertension , diabetes mellitus , cardiac disease , bleeding disorders , previous anesthetic complications and any medication history including anti - platelet / anticoagulant therapy were obtained . patients with systemic hypertension were advised to take their morning dose of anti - hypertensive medications at 5 a.m. on the day of procedure with a sip of water . in known diabetic patients , morning dose of anti - diabetic medications were skipped . hemoglobin , platelet counts , ecg and prothrombin time values were available for all patients . all patients had undergone diagnostic thoracentesis [ total and differential cell count , protein , glucose , ada ( adenosine deaminase ) , acid fast stain , gram stain and bacterial cultures and three pleural fluid cytology examinations ( whole amount of pleural fluid ) ] , prior to the pleuroscopy procedure . closed pleural biopsy ( not a routine practice at our center ) prior to thoracoscopy had been performed in two patients and had been non - contributory . pre - procedure thoracic ultrasound examination ( to assess the amount of pleural fluid , loculations and for optimum site of entry ) was performed for all patients before the procedure . other relevant investigations as appropriate for the underlying clinical condition were performed in all patients . peripheral venous access was secured on forearm on the side of pleural effusion and low - flow oxygen was administered via nasal cannula . comprised a combination of short - acting benzodiazepine ( midazolam ) and an opioid ( pentazocine / fentanyl ) administered by the bronchoscopy nurse under supervision of the operating team . heart rate , blood pressure , continuous electrocardiographic monitoring and pulse oximetric saturation measurement were observed throughout the procedure and in the post - procedure period for 2 hours . after the procedure , patients were observed for recovery from anesthesia and were discharged from the hospital ( usually on the same day ) and were followed up in the outpatient clinic . the operating team usually comprised of one / two teaching faculty , one pulmonary fellow ( resident doctor ) and two nurses ( one to administer the intravenous medications and other to assist with the procedure proper ) . site of thoracoscope entry ( port ) was selected based on the pre - procedure chest ultrasound examination . the usual site was in either one of the 4 to 7 intercostal space in the mid axillary line . a lower intercostal space was usually chosen in patients with smaller sized effusions or where , ct thorax indicated a basal predominance of pleural abnormality / nodules . patient was positioned on the operating table in lateral decubitus position with the side of pleural effusion upward and the normal side being dependent . the incision site was exposed , marked and prepared aseptically covering the area from the shoulder level till the iliac crest using 10% povidone iodine . local anesthetic [ 2% lignocaine ( 21.6 mg / ml ) , usually maximum of 10 ml and taking into consideration the body weight ] was infiltrated into the selected intercostal space from the skin level to the parietal pleura . a linear incision , approximately 1 cm in size ( usual site being in the mid axillary line or slightly anteriorly ) was given and blunt dissection was performed to gain entry to the pleural space . semi - rigid thoracoscopy was performed using the olympus , ltf - type 160 semi - rigid thoracoscope . the scope essentially resembles a flexible bronchoscope and is compatible with the light source and video processor used for the flexible video - bronchoscope of corresponding make . the scope has a 22 cm rigid insertion shaft with a 5 cm flexible tip and has a 2.8 mm internal working channel for introduction of forceps and other accessories . the angulation range of flexible tip is up 160/down 130. after entry of the scope into the pleural space , pleural fluid was aspirated slowly so as to allow maximum possible visualization of pleural cavity . suction was intermittently stopped so to allow air to enter into the pleural cavity to replace the pleural fluid and to facilitate the lung collapse in order to minimize the likelihood of development of re - expansion pulmonary edema . the entire pleural space was then systematically inspected and visualized abnormalities were noted such as pleural adhesions ( thick , thin or both ) , pleural nodules and distribution and pleural infiltration ( visceral / parietal ) . multiple pleural biopsies ( about 10 to 12 biopsy samples ) were taken from the visualized abnormal areas and the samples were collected in formalin ( for histopathological examination ) and in saline ( for mycobacterial cultures ) , if tb was clinically suspected . in those patients with no visualized pleural abnormality on inspection findings , biopsies were taken from the parietal pleura and sent for histopathological examination . in patients with a clinical diagnosis of lung cancer and thoracoscopic appearance suggestive of malignant pleural involvement , samples were also additionally obtained for epidermal growth factor receptor / anaplastic lymphoma kinase ( egfr / alk ) mutation analysis . in those patients with proven lung cancer or a high likelihood of extensive malignant pleural infiltration , pleurodesis was performed either as intraprocedural talc insufflation ( 4 grams of graded large particle size talc , [ steritalc , novatech , france ] ) or iodopovidone pleurodesis ( 20 cc of 10% iodopovidone ) later . after the procedure , intercostal tube ( size 28 f or 30 f ) was inserted and secured using non - absorbable silk suture . intercostal tube was removed on the day following the procedure , or usually within 34 days time if there was increasing fluid drainage initially . pleural biopsies were subjected to detailed histopathological analysis / immunohistochemistry and interpreted by experienced pathologists . adhesiolysis if required was performed using the 2 mm biopsy forceps inserted through the working channel of the semi - rigid thoracoscope . due to inherent limitations of the small forceps size , adhesiolysis was partial in most of the cases . in case of failure of lung expansion ( hydropneumothorax ) or air leak , it was planned to keep the intercostal drain ( icd ) in situ for a longer duration . in most of the patients , semi - rigid thoracoscopy procedure was considered diagnostic , if the pleural biopsy showed definitive features such as malignancy or granulomatous inflammation suggestive of tb or positive microbiological investigations . procedure was considered non - diagnostic , if pleural biopsy showed features of non - specific pleuritis or normal pleura . patients with clinico - radiological features suggestive of tb and pleural biopsy suggestive of non - specific pleuritis were started on anti - tuberculosis therapy and were followed up . categorical variables were expressed as frequency ( percentages ) and quantitative variables were expressed as mean ( sd ) or median ( iqr ) . we searched the pubmed and embase databases for studies and systematic reviews in order to identify the indian studies reporting the diagnostic efficacy of semi - rigid thoracoscopy . patients were asked to report fasting ( for solids and liquids ) for at least 8 hours prior to the thoracoscopy procedure . history of systemic hypertension , diabetes mellitus , cardiac disease , bleeding disorders , previous anesthetic complications and any medication history including anti - platelet / anticoagulant therapy were obtained . patients with systemic hypertension were advised to take their morning dose of anti - hypertensive medications at 5 a.m. on the day of procedure with a sip of water . in known diabetic patients , morning dose of anti - diabetic medications were skipped . hemoglobin , platelet counts , ecg and prothrombin time values were available for all patients . all patients had undergone diagnostic thoracentesis [ total and differential cell count , protein , glucose , ada ( adenosine deaminase ) , acid fast stain , gram stain and bacterial cultures and three pleural fluid cytology examinations ( whole amount of pleural fluid ) ] , prior to the pleuroscopy procedure . closed pleural biopsy ( not a routine practice at our center ) prior to thoracoscopy had been performed in two patients and had been non - contributory . pre - procedure thoracic ultrasound examination ( to assess the amount of pleural fluid , loculations and for optimum site of entry ) was performed for all patients before the procedure . other relevant investigations as appropriate for the underlying clinical condition were performed in all patients . the procedures were performed in the bronchoscopy suite . peripheral venous access was secured on forearm on the side of pleural effusion and low - flow oxygen was administered via nasal cannula . comprised a combination of short - acting benzodiazepine ( midazolam ) and an opioid ( pentazocine / fentanyl ) administered by the bronchoscopy nurse under supervision of the operating team . heart rate , blood pressure , continuous electrocardiographic monitoring and pulse oximetric saturation measurement were observed throughout the procedure and in the post - procedure period for 2 hours . after the procedure , patients were observed for recovery from anesthesia and were discharged from the hospital ( usually on the same day ) and were followed up in the outpatient clinic . the operating team usually comprised of one / two teaching faculty , one pulmonary fellow ( resident doctor ) and two nurses ( one to administer the intravenous medications and other to assist with the procedure proper ) . site of thoracoscope entry ( port ) was selected based on the pre - procedure chest ultrasound examination . the usual site was in either one of the 4 to 7 intercostal space in the mid axillary line . a lower intercostal space was usually chosen in patients with smaller sized effusions or where , ct thorax indicated a basal predominance of pleural abnormality / nodules . patient was positioned on the operating table in lateral decubitus position with the side of pleural effusion upward and the normal side being dependent . the incision site was exposed , marked and prepared aseptically covering the area from the shoulder level till the iliac crest using 10% povidone iodine . local anesthetic [ 2% lignocaine ( 21.6 mg / ml ) , usually maximum of 10 ml and taking into consideration the body weight ] was infiltrated into the selected intercostal space from the skin level to the parietal pleura . a linear incision , approximately 1 cm in size ( usual site being in the mid axillary line or slightly anteriorly ) was given and blunt dissection was performed to gain entry to the pleural space . semi - rigid thoracoscopy was performed using the olympus , ltf - type 160 semi - rigid thoracoscope . the scope essentially resembles a flexible bronchoscope and is compatible with the light source and video processor used for the flexible video - bronchoscope of corresponding make . the scope has a 22 cm rigid insertion shaft with a 5 cm flexible tip and has a 2.8 mm internal working channel for introduction of forceps and other accessories . the angulation range of flexible tip is up 160/down 130. after entry of the scope into the pleural space , pleural fluid was aspirated slowly so as to allow maximum possible visualization of pleural cavity . suction was intermittently stopped so to allow air to enter into the pleural cavity to replace the pleural fluid and to facilitate the lung collapse in order to minimize the likelihood of development of re - expansion pulmonary edema . the entire pleural space was then systematically inspected and visualized abnormalities were noted such as pleural adhesions ( thick , thin or both ) , pleural nodules and distribution and pleural infiltration ( visceral / parietal ) . multiple pleural biopsies ( about 10 to 12 biopsy samples ) were taken from the visualized abnormal areas and the samples were collected in formalin ( for histopathological examination ) and in saline ( for mycobacterial cultures ) , if tb was clinically suspected . in those patients with no visualized pleural abnormality on inspection findings , biopsies were taken from the parietal pleura and sent for histopathological examination . in patients with a clinical diagnosis of lung cancer and thoracoscopic appearance suggestive of malignant pleural involvement , samples were also additionally obtained for epidermal growth factor receptor / anaplastic lymphoma kinase ( egfr / alk ) mutation analysis . in those patients with proven lung cancer or a high likelihood of extensive malignant pleural infiltration , pleurodesis was performed either as intraprocedural talc insufflation ( 4 grams of graded large particle size talc , [ steritalc , novatech , france ] ) or iodopovidone pleurodesis ( 20 cc of 10% iodopovidone ) later . after the procedure , intercostal tube ( size 28 f or 30 f ) was inserted and secured using non - absorbable silk suture . intercostal tube was removed on the day following the procedure , or usually within 34 days time if there was increasing fluid drainage initially . pleural biopsies were subjected to detailed histopathological analysis / immunohistochemistry and interpreted by experienced pathologists . adhesiolysis if required was performed using the 2 mm biopsy forceps inserted through the working channel of the semi - rigid thoracoscope . due to inherent limitations of the small forceps size , adhesiolysis was partial in most of the cases . in case of failure of lung expansion ( hydropneumothorax ) or air leak , it was planned to keep the intercostal drain ( icd ) in situ for a longer duration . in most of the patients , semi - rigid thoracoscopy procedure was considered diagnostic , if the pleural biopsy showed definitive features such as malignancy or granulomatous inflammation suggestive of tb or positive microbiological investigations . procedure was considered non - diagnostic , if pleural biopsy showed features of non - specific pleuritis or normal pleura . patients with clinico - radiological features suggestive of tb and pleural biopsy suggestive of non - specific pleuritis were started on anti - tuberculosis therapy and were followed up . categorical variables were expressed as frequency ( percentages ) and quantitative variables were expressed as mean ( sd ) or median ( iqr ) . we searched the pubmed and embase databases for studies and systematic reviews in order to identify the indian studies reporting the diagnostic efficacy of semi - rigid thoracoscopy . forty - eight patients with undiagnosed pleural effusion underwent semi - rigid thoracoscopy during the period august 2012 to december 2013 , at our center . the mean age of the patients was 50.9 14.1 years ( range 1778 years ) . 36 ( 75% ) patients were clinically suspected to have malignant pleural effusion and in 10 ( 20.83% ) patients , a clinical possibility of pleural tb had been considered . pre - procedure diagnostic possibility was based on the clinical , radiological and pleural fluid analysis profile according to the treating physician . two ( 4.17% ) patients with empyema underwent semi - rigid medical thoracoscopy for pleural biopsy , optimal placement of intercostal tube and adhesiolysis . twenty - five ( 52.08% ) patients had right - sided pleural effusion and 23 ( 47.92% ) had left - sided pleural effusion . small , moderate , large pleural effusion were present in 23 ( 47.92% ) , 18 ( 37.50% ) , 7 ( 14.58% ) patients , respectively . eighteen ( 37.5% ) patients had associated co - morbidities [ table 1 ] . thoracic ct examination showed following features : pleural nodularity 14 ( 29.79% ) patients , lung mass 18 ( 38.30% ) patients , mediastinal lymphadenopathy 11 ( 23.40% ) patients and pleural fluid loculations 13 ( 27.66% ) patients . all patients had exudative pleural effusion with mean pleural fluid protein of 4.58 0.74 ( mean sd ) g% . baseline clinico - radiological and pleural fluid characteristics of the study group are shown in table 1 . baseline characteristics of patients with undiagnosed pleural effusion undergoing semi - rigid thoracoscopy findings observed during pleural visualization included adhesions ( thick and/or thin adhesions ) in 35 ( 72.92% ) patients ; pleural nodularity and pleural infiltration in 29 ( 60.42% ) patients each ; diaphragmatic nodules in 13 ( 27.08% ) patients and visceral pleural infiltration in 19 ( 39.58% ) patients . performance characteristics of semi - rigid thoracoscopy in undiagnosed exudative pleural effusions performance characteristics of semi - rigid thoracoscopy procedures in our study in undiagnosed exudative pleural effusions are depicted in table 2 . a definitive diagnosis with a thoracoscopic pleural biopsy was obtained in 32 ( 66.67% ) out of 48 patients . malignant pleural involvement was diagnosed in 30 ( 62.5% ) patients , non - specific pleuritis in 14 ( 29.17% ) patients and tb and normal pleura were diagnosed in 2 ( 4.17% ) patients each . out of the two patients with empyema who underwent the procedure , one patient had normal pleura and the other patient had non - specific pleuritis . adenocarcinoma ( lung ) was the most common histological pleural malignancy seen in our study group ( 18 patients ) followed by small - cell lung carcinoma and metastatic carcinoma breast in three ( 6.25% ) patients each . squamous cell lung carcinoma , metastatic papillary thyroid carcinoma , poorly differentiated malignant tumor , germ cell tumor , leiomyosarcoma and adenocarcinoma with unknown primary were seen in one patient each [ table 2 ] . representative images of the radiological and thoracoscopic pleural abnormalities in patients with malignant pleural effusion are shown in figure 1 . radiological ( ct ) , thoracoscopy and histopathological appearance of patients with malignant pleural effusion ( a ) small cell carcinoma - ct - large left pleural effusion with extensive areas of pleural nodularity and pleural thickening seen . histopathological examination - photomicrograph showing sheets of small round cells with scant cytoplasm and hyper chromatic nuclei infiltrating in between fibrocollagenous tissue . ( h and e , 100 ) ( b ) metastatic papillary thyroid carcinoma - ct - large right pleural effusion with few areas of pleural nodularity seen over the posterior and basal aspects . thoracoscopy - a large sized discrete area of parietal pleural nodularity . ( ihc , 400 ) ( c ) metastatic breast carcinoma - ct - bilateral pleural effusion is seen right > left without any obvious areas of pleural thickening . histopathological examination - photomicrograph showing breast carcinoma cells infiltrating the fibrocollagenous tissue in small nests and singly . ( h and e , 400 ) sixteen ( 33.33% ) patients remained undiagnosed after thoracoscopic pleural biopsy [ pathological findings of non - specific pleuritis ( 14 ) or normal pleura ( 2 ) ] . among 14 patients with non - specific pleuritis , 5 patients were started on anti - tuberculosis therapy ( att ) based on a clinical possibility of underlying tb . two patients had complete response ( resolution of effusion ) with att , one patient is currently on att ( under follow - up and showing improvement ) and one patient was lost follow - up . the remaining fifth patient , who was initially started on att , was later diagnosed to have adenocarcinoma lung on follow - up . the remaining three patients with non - specific pleuritis were lost to follow - up . pleural biopsy samples were sent for mycobacterial cultures in 19 ( 40.43% ) patients and all were negative . the representative appearances of non - specific pleuritis and tb are shown in figures 2 and 3 , respectively . left panel - cect examination of the thorax demonstrating moderate left pleural effusion . . histopathological examination of pleural biopsy from the nodule demonstrated granulomatous inflammation compatible with tuberculosis left panel - cect examination of the thorax in a patient with rheumatoid arthritis demonstrating a large loculated right pleural effusion . right panel : thoracoscopic appearance of a large area of plaque like erythema over the visualized parietal pleura from which pleural biopsy is being obtained . histopathological examination of pleural biosy demonstrated non specific chronic inflammation egfr mutations were positive in 6 ( 12.50% ) out of 19 patients with adenocarcinoma . pleurodesis using iodopovidone and talc were performed in 25 ( 52.08% ) and 4 ( 8.33% ) patients , respectively . minor procedure - related complications included localized subcutaneous emphysema ( 8 patients ) , mild bleeding ( 2 patients ) and minor visceral pleural injury ( 1 patient ) . the patient with visceral pleural injury had forced expiratory air leak which resolved with autologous blood patch . combined overall sensitivity , specificity , positive predictive value and negative predictive value of thoracoscopic pleural biopsy for malignant pleural effusion were 96.77% , 100% , 100% and 66.67% , respectively . on performing the systematic review , four studies reporting the utility of semi - rigid thoracoscopy from india were identified which met the inclusion criteria . on performing the systematic review , four studies reporting the utility of semi - rigid thoracoscopy from india were identified which met the inclusion criteria . traditionally , medical thoracoscopy has been performed using rigid instruments and the same continued to be the case till the introduction of the semi - rigid thoracoscope . the near complete similarity of the semi - rigid thoracoscope with the flexible bronchoscope has made its adoption easy for the pulmonologists familiar with performance of flexible bronchoscopy . the efficacy , safety and high diagnostic yield of semi - rigid thoracoscopy has been reported in multiple reports and this had led to the development of this technique into a useful adjunctive diagnostic modality in patients with undiagnosed pleural effusion . the distal flexible tip ( 5 cm ) of the instrument and the better maneuverability makes easy to obtain multiple pleural biopsy samples from all accessible pleural aspects . in addition , it allows removal of thin pleural adhesions and can also guide the correct positioning of the intercostal drains . forty - eight patients with undiagnosed exudative pleural effusion underwent single port semi - rigid thoracoscopy procedure under local anesthesia and conscious sedation in our study . all patients who had pleural nodularity on ct chest were detected to have pleural nodules during the thoracoscopy procedures and were positive for malignancy on histopathological examination of the pleural biopsy . a flexible bronchoscopy examination was usually performed prior to thoracoscopy in patients with suspected malignant pleural effusion with possibility of endobronchial obstruction . in none of the patients with a contralateral mediastinal shift ( 8 ( 17.02% ) patients ) seen on ct / chest x ray did fiber optic flexible bronchoscopy demonstrate any endobronchial lesion . as has been described in the literature , contralateral shift of the mediastinum in a patient with pleural effusion essentially rules out the presence of significant ipsilateral major bronchial obstruction . in patients with malignant pleural effusion , closed pleural biopsy has been reported to be diagnostic in 44% , pleural fluid cytology in 62% , and yield is 74% when both these modalities are combined . as a standalone modality , medical thoracoscopy is diagnostic in 95% cases and 97% when all these three modalities are combined . overall sensitivity and specificity of semi - rigid thoracoscopy for malignant pleural effusion in our study were 96.8% and 100% , respectively . pleural fluid cytology examination was performed three times prior to pleuroscopy in all our patients . out of the 36 patients who were initially suspected to have malignant pleural effusion on clinico - radiological basis , malignant pleural involvement was confirmed on histopathology in 30 patients . non - specific pleuritis was diagnosed in five patients and normal pleura in one patient . out of the 10 patients with clinico - radiological suspicion of tb , 2 patients had histopathologically confirmed tb and remaining 8 patients had non - specific pleuritis . of the two patients with empyema who underwent semi - rigid thoracoscopy , one patient had normal pleura and the other had non - specific pleuritis on pleural biopsy . the results of previously reported studies from india describing the utility of semi - rigid thoracoscopy have been summarized in the table 3 . a surprisingly low incidence of tb on a thoracoscopic pleural biopsy was found in our study ( 4.17% ) . this is in contrast to high incidence of tb reported by other studies with thoracoscopic pleural biopsy in tb endemic populations such as by kannan , et al . the low incidence of tb on a thoracoscopic pleural biopsy in our study can be explained by referral bias . most of the patients with a clinical diagnosis of pleural effusion are often started on anti - tuberculous medications ( often without a pleural fluid examination also ) . therefore , many patients who present to us for evaluation of undiagnosed pleural effusion have often received some combination of anti - tb medications . therefore , patients with tuberculous pleural effusion , most of whom improve with anti - tb drugs are less likely to be referred to us . also , the sensitivity and specificity of pleural fluid ada for tubercular pleural effusions in high prevalence populations is good and most the patients in our setting receive successful treatment for tb without a pleural biopsy . notwithstanding , the diagnostic utility of thoracoscopic pleural biopsy for pleural tuberculosis has been reported to be excellent . though we observed diaphragmatic nodules in 13 patients , they were not biopsied in most of the patients as adequate pleural biopsies could be obtained from other sites of parietal pleural abnormalities . out of the 30 pleural biopsies confirmatory of malignancy , lung adenocarcinoma was the most common histology seen in our group ( 18 patients ) as has been reported in other studies also . non - specific pleuritis was seen in 14 ( 29.17% ) patients in our study group . in a retrospective study of 142 patients undergoing thoracoscopic pleural biopsy , davies , et al . all patients with the diagnosis of non - specific pleuritis were kept under close clinical follow - up in our study . out of these , five patients were started on att based on the clinical and radiological suspicion and out of them , two patients completely responded to the therapy , one patient improved ( on treatment till the time of writing the manuscript ) and one patient was lost on follow - up . the last remaining patient was later diagnosed with adenocarcinoma lung on follow - up after 5 months of att . it has been proposed that deep pleural biopsies should be obtained in patients where mesothelioma is suspected . we attempted to obtain deep pleural biopsies in all the patients but there are inherent limitations in obtaining deep pleural biopsies using the 2 mm flexible forceps . the causes of death in these patients were likely related to their associated co - morbidities and not with pleural effusion per se . venekamp , et al . followed 75 patients with non - specific pleuritis and among these , 8.3% eventually developed a malignancy during the follow - up period and in the remaining patients ( 91.7% ) , the clinical evolution followed a benign course . in the same study , true idiopathic pleuritis was seen in 25% of patients with the histological diagnosis of non - specific pleuritis . the procedure - related complications were minor in our study and there was no procedure - related mortality in our study population . no procedure was prematurely terminated because of any procedural complications . on follow - up , no patients had malignant infiltration of the thoracoscopic port site . this may be due to absence of any mesothelioma in our study group in which tumor seedling of the tract is more common . medical thoracoscopy is an extremely useful diagnostic modality that can often contribute crucially to accurate clinical decision - making in patients with undiagnosed pleural effusion . in patient where a successful biopsy can be obtained , the yield of medical thoracoscopy performed by either rigid or semi - rigid thoracoscopy instruments has been reported to be similar in a randomized comparison between the two techniques . in a prospective randomized study comparing the size , quality and diagnostic adequacy of biopsy specimens obtained by semi - rigid and rigid thoracoscope , it was demonstrated that there were no differences in the quality and interpretability of the specimens obtained by both the procedures . the diagnostic accuracy was 100% for the rigid thoracoscope and 97.6% for the semi - rigid thoracoscope . although the specimens obtained by semi - rigid thoracoscope were smaller , they were still of adequate quality and the diagnostic accuracy was comparable with that of rigid thoracoscopy in the evaluation of pleural effusion of undiagnosed etiology . rigid thoracoscope allows obtaining pleural biopsies of larger size and deep pleural biopsies which possibly may be of added utility in mesothelioma . it is definitely superior compared with the semi - rigid thoracoscope when performing adhesiolysis is the aim . in cases where an aggressive adhesiolysis is not the aim , semi - rigid thoracoscope offers particular advantages in terms of the procedure being less painful , lesser requirements of analgesic drugs and a smaller scar size . the greatest advantage , however , is the ease of adoption of the semi - rigid thoracoscope by bronchoscopist as the handling of the instrument essentially resembles that of a flexible bronchoscope . semi - rigid thoracoscopy is a safe procedure with high diagnostic yield in undiagnosed pleural effusion . rigid thoracoscope is preferable when performing adhesiolysis is the aim and for obtaining deep pleural biopsies in suspected mesothelioma . patients with non - specific pleuritis require close clinical follow - up and other ancillary investigations to ascertain the underlying cause for pleural effusion .

background : semi - rigid thoracoscopy is a safe and efficacious procedure in patients with undiagnosed pleural effusion . literature on its utility from developing countries is limited . we herein describe our initial experience on the utility of semi - rigid thoracoscopy from a tertiary care teaching and referral center in north india . we also perform a systematic review of studies reporting the utility of semi - rigid thoracoscopy from india.patients and methods : the primary objective was to evaluate the diagnostic utility of semi - rigid thoracoscopy in patients with undiagnosed exudative pleural effusion . semi - rigid thoracoscopy was performed under local anesthesia and conscious sedation in the bronchoscopy suite.results:a total of 48 patients underwent semi - rigid thoracoscopy between august 2012 and december 2013 for undiagnosed pleural effusion . mean age was 50.9 14.1 years ( range : 1778 years ) . pre - procedure clinico - radiological diagnoses were malignant pleural effusion [ 36 patients ( 75% ) ] , tuberculosis ( tb ) [ 10 ( 20.83% ) patients ] , and empyema [ 2 patients ( 4.17% ) ] . patients with empyema underwent the procedure for pleural biopsy , optimal placement of intercostal tube and adhesiolysis . thoracoscopic pleural biopsy diagnosed pleural malignancy in 30 ( 62.5% ) patients and tb in 2 ( 4.17% ) patients . fourteen ( 29.17% ) patients were diagnosed with non - specific pleuritis and normal pleura was diagnosed on a pleural biopsy in 2 ( 4.17% ) patients . overall , a definitive diagnosis of either pleural malignancy or tb was obtained in 32 ( 66.7% ) patients . combined overall sensitivity , specificity , positive predictive value and negative predictive value of thoracoscopic pleural biopsy for malignant pleural effusion were 96.77% , 100% , 100% and 66.67% , respectively . there was no procedure - related mortality . on performing a systematic review of literature , four studies on semi - rigid thoracoscopy from india were identified.conclusion:semi-rigid thoracoscopy is a safe and efficacious procedure in patients with undiagnosed exudative pleural effusions .

INTRODUCTION PATIENTS AND METHODS Patient preparation Premedication and anesthesia Semi-rigid thoracoscopy procedure Data analysis Systematic review RESULTS Systematic review DISCUSSION CONCLUSION

pleural effusion results from an abnormal collection of fluid due to excessive production or decreased pleural fluid absorption and can be transudative or exudative depending on the fluid composition . for a definitive etiological diagnosis in pleural effusions which remain undiagnosed after diagnostic thoracentesis medical thoracoscopy / pleuroscopy is a minimally invasive procedure that allows complete visualization of the pleural space using a combination of viewing and working instruments enabling the diagnostic and the therapeutic procedures , like pleural biopsy and talc insufflation for pleurodesis to be performed safely . the procedure which can be performed using either rigid or semi - rigid instruments provides minimally invasive access to the pleural space , mediastinum and the lungs . development of the technique of semi - rigid thoracoscopy provided an alternate method to access the pleura using a scope which essentially resembles the flexible bronchoscope . over the past decade , the use of semi - rigid thoracoscope for pleuroscopy has been increasingly evaluated . the aim of our study was to evaluate the diagnostic utility and safety of semi - rigid thoracoscopy in patients with undiagnosed exudative pleural effusions presenting to our center . medical thoracoscopy ( semi - rigid thoracoscopy ) procedures were performed as part of the routine clinical protocol . closed pleural biopsy ( not a routine practice at our center ) prior to thoracoscopy had been performed in two patients and had been non - contributory . pre - procedure thoracic ultrasound examination ( to assess the amount of pleural fluid , loculations and for optimum site of entry ) was performed for all patients before the procedure . peripheral venous access was secured on forearm on the side of pleural effusion and low - flow oxygen was administered via nasal cannula . heart rate , blood pressure , continuous electrocardiographic monitoring and pulse oximetric saturation measurement were observed throughout the procedure and in the post - procedure period for 2 hours . after the procedure , patients were observed for recovery from anesthesia and were discharged from the hospital ( usually on the same day ) and were followed up in the outpatient clinic . site of thoracoscope entry ( port ) was selected based on the pre - procedure chest ultrasound examination . patient was positioned on the operating table in lateral decubitus position with the side of pleural effusion upward and the normal side being dependent . a linear incision , approximately 1 cm in size ( usual site being in the mid axillary line or slightly anteriorly ) was given and blunt dissection was performed to gain entry to the pleural space . semi - rigid thoracoscopy was performed using the olympus , ltf - type 160 semi - rigid thoracoscope . in patients with a clinical diagnosis of lung cancer and thoracoscopic appearance suggestive of malignant pleural involvement , samples were also additionally obtained for epidermal growth factor receptor / anaplastic lymphoma kinase ( egfr / alk ) mutation analysis . in those patients with proven lung cancer or a high likelihood of extensive malignant pleural infiltration , pleurodesis was performed either as intraprocedural talc insufflation ( 4 grams of graded large particle size talc , [ steritalc , novatech , france ] ) or iodopovidone pleurodesis ( 20 cc of 10% iodopovidone ) later . after the procedure , intercostal tube ( size 28 f or 30 f ) was inserted and secured using non - absorbable silk suture . intercostal tube was removed on the day following the procedure , or usually within 34 days time if there was increasing fluid drainage initially . adhesiolysis if required was performed using the 2 mm biopsy forceps inserted through the working channel of the semi - rigid thoracoscope . in most of the patients , semi - rigid thoracoscopy procedure was considered diagnostic , if the pleural biopsy showed definitive features such as malignancy or granulomatous inflammation suggestive of tb or positive microbiological investigations . procedure was considered non - diagnostic , if pleural biopsy showed features of non - specific pleuritis or normal pleura . patients with clinico - radiological features suggestive of tb and pleural biopsy suggestive of non - specific pleuritis were started on anti - tuberculosis therapy and were followed up . we searched the pubmed and embase databases for studies and systematic reviews in order to identify the indian studies reporting the diagnostic efficacy of semi - rigid thoracoscopy . patients with systemic hypertension were advised to take their morning dose of anti - hypertensive medications at 5 a.m. on the day of procedure with a sip of water . all patients had undergone diagnostic thoracentesis [ total and differential cell count , protein , glucose , ada ( adenosine deaminase ) , acid fast stain , gram stain and bacterial cultures and three pleural fluid cytology examinations ( whole amount of pleural fluid ) ] , prior to the pleuroscopy procedure . closed pleural biopsy ( not a routine practice at our center ) prior to thoracoscopy had been performed in two patients and had been non - contributory . pre - procedure thoracic ultrasound examination ( to assess the amount of pleural fluid , loculations and for optimum site of entry ) was performed for all patients before the procedure . the procedures were performed in the bronchoscopy suite . peripheral venous access was secured on forearm on the side of pleural effusion and low - flow oxygen was administered via nasal cannula . heart rate , blood pressure , continuous electrocardiographic monitoring and pulse oximetric saturation measurement were observed throughout the procedure and in the post - procedure period for 2 hours . after the procedure , patients were observed for recovery from anesthesia and were discharged from the hospital ( usually on the same day ) and were followed up in the outpatient clinic . site of thoracoscope entry ( port ) was selected based on the pre - procedure chest ultrasound examination . a lower intercostal space was usually chosen in patients with smaller sized effusions or where , ct thorax indicated a basal predominance of pleural abnormality / nodules . patient was positioned on the operating table in lateral decubitus position with the side of pleural effusion upward and the normal side being dependent . semi - rigid thoracoscopy was performed using the olympus , ltf - type 160 semi - rigid thoracoscope . in patients with a clinical diagnosis of lung cancer and thoracoscopic appearance suggestive of malignant pleural involvement , samples were also additionally obtained for epidermal growth factor receptor / anaplastic lymphoma kinase ( egfr / alk ) mutation analysis . in those patients with proven lung cancer or a high likelihood of extensive malignant pleural infiltration , pleurodesis was performed either as intraprocedural talc insufflation ( 4 grams of graded large particle size talc , [ steritalc , novatech , france ] ) or iodopovidone pleurodesis ( 20 cc of 10% iodopovidone ) later . after the procedure , intercostal tube ( size 28 f or 30 f ) was inserted and secured using non - absorbable silk suture . intercostal tube was removed on the day following the procedure , or usually within 34 days time if there was increasing fluid drainage initially . adhesiolysis if required was performed using the 2 mm biopsy forceps inserted through the working channel of the semi - rigid thoracoscope . in most of the patients , semi - rigid thoracoscopy procedure was considered diagnostic , if the pleural biopsy showed definitive features such as malignancy or granulomatous inflammation suggestive of tb or positive microbiological investigations . procedure was considered non - diagnostic , if pleural biopsy showed features of non - specific pleuritis or normal pleura . patients with clinico - radiological features suggestive of tb and pleural biopsy suggestive of non - specific pleuritis were started on anti - tuberculosis therapy and were followed up . we searched the pubmed and embase databases for studies and systematic reviews in order to identify the indian studies reporting the diagnostic efficacy of semi - rigid thoracoscopy . forty - eight patients with undiagnosed pleural effusion underwent semi - rigid thoracoscopy during the period august 2012 to december 2013 , at our center . the mean age of the patients was 50.9 14.1 years ( range 1778 years ) . 36 ( 75% ) patients were clinically suspected to have malignant pleural effusion and in 10 ( 20.83% ) patients , a clinical possibility of pleural tb had been considered . pre - procedure diagnostic possibility was based on the clinical , radiological and pleural fluid analysis profile according to the treating physician . two ( 4.17% ) patients with empyema underwent semi - rigid medical thoracoscopy for pleural biopsy , optimal placement of intercostal tube and adhesiolysis . small , moderate , large pleural effusion were present in 23 ( 47.92% ) , 18 ( 37.50% ) , 7 ( 14.58% ) patients , respectively . thoracic ct examination showed following features : pleural nodularity 14 ( 29.79% ) patients , lung mass 18 ( 38.30% ) patients , mediastinal lymphadenopathy 11 ( 23.40% ) patients and pleural fluid loculations 13 ( 27.66% ) patients . all patients had exudative pleural effusion with mean pleural fluid protein of 4.58 0.74 ( mean sd ) g% . baseline clinico - radiological and pleural fluid characteristics of the study group are shown in table 1 . baseline characteristics of patients with undiagnosed pleural effusion undergoing semi - rigid thoracoscopy findings observed during pleural visualization included adhesions ( thick and/or thin adhesions ) in 35 ( 72.92% ) patients ; pleural nodularity and pleural infiltration in 29 ( 60.42% ) patients each ; diaphragmatic nodules in 13 ( 27.08% ) patients and visceral pleural infiltration in 19 ( 39.58% ) patients . performance characteristics of semi - rigid thoracoscopy in undiagnosed exudative pleural effusions performance characteristics of semi - rigid thoracoscopy procedures in our study in undiagnosed exudative pleural effusions are depicted in table 2 . a definitive diagnosis with a thoracoscopic pleural biopsy was obtained in 32 ( 66.67% ) out of 48 patients . malignant pleural involvement was diagnosed in 30 ( 62.5% ) patients , non - specific pleuritis in 14 ( 29.17% ) patients and tb and normal pleura were diagnosed in 2 ( 4.17% ) patients each . out of the two patients with empyema who underwent the procedure , one patient had normal pleura and the other patient had non - specific pleuritis . adenocarcinoma ( lung ) was the most common histological pleural malignancy seen in our study group ( 18 patients ) followed by small - cell lung carcinoma and metastatic carcinoma breast in three ( 6.25% ) patients each . representative images of the radiological and thoracoscopic pleural abnormalities in patients with malignant pleural effusion are shown in figure 1 . radiological ( ct ) , thoracoscopy and histopathological appearance of patients with malignant pleural effusion ( a ) small cell carcinoma - ct - large left pleural effusion with extensive areas of pleural nodularity and pleural thickening seen . ( h and e , 400 ) sixteen ( 33.33% ) patients remained undiagnosed after thoracoscopic pleural biopsy [ pathological findings of non - specific pleuritis ( 14 ) or normal pleura ( 2 ) ] . among 14 patients with non - specific pleuritis , 5 patients were started on anti - tuberculosis therapy ( att ) based on a clinical possibility of underlying tb . the remaining three patients with non - specific pleuritis were lost to follow - up . pleural biopsy samples were sent for mycobacterial cultures in 19 ( 40.43% ) patients and all were negative . the representative appearances of non - specific pleuritis and tb are shown in figures 2 and 3 , respectively . histopathological examination of pleural biopsy from the nodule demonstrated granulomatous inflammation compatible with tuberculosis left panel - cect examination of the thorax in a patient with rheumatoid arthritis demonstrating a large loculated right pleural effusion . pleurodesis using iodopovidone and talc were performed in 25 ( 52.08% ) and 4 ( 8.33% ) patients , respectively . minor procedure - related complications included localized subcutaneous emphysema ( 8 patients ) , mild bleeding ( 2 patients ) and minor visceral pleural injury ( 1 patient ) . combined overall sensitivity , specificity , positive predictive value and negative predictive value of thoracoscopic pleural biopsy for malignant pleural effusion were 96.77% , 100% , 100% and 66.67% , respectively . on performing the systematic review , four studies reporting the utility of semi - rigid thoracoscopy from india were identified which met the inclusion criteria . on performing the systematic review , four studies reporting the utility of semi - rigid thoracoscopy from india were identified which met the inclusion criteria . the efficacy , safety and high diagnostic yield of semi - rigid thoracoscopy has been reported in multiple reports and this had led to the development of this technique into a useful adjunctive diagnostic modality in patients with undiagnosed pleural effusion . forty - eight patients with undiagnosed exudative pleural effusion underwent single port semi - rigid thoracoscopy procedure under local anesthesia and conscious sedation in our study . a flexible bronchoscopy examination was usually performed prior to thoracoscopy in patients with suspected malignant pleural effusion with possibility of endobronchial obstruction . as has been described in the literature , contralateral shift of the mediastinum in a patient with pleural effusion essentially rules out the presence of significant ipsilateral major bronchial obstruction . in patients with malignant pleural effusion , closed pleural biopsy has been reported to be diagnostic in 44% , pleural fluid cytology in 62% , and yield is 74% when both these modalities are combined . overall sensitivity and specificity of semi - rigid thoracoscopy for malignant pleural effusion in our study were 96.8% and 100% , respectively . out of the 36 patients who were initially suspected to have malignant pleural effusion on clinico - radiological basis , malignant pleural involvement was confirmed on histopathology in 30 patients . non - specific pleuritis was diagnosed in five patients and normal pleura in one patient . out of the 10 patients with clinico - radiological suspicion of tb , 2 patients had histopathologically confirmed tb and remaining 8 patients had non - specific pleuritis . of the two patients with empyema who underwent semi - rigid thoracoscopy , one patient had normal pleura and the other had non - specific pleuritis on pleural biopsy . the results of previously reported studies from india describing the utility of semi - rigid thoracoscopy have been summarized in the table 3 . a surprisingly low incidence of tb on a thoracoscopic pleural biopsy was found in our study ( 4.17% ) . this is in contrast to high incidence of tb reported by other studies with thoracoscopic pleural biopsy in tb endemic populations such as by kannan , et al . the low incidence of tb on a thoracoscopic pleural biopsy in our study can be explained by referral bias . most of the patients with a clinical diagnosis of pleural effusion are often started on anti - tuberculous medications ( often without a pleural fluid examination also ) . therefore , many patients who present to us for evaluation of undiagnosed pleural effusion have often received some combination of anti - tb medications . also , the sensitivity and specificity of pleural fluid ada for tubercular pleural effusions in high prevalence populations is good and most the patients in our setting receive successful treatment for tb without a pleural biopsy . notwithstanding , the diagnostic utility of thoracoscopic pleural biopsy for pleural tuberculosis has been reported to be excellent . non - specific pleuritis was seen in 14 ( 29.17% ) patients in our study group . in a retrospective study of 142 patients undergoing thoracoscopic pleural biopsy , davies , et al . all patients with the diagnosis of non - specific pleuritis were kept under close clinical follow - up in our study . out of these , five patients were started on att based on the clinical and radiological suspicion and out of them , two patients completely responded to the therapy , one patient improved ( on treatment till the time of writing the manuscript ) and one patient was lost on follow - up . the causes of death in these patients were likely related to their associated co - morbidities and not with pleural effusion per se . followed 75 patients with non - specific pleuritis and among these , 8.3% eventually developed a malignancy during the follow - up period and in the remaining patients ( 91.7% ) , the clinical evolution followed a benign course . in the same study , true idiopathic pleuritis was seen in 25% of patients with the histological diagnosis of non - specific pleuritis . the procedure - related complications were minor in our study and there was no procedure - related mortality in our study population . medical thoracoscopy is an extremely useful diagnostic modality that can often contribute crucially to accurate clinical decision - making in patients with undiagnosed pleural effusion . in patient where a successful biopsy can be obtained , the yield of medical thoracoscopy performed by either rigid or semi - rigid thoracoscopy instruments has been reported to be similar in a randomized comparison between the two techniques . in a prospective randomized study comparing the size , quality and diagnostic adequacy of biopsy specimens obtained by semi - rigid and rigid thoracoscope , it was demonstrated that there were no differences in the quality and interpretability of the specimens obtained by both the procedures . the diagnostic accuracy was 100% for the rigid thoracoscope and 97.6% for the semi - rigid thoracoscope . although the specimens obtained by semi - rigid thoracoscope were smaller , they were still of adequate quality and the diagnostic accuracy was comparable with that of rigid thoracoscopy in the evaluation of pleural effusion of undiagnosed etiology . it is definitely superior compared with the semi - rigid thoracoscope when performing adhesiolysis is the aim . in cases where an aggressive adhesiolysis is not the aim , semi - rigid thoracoscope offers particular advantages in terms of the procedure being less painful , lesser requirements of analgesic drugs and a smaller scar size . the greatest advantage , however , is the ease of adoption of the semi - rigid thoracoscope by bronchoscopist as the handling of the instrument essentially resembles that of a flexible bronchoscope . semi - rigid thoracoscopy is a safe procedure with high diagnostic yield in undiagnosed pleural effusion . patients with non - specific pleuritis require close clinical follow - up and other ancillary investigations to ascertain the underlying cause for pleural effusion .

gaucher disease ( gd ) is the most common lysosomal storage disorder and is caused by a deficiency in the enzyme glucocerebrosidase ( gcase ) . it is an autosomal recessive disorder that primarily affects the mononuclear phagocyte system by lipid accumulation ( tayebi et al . patients typically manifest hepatosplenomegaly , hematological changes , anemia and orthopedic complications ( grabowski 2008 ; choy and campbell 2011 ) . gd is classified into three types , on the basis of the presence or absence of neurological involvement : type 1 , the most common form , has no associated neurological symptoms ; type 2 , or acute neuronopathic disease , displays severe neurological involvement leading to death within the first years of life ; type 3 , or chronic neuronopathic gd , exhibits varying degrees of systemic involvement with at least one neurological manifestation ( hruska et al . the gene encoding gcase , gba , is located on chromosome 1q2122 and comprises 11 exons encoding a 497 amino acid protein . so far , about 300 mutations of this gene have been identified , including frame - shift , point and splice site mutations as well as deletions , insertions and recombinant alleles ( choy et al . 2007 ; hruska et al . 2008 ; choy and campbell 2011 ) . regarding the phenotype , null , mild or severe , depending on the resulting level of gcase production . null mutations , such as c.84dupg ( 84gg ) , do not lead to any enzyme production . mild mutations , such as c.1226a > g ( n370s ) , are only associated with type 1 disease . severe mutations , such as c.1448t > c ( l444p ) , result in enzyme production and are usually associated with type 2 or 3 disease ( beutler et al . 2005 ; campbell and choy 2012 ) . findings from more studies have shown that gba is an important risk factor for parkinson s disease ( pd ) . a small subset of gd patients with gba carriers developed parkinsonian symptoms including tremor , rigidity and bradykinesia ( bembi et al . 2003 ) and were also associated with diffuse lewy body ( lb ) pathology in the brain ( nishioka et al . this article reviews the present knowledge on the interaction of synuclein and gcase , with an emphasis on the pathogenesis and mechanism of gba - associated parkinsonism . parkinson s disease is the second most common neurodegenerative disorder , affecting 1 % of people over the age of 65 worldwide ( depaolo et al . it is generally diagnosed after the age of 60 and causes motor dysfunctions , such as bradykinesia , resting tremor , rigidity and postural instability , but also affects autonomic functions and cognition ( poewe 2008 ) . the two pathological features of pd are the death of neurons in certain regions of the brain and accumulation of proteins and lipids into structures . the former leads to the clinical picture of tremor , stiffness , slowness and difficulties with posture , and the latter generates lbs inside surviving neurons ( gai et al . these neuropathologic aggregates are also associated with other neurodegenerative disorders including dementia with lbs ( dlb ) and multiple system atrophy ( msa ) ( dev et al . the pathogenesis of pd is unknown , although age and neurotoxins are associated risk factors . in recent decades , several causative genes have been identified , including three missense mutations , a30p ( krger et al . 1998 ) , e46k ( zarranz et al . 2004 ) and a53 t ( polymeropoulos et al . 1997 ) of -syn , which is strongly associated with early - onset pd ( singleton et al . susceptibility studies suggest that contributing factors to pd include abnormal handling of misfolded proteins by the ubiquitin - proteasome system ( ups ) and autophagy - lysosomal pathway ( alp ) , increased oxidative stress , and mitochondrial , lysosomal and other pathogenic dysfunctions ( lesage and brice 2009 ) . multiple independent studies have reported the association between gba mutations and parkinsonism with an increased frequency of heterozygous gba mutations in various cohorts of patients with parkinsonism ( westbroek et al . furthermore , gba mutation carriers exhibit parkinsonian phenotypes and present a diffuse pattern of lb distribution in the cerebral cortex ( do rosrio almeida 2012 ) . the initial finding was that certain patients with gd exhibited parkinsonian symptoms and lb in the clinic , and additional reports were subsequently published ( bembi et al . 2003 ; singleton et al . 2003 ; chartier - harlin et al . 2004 ; westbroek et al . . mutations in the gba gene were identified as from 4 to 28 % in cases with pd and lb disorders ( goker - alpan et al . 2010 ; clark et al . 2005 ) . in 2003 , a group was assembled of 17 such individuals of various ethnicities , including ashkenazi jewish patients . the patients in this series had relatively mild gaucher manifestations with a mean age at diagnosis of 35 years ( tayebi et al . their parkinsonian manifestations were similar to those noted in sporadic parkinson s disease , although many had an age of onset in their 40s , and most were younger than that for sporadic pd , although not all responded favorably to levodopa . these individuals exhibited asymmetric tremor , rigidity , akinesia and , at times , dementia , showing that cognitive changes had also occurred . autopsies were carried out in the brain and showed lb inclusions , appearing gcase - positive on immunostains , with a more diffuse distribution involving in the cerebral cortex , hippocampus and brainstem / limbic regions ( nishioka et al . gba mutation carriers have diverse lb pathologies in brain regions and phenotypes compared to sporadic pd patients . it was noted that gd patients with parkinsonism frequently had relatives with parkinsonism who were heterozygous for gba mutations ( goker - alpan et al . in immunological analyses of brain tissue from gd patients and gd carriers with parkinsonism , all the samples with gba mutations showed lb pathology ( shachar et al . many other studies in different populations further support the link among gd , pd and lb disorders ( clark et al . 2008 ; kalinderi et al . 2009 ; sidransky et al . 2009 ; mitsui et al . 2009 ; lesage et al . 2010 ; mao et al . 2010 ) . a study of gba in 57 brain bank samples from subjects with pathologically confirmed pd demonstrated that 12 % carried a mutation , which was significantly higher than the mutation frequency even in the at - risk ashkenazi jewish population ( lwin et al . family studies revealed that among relatives of gaucher probands , there was an increased number of individuals affected with parkinsonism ( goker - alpan et al . importantly , several additional genetic studies in large patient cohorts demonstrated that patients with parkinsonism have an increased incidence of gba mutations . in 2009 , sidransky et al . published a study that consisted of a collective analysis of 5691 patients with pd , complemented by 4898 controls , from 16 centers and across 12 countries . the full gba coding region was screened , and loss - of - function mutations were observed in 6.9 % of cases and 1.3 % of controls . among the ashkenazi jewish subset , higher mutation frequencies were seen : 19.3 % in cases and 4.1 % in controls ( sidransky et al . subsequent studies indicated an increased incidence of pd in relatives of gd patients , many of whom were carriers of gba mutations ( goker - alpan et al . 2006 ) , making gba the most common known genetic risk factor for pd to date . furthermore , it is reported that non - motor symptoms ( nmss ) are more frequent in pd patients with heterozygous glucocerebrosidase mutations ( pd - gba ) ( brockmann et al . the overall nms score was higher for pd - gba than sporadic pd patients , and pd - gba patients showed more severe cognitive dysfunction , apathy , depression and anxiety ( mcneill et al . importantly , patients with gba - positive pd had significantly less precise memory compared to cases with gba - negative pd as well as gba - positive individuals without pd ( zokaei et al . meanwhile , among gd patients and heterozygous carriers , hyposmia , cognitive dysfunction and parkinsonian motor signs are prevalent . hyposmia is the most common prodromal marker , 10 % in gd patients and gba carriers , suggesting that hyposmia develops before cognitive dysfunction or motor abnormalities ( mcneill et al . hyposmia also occurs in lbd , in which olfactory dysfunction in lbd and pd is associated with lb deposition in the olfactory bulb , nucleus and cortex ( williams et al . it proposes that hyposmia may be the most sensitive , but not a specific marker for identifying gd patients and carriers at greatest risk of developing pd . the mechanisms underlying the relation between gba mutations and the development of parkinson s disease remain elusive . however , recent studies provide some new perspectives . generally , in autosomal recessive forms of parkinson s disease , such as those involving park2 , dj-1 and pink1 , loss - of - function mutations are implicated , and these patients have an early onset of disease manifestation . by contrast , gain - of - function mutations , for example those in scna and lrrk2 , are usually associated with autosomal dominant forms of parkinson s disease ( hardy et al . the recognition of the link among gba mutants , gcase activity , snca and pd has made an important contribution to our thinking on the pathogenetic pathways in pd and dementia with lbs ( lwin et al . support for gain - of - function mutations in gba is mainly from the following findings . most gba mutations are missense mutations , which result in a misfolded protein . in keeping with this is the finding that mutant gcase is present within a significant proportion of -syn inclusions in pd and gd brains ( sardi et al . 2011 , 2013 ) , suggesting that this abnormal conformation could contribute to the development of parkinsonism by increasing -syn aggregation or directly interfering with the cellular autophagic - lysosomal mechanisms that mediate -syn degradation ( cuervo et al . 2004 ) . furthermore , mutant gcase can accumulate in the endoplasmic reticulum , overwhelming the ubiquitin - proteasome pathway , burdening the lysosomal system or causing impairment of autophagy . some gba mutations encountered in patients with parkinsonism are null mutations a finding in conflict with the gain - of - function hypothesis . in fact , carriers of null alleles might have an even higher risk of developing parkinsonism ( neumann et al . the potential role of lipid homoeostasis in causing parkinson s disease is a topic of great interest . parkinsonism could arise as a consequence of gcase deficiency because of the loss of function of the enzyme . -syn can bind to the lipid raft - associated ganglioside gm1s ( martinez et al . 2007 ; wei et al . 2009 ; yap et al . 2013 ) , so disordered glucosylceramide ( glccer ) metabolism is postulated to affect proper trafficking of -syn to presynaptic membranes or to directly affect its fibrillization , leading to an accumulation of toxic -syn species . hence , lipid storage could cause changes in lipid homoeostasis , with subsequent alterations in synuclein processing ( fortin et al . 2004 ; wei et al . 2009 ) . in gcase ko mice markers of neurodegeneration , p62/sqstm1 , ubiquitinated proteins and insoluble mitochondria were dysfunctional and fragmented , with impaired respiration and reduced respiratory chain complex activities . thus , a primary lysosomal defect causes accumulation of dysfunctional mitochondria as a result of impaired autophagy and dysfunctional proteasomal pathways ( osellame et al . gcase enzyme activity was reduced in the substantia nigra and cerebellum in sporadic pd brains , and gcase deficiency was significantly observed in pd with gba brain areas . for the most part , gcase protein expression was lower in pd with gba and pd brains , as well also decreased in sh - sy5y cells overexpressing -syn or with pten - induced putative kinase 1 ( pink1 ) deficiency ( gegg et al . 2012 ) . similar results were observed in fibroblasts from patients with gd and heterozygous mutation carriers with and without parkinson s disease compared with controls ( mcneill et al . the clinical finding that most patients with gd never develop parkinson s disease , despite low gcase activity , argues against a solely loss - of - function mechanism . this theory is supported by the low frequency of parkinson s disease reported in the large gaucher cohort in the international collaborative gaucher group ( icgg ) gaucher registry ( rosenbloom et al . thus , in most cases , the enzymatic deficiency can not predict parkinson s disease . -syn , a kind of presynaptic protein , is widely recognized for its role in pd . findings from recent studies have further suggested that -syn is involved in the pathogenesis of gba - associated parkinsonism . in the early stages of parkinson s disease , gcase deficits in sporadic parkinson s disease are related to the abnormal accumulation of -syn ( murphy et al . recently , a selective interaction between -syn and gcase was determined ( yap et al . 2011 ) . the interaction occurred under lysosomal solution conditions ( ph 5.5 ) , and no significant interaction was noted at ph 7.4 . residue level mapping showed that the interaction site was at the -syn c - terminal residues , 118137 . immunoprecipitation and immunofluorescence studies verified this interaction in human tissue and neuronal cell culture , respectively . intriguingly , the n370s mutant form of gcase has a reduced affinity for -syn , so this binding at lysosomal ph might play an important role in facilitating -syn degradation or preventing aggregation . much of the processing of -syn occurs in the cytoplasm , where this interaction was unexpected , but it offers a possibility for lysosomal pathways in -syn degradation . when gba is mutated or absent , the beneficial role of the interaction in -syn processing is disrupted . these findings suggest that the -syn - gcase association is favored in the lysosome and that understanding this noncovalent interaction provides the groundwork to explore molecular mechanisms linking pd with mutant gba alleles . studied the association between -syn pathology and gcase from a different perspective ( mazzulli et al . 2011 ) . they performed a small hairpin rna ( shrna)-mediated knockdown of gcase by lentiviral infection , which resulted in a 50 % reduction in gcase protein levels . reduced concentrations of endoglycosidase h - resistant gcase were also reported , ascribed to the depletion of the lysosomal form of the enzyme . gcase knockdown increased the steady - state levels of -syn 1.8-fold relative to controls , while the levels of tau , another disease - associated aggregation - prone protein , did not change . the same group then analyzed the dopaminergic neurons that were generated from induced pluripotent stem cells made from reprogrammed fibroblasts of a patient with gd . the results showed that these neurons also had an accumulation of -syn that resulted in neurotoxicity attributed to aggregation - dependent mechanisms . by working with a mouse model and human brain samples , they concluded that intracellular glccer levels control the formation of soluble toxic -syn oligomers and fibrils in cultured neurons , and mouse and human brain , leading to neurodegeneration . the promoted formation of -syn assemblies further contributes to a pathogenic cycle by inhibiting the lysosomal maturation and activity of normal gcase , resulting in additional glccer accumulation and elevated -syn oligomer formation . however , studies of brain samples indicate that aggregation of -syn is not related to gcase deficiency alone . proteins extracted from cerebral cortex samples from patients with synucleinopathies with and without gba mutations , as well as samples from control individuals and patients with gd , were studied by western blotting . patients with gba - associated parkinsonism showed aggregated oligomeric forms of -syn in the insoluble fraction , whereas only monomeric -syn was noted in patients with gba mutations without parkinsonism , including samples from patients with neuropathic gd ( choi et al . 2011 ) . recent evidence in saccharomyces cerevisiae and cell lines indicates that overexpression of -syn has the ability to block endoplasmic reticulum - golgi trafficking of proteins by inhibiting the formation of soluble n - ethylmaleimide - sensitive factor attachment protein receptor ( snare ) protein complexes ( cooper et al . furthermore , mazzulli et al . used primary cortical neurons to demonstrate that overexpression of either wild - type or a53t - mutant -syn resulted in retention of gcase in the er , associated with reduced lysosomal gcase activity ( mazzulli et al . 2011 ) . they then confirmed this in vitro by studying gcase glycosylation patterns in pd and healthy control brain tissue . in addition , the same group analyzed human cortical material by gcase western blot and showed that normal variation of -syn protein levels modulates the lysosomal maturation and activity of gcase in vivo . taken together , these data suggest that elevated levels of -syn observed in pd and other synucleinopathies leads to decreased lysosomal activity of normal gcase ( mazzulli et al . support for gain - of - function mutations in gba is mainly from the following findings . most gba mutations are missense mutations , which result in a misfolded protein . in keeping with this is the finding that mutant gcase is present within a significant proportion of -syn inclusions in pd and gd brains ( sardi et al . 2011 , 2013 ) , suggesting that this abnormal conformation could contribute to the development of parkinsonism by increasing -syn aggregation or directly interfering with the cellular autophagic - lysosomal mechanisms that mediate -syn degradation ( cuervo et al . 2004 ) . furthermore , mutant gcase can accumulate in the endoplasmic reticulum , overwhelming the ubiquitin - proteasome pathway , burdening the lysosomal system or causing impairment of autophagy . some gba mutations encountered in patients with parkinsonism are null mutations a finding in conflict with the gain - of - function hypothesis . in fact , carriers of null alleles might have an even higher risk of developing parkinsonism ( neumann et al . the potential role of lipid homoeostasis in causing parkinson s disease is a topic of great interest . parkinsonism could arise as a consequence of gcase deficiency because of the loss of function of the enzyme . -syn can bind to the lipid raft - associated ganglioside gm1s ( martinez et al . 2007 ; wei et al . 2009 ; yap et al . 2013 ) , so disordered glucosylceramide ( glccer ) metabolism is postulated to affect proper trafficking of -syn to presynaptic membranes or to directly affect its fibrillization , leading to an accumulation of toxic -syn species . hence , lipid storage could cause changes in lipid homoeostasis , with subsequent alterations in synuclein processing ( fortin et al . 2009 ) . in gcase ko mice markers of neurodegeneration , p62/sqstm1 , ubiquitinated proteins and insoluble mitochondria were dysfunctional and fragmented , with impaired respiration and reduced respiratory chain complex activities . thus , a primary lysosomal defect causes accumulation of dysfunctional mitochondria as a result of impaired autophagy and dysfunctional proteasomal pathways ( osellame et al . gcase enzyme activity was reduced in the substantia nigra and cerebellum in sporadic pd brains , and gcase deficiency was significantly observed in pd with gba brain areas . for the most part , gcase protein expression was lower in pd with gba and pd brains , as well also decreased in sh - sy5y cells overexpressing -syn or with pten - induced putative kinase 1 ( pink1 ) deficiency ( gegg et al . 2012 ) . similar results were observed in fibroblasts from patients with gd and heterozygous mutation carriers with and without parkinson s disease compared with controls ( mcneill et al . the clinical finding that most patients with gd never develop parkinson s disease , despite low gcase activity , argues against a solely loss - of - function mechanism . this theory is supported by the low frequency of parkinson s disease reported in the large gaucher cohort in the international collaborative gaucher group ( icgg ) gaucher registry ( rosenbloom et al . thus , in most cases , the enzymatic deficiency can not predict parkinson s disease . -syn , a kind of presynaptic protein , is widely recognized for its role in pd . findings from recent studies have further suggested that -syn is involved in the pathogenesis of gba - associated parkinsonism . in the early stages of parkinson s disease , gcase deficits in sporadic parkinson s disease are related to the abnormal accumulation of -syn ( murphy et al . recently , a selective interaction between -syn and gcase was determined ( yap et al . 2011 ) . the interaction occurred under lysosomal solution conditions ( ph 5.5 ) , and no significant interaction was noted at ph 7.4 . residue level mapping showed that the interaction site was at the -syn c - terminal residues , 118137 . immunoprecipitation and immunofluorescence studies verified this interaction in human tissue and neuronal cell culture , respectively . intriguingly , the n370s mutant form of gcase has a reduced affinity for -syn , so this binding at lysosomal ph might play an important role in facilitating -syn degradation or preventing aggregation . much of the processing of -syn occurs in the cytoplasm , where this interaction was unexpected , but it offers a possibility for lysosomal pathways in -syn degradation . when gba is mutated or absent , the beneficial role of the interaction in -syn processing is disrupted . these findings suggest that the -syn - gcase association is favored in the lysosome and that understanding this noncovalent interaction provides the groundwork to explore molecular mechanisms linking pd with mutant gba alleles . studied the association between -syn pathology and gcase from a different perspective ( mazzulli et al . 2011 ) . they performed a small hairpin rna ( shrna)-mediated knockdown of gcase by lentiviral infection , which resulted in a 50 % reduction in gcase protein levels . reduced concentrations of endoglycosidase h - resistant gcase were also reported , ascribed to the depletion of the lysosomal form of the enzyme . gcase knockdown increased the steady - state levels of -syn 1.8-fold relative to controls , while the levels of tau , another disease - associated aggregation - prone protein , did not change . the same group then analyzed the dopaminergic neurons that were generated from induced pluripotent stem cells made from reprogrammed fibroblasts of a patient with gd . the results showed that these neurons also had an accumulation of -syn that resulted in neurotoxicity attributed to aggregation - dependent mechanisms . by working with a mouse model and human brain samples , they concluded that intracellular glccer levels control the formation of soluble toxic -syn oligomers and fibrils in cultured neurons , and mouse and human brain , leading to neurodegeneration . the promoted formation of -syn assemblies further contributes to a pathogenic cycle by inhibiting the lysosomal maturation and activity of normal gcase , resulting in additional glccer accumulation and elevated -syn oligomer formation . however , studies of brain samples indicate that aggregation of -syn is not related to gcase deficiency alone . proteins extracted from cerebral cortex samples from patients with synucleinopathies with and without gba mutations , as well as samples from control individuals and patients with gd , were studied by western blotting . patients with gba - associated parkinsonism showed aggregated oligomeric forms of -syn in the insoluble fraction , whereas only monomeric -syn was noted in patients with gba mutations without parkinsonism , including samples from patients with neuropathic gd ( choi et al . 2011 ) . recent evidence in saccharomyces cerevisiae and cell lines indicates that overexpression of -syn has the ability to block endoplasmic reticulum - golgi trafficking of proteins by inhibiting the formation of soluble n - ethylmaleimide - sensitive factor attachment protein receptor ( snare ) protein complexes ( cooper et al . furthermore , mazzulli et al . used primary cortical neurons to demonstrate that overexpression of either wild - type or a53t - mutant -syn resulted in retention of gcase in the er , associated with reduced lysosomal gcase activity ( mazzulli et al . 2011 ) . they then confirmed this in vitro by studying gcase glycosylation patterns in pd and healthy control brain tissue . in addition , the same group analyzed human cortical material by gcase western blot and showed that normal variation of -syn protein levels modulates the lysosomal maturation and activity of gcase in vivo . taken together , these data suggest that elevated levels of -syn observed in pd and other synucleinopathies leads to decreased lysosomal activity of normal gcase ( mazzulli et al . the proposed gain - of - function or loss - of - function mechanisms do not perfectly explain the association between pd and gd , and there are some clinical exceptions for each of them . therefore , the focus should be on the altered function of -syn in the observed association instead of a single gain or loss of function . a newly identified interaction between -syn and gcase was reported that might influence cellular levels of -syn by either promoting protein degradation and/or preventing aggregation ( sybertz and krainc 2014 ; yap et al . the authors also demonstrated that membrane - bound -syn interacted with gcase and that this complex formation inhibited enzyme function . the interaction of -syn and gcase in lysosomes may be beneficial to the degradation of -syn , while intracellular glccer levels control the formation of soluble toxic -syn oligomers and fibrils ( mazzulli et al . in other words , the interactions between -syn and gcase and between -syn and glccer have opposite effects on the aggregation of -syn . some gba mutations result in misfolded gcase , which has no activity but can still interact with -syn in lysosomes and contribute to the degradation . thus , the aggregation of -syn caused by glccer is compromised , which may explain why gd patients with low gcase activity will not develop parkinsonism . when gba mutations affect the interaction site , like n370s , the beneficial role of the interaction in -syn processing is disrupted , and the balance between aggregation and degradation is broken . this may be why carriers of null alleles might have an even higher risk of developing parkinsonism ( neumann et al . , if the aggregation of -syn can not be cleared , it will further inhibit the lysosomal maturation and gcase activity , resulting in additional glccer accumulation and elevated -syn . recent data show that increasing gcase activity with adeno - associated viral vector ( aav ) delivery of the enzyme into the brain of a gd mouse model can reduce the accumulation of snca , tau and ubiquitin ( sardi et al . 2013 ) . it suggested that increasing the glycosidase activity can modulate -syn processing and may modulate the progression of -synucleinopathies . in addition , neuronal glucosylceramide ( glccer ) accumulates to a certain threshold ; a series of second events is triggered , which includes neuroinflammation and neurodegeneration , then subsequently causes neuronal death ( farfel - becker et al . 2014 ) . hence , increasing the gcase activity in the cns represents a potential therapeutic strategy for gba - related and non - gba - associated synucleinopathies , including pd ( schapira and gegg 2013 ) . since a reduction of glucosylceramidase protein levels and activity occurs in the brain of gd and pd patients with gba mutation carriers , gcaes is a potential treatment target for gd therapy . ambroxol hydrochloride , a small molecule , reduces alpha - synuclein levels in overexpressing neuroblastoma cells and markers of oxidative and endoplasmic reticulum stress in cells bearing gcase mutations . ambroxol treatment results in a significant elevation of glucosylceramidase protein and activity levels in gd and pd with gba mutations by activating the coordinated lysosomal expression and regulation network ( mcneill et al . in addition , lysosomal activity of gc is tightly linked to expression of its trafficking receptor , the lysosomal integral membrane protein type-2 ( limp-2 ) , which acts as a chaperone helping to traffic glucosylceramidase from the endoplasmic reticulum to the lysosome ; therefore , manipulating limp-2 expression to increase lysosomal gc activity is also a promising strategy for the treatment of synucleinopathies and gba mutation carriers . heterologous expression of limp-2 accelerated clearance of overexpressed -synuclein and promoted autophagic / lysosomal function , possibly by increasing lysosomal gc activity ( rothaug et al . additionally , the receptor - interacting protein kinases ( ripks ) , a class of serine / threonine protein kinases , are directly involved in the pathway of pathological events in severe forms of gd . the significant extension of lifespan and improvements of motor coordination in ripk3 knockout mice support the notion that ripk3 is not only a key activator of necrotic cell death , but also the ripk3 pathway might be a molecular target for therapeutic intervention in gd ( vitner et al . further studies using techniques in cell biology , neuropathology and genetics are needed to better reveal the mechanisms that contribute to gba - associated parkinsonism . the role of -syn and gcase will continue to be a source of considerable interest in the field . elucidating the factors underlying this association is necessary to improve genetic counseling for people who carry mutations in this gene . moreover , a better understanding of the role of gcase in the development of parkinsonism will facilitate the development of new therapeutic strategies for gba - associated parkinsonism .

gaucher disease is associated with parkinson s disease ( pd ) by mutations in glucocerebrosidase ( gcase ) . the gene encoding gcase , glucosidase beta acid ( gba ) , is an important risk factor for pd . findings from large studies have shown that patients with pd have an increased frequency of mutations in gba and that gba mutation carriers exhibit diverse parkinsonian phenotypes and lewy body pathology . although the mechanism for this association remains elusive , some hypotheses have been proposed to explain it , including gain of function caused by gba mutations , which increases -synuclein ( -syn ) aggregation , loss of function due to lysosomal enzyme deficiency , which affects -syn clearance , and even a bidirectional feedback loop , but each of these hypotheses has its limitations . it is also worth noting that many findings have implicated the interaction between -syn and gcase , indicating the essential role of the interaction in the pathogenesis of gba - associated parkinsonism . therefore , the current review focuses on -syn and gcase , and it provides some new thoughts that may be helpful for understanding the -syn - gcase interaction and unraveling the exact mechanism underlying gba - associated parkinsonism .

Introduction Gaucher Disease with Parkinsonism and Parkinsons Disease in Proposed Mechanism for Gain of Function Loss of Function The Role of -Synuclein in Parkinsonism with Therapeutic Implication in -syn and GCase Interaction

gaucher disease ( gd ) is the most common lysosomal storage disorder and is caused by a deficiency in the enzyme glucocerebrosidase ( gcase ) . it is an autosomal recessive disorder that primarily affects the mononuclear phagocyte system by lipid accumulation ( tayebi et al . gd is classified into three types , on the basis of the presence or absence of neurological involvement : type 1 , the most common form , has no associated neurological symptoms ; type 2 , or acute neuronopathic disease , displays severe neurological involvement leading to death within the first years of life ; type 3 , or chronic neuronopathic gd , exhibits varying degrees of systemic involvement with at least one neurological manifestation ( hruska et al . the gene encoding gcase , gba , is located on chromosome 1q2122 and comprises 11 exons encoding a 497 amino acid protein . so far , about 300 mutations of this gene have been identified , including frame - shift , point and splice site mutations as well as deletions , insertions and recombinant alleles ( choy et al . null mutations , such as c.84dupg ( 84gg ) , do not lead to any enzyme production . mild mutations , such as c.1226a > g ( n370s ) , are only associated with type 1 disease . severe mutations , such as c.1448t > c ( l444p ) , result in enzyme production and are usually associated with type 2 or 3 disease ( beutler et al . findings from more studies have shown that gba is an important risk factor for parkinson s disease ( pd ) . 2003 ) and were also associated with diffuse lewy body ( lb ) pathology in the brain ( nishioka et al . this article reviews the present knowledge on the interaction of synuclein and gcase , with an emphasis on the pathogenesis and mechanism of gba - associated parkinsonism . parkinson s disease is the second most common neurodegenerative disorder , affecting 1 % of people over the age of 65 worldwide ( depaolo et al . it is generally diagnosed after the age of 60 and causes motor dysfunctions , such as bradykinesia , resting tremor , rigidity and postural instability , but also affects autonomic functions and cognition ( poewe 2008 ) . the two pathological features of pd are the death of neurons in certain regions of the brain and accumulation of proteins and lipids into structures . the former leads to the clinical picture of tremor , stiffness , slowness and difficulties with posture , and the latter generates lbs inside surviving neurons ( gai et al . these neuropathologic aggregates are also associated with other neurodegenerative disorders including dementia with lbs ( dlb ) and multiple system atrophy ( msa ) ( dev et al . the pathogenesis of pd is unknown , although age and neurotoxins are associated risk factors . in recent decades , several causative genes have been identified , including three missense mutations , a30p ( krger et al . 1997 ) of -syn , which is strongly associated with early - onset pd ( singleton et al . susceptibility studies suggest that contributing factors to pd include abnormal handling of misfolded proteins by the ubiquitin - proteasome system ( ups ) and autophagy - lysosomal pathway ( alp ) , increased oxidative stress , and mitochondrial , lysosomal and other pathogenic dysfunctions ( lesage and brice 2009 ) . multiple independent studies have reported the association between gba mutations and parkinsonism with an increased frequency of heterozygous gba mutations in various cohorts of patients with parkinsonism ( westbroek et al . furthermore , gba mutation carriers exhibit parkinsonian phenotypes and present a diffuse pattern of lb distribution in the cerebral cortex ( do rosrio almeida 2012 ) . the initial finding was that certain patients with gd exhibited parkinsonian symptoms and lb in the clinic , and additional reports were subsequently published ( bembi et al . mutations in the gba gene were identified as from 4 to 28 % in cases with pd and lb disorders ( goker - alpan et al . their parkinsonian manifestations were similar to those noted in sporadic parkinson s disease , although many had an age of onset in their 40s , and most were younger than that for sporadic pd , although not all responded favorably to levodopa . gba mutation carriers have diverse lb pathologies in brain regions and phenotypes compared to sporadic pd patients . it was noted that gd patients with parkinsonism frequently had relatives with parkinsonism who were heterozygous for gba mutations ( goker - alpan et al . a study of gba in 57 brain bank samples from subjects with pathologically confirmed pd demonstrated that 12 % carried a mutation , which was significantly higher than the mutation frequency even in the at - risk ashkenazi jewish population ( lwin et al . importantly , several additional genetic studies in large patient cohorts demonstrated that patients with parkinsonism have an increased incidence of gba mutations . published a study that consisted of a collective analysis of 5691 patients with pd , complemented by 4898 controls , from 16 centers and across 12 countries . the full gba coding region was screened , and loss - of - function mutations were observed in 6.9 % of cases and 1.3 % of controls . subsequent studies indicated an increased incidence of pd in relatives of gd patients , many of whom were carriers of gba mutations ( goker - alpan et al . 2006 ) , making gba the most common known genetic risk factor for pd to date . furthermore , it is reported that non - motor symptoms ( nmss ) are more frequent in pd patients with heterozygous glucocerebrosidase mutations ( pd - gba ) ( brockmann et al . the overall nms score was higher for pd - gba than sporadic pd patients , and pd - gba patients showed more severe cognitive dysfunction , apathy , depression and anxiety ( mcneill et al . importantly , patients with gba - positive pd had significantly less precise memory compared to cases with gba - negative pd as well as gba - positive individuals without pd ( zokaei et al . hyposmia also occurs in lbd , in which olfactory dysfunction in lbd and pd is associated with lb deposition in the olfactory bulb , nucleus and cortex ( williams et al . it proposes that hyposmia may be the most sensitive , but not a specific marker for identifying gd patients and carriers at greatest risk of developing pd . the mechanisms underlying the relation between gba mutations and the development of parkinson s disease remain elusive . generally , in autosomal recessive forms of parkinson s disease , such as those involving park2 , dj-1 and pink1 , loss - of - function mutations are implicated , and these patients have an early onset of disease manifestation . by contrast , gain - of - function mutations , for example those in scna and lrrk2 , are usually associated with autosomal dominant forms of parkinson s disease ( hardy et al . the recognition of the link among gba mutants , gcase activity , snca and pd has made an important contribution to our thinking on the pathogenetic pathways in pd and dementia with lbs ( lwin et al . support for gain - of - function mutations in gba is mainly from the following findings . most gba mutations are missense mutations , which result in a misfolded protein . some gba mutations encountered in patients with parkinsonism are null mutations a finding in conflict with the gain - of - function hypothesis . the potential role of lipid homoeostasis in causing parkinson s disease is a topic of great interest . parkinsonism could arise as a consequence of gcase deficiency because of the loss of function of the enzyme . gcase enzyme activity was reduced in the substantia nigra and cerebellum in sporadic pd brains , and gcase deficiency was significantly observed in pd with gba brain areas . similar results were observed in fibroblasts from patients with gd and heterozygous mutation carriers with and without parkinson s disease compared with controls ( mcneill et al . the clinical finding that most patients with gd never develop parkinson s disease , despite low gcase activity , argues against a solely loss - of - function mechanism . this theory is supported by the low frequency of parkinson s disease reported in the large gaucher cohort in the international collaborative gaucher group ( icgg ) gaucher registry ( rosenbloom et al . thus , in most cases , the enzymatic deficiency can not predict parkinson s disease . findings from recent studies have further suggested that -syn is involved in the pathogenesis of gba - associated parkinsonism . in the early stages of parkinson s disease , gcase deficits in sporadic parkinson s disease are related to the abnormal accumulation of -syn ( murphy et al . recently , a selective interaction between -syn and gcase was determined ( yap et al . the interaction occurred under lysosomal solution conditions ( ph 5.5 ) , and no significant interaction was noted at ph 7.4 . residue level mapping showed that the interaction site was at the -syn c - terminal residues , 118137 . intriguingly , the n370s mutant form of gcase has a reduced affinity for -syn , so this binding at lysosomal ph might play an important role in facilitating -syn degradation or preventing aggregation . much of the processing of -syn occurs in the cytoplasm , where this interaction was unexpected , but it offers a possibility for lysosomal pathways in -syn degradation . when gba is mutated or absent , the beneficial role of the interaction in -syn processing is disrupted . these findings suggest that the -syn - gcase association is favored in the lysosome and that understanding this noncovalent interaction provides the groundwork to explore molecular mechanisms linking pd with mutant gba alleles . studied the association between -syn pathology and gcase from a different perspective ( mazzulli et al . reduced concentrations of endoglycosidase h - resistant gcase were also reported , ascribed to the depletion of the lysosomal form of the enzyme . the promoted formation of -syn assemblies further contributes to a pathogenic cycle by inhibiting the lysosomal maturation and activity of normal gcase , resulting in additional glccer accumulation and elevated -syn oligomer formation . proteins extracted from cerebral cortex samples from patients with synucleinopathies with and without gba mutations , as well as samples from control individuals and patients with gd , were studied by western blotting . patients with gba - associated parkinsonism showed aggregated oligomeric forms of -syn in the insoluble fraction , whereas only monomeric -syn was noted in patients with gba mutations without parkinsonism , including samples from patients with neuropathic gd ( choi et al . used primary cortical neurons to demonstrate that overexpression of either wild - type or a53t - mutant -syn resulted in retention of gcase in the er , associated with reduced lysosomal gcase activity ( mazzulli et al . support for gain - of - function mutations in gba is mainly from the following findings . most gba mutations are missense mutations , which result in a misfolded protein . some gba mutations encountered in patients with parkinsonism are null mutations a finding in conflict with the gain - of - function hypothesis . in fact , carriers of null alleles might have an even higher risk of developing parkinsonism ( neumann et al . the potential role of lipid homoeostasis in causing parkinson s disease is a topic of great interest . parkinsonism could arise as a consequence of gcase deficiency because of the loss of function of the enzyme . -syn can bind to the lipid raft - associated ganglioside gm1s ( martinez et al . gcase enzyme activity was reduced in the substantia nigra and cerebellum in sporadic pd brains , and gcase deficiency was significantly observed in pd with gba brain areas . similar results were observed in fibroblasts from patients with gd and heterozygous mutation carriers with and without parkinson s disease compared with controls ( mcneill et al . the clinical finding that most patients with gd never develop parkinson s disease , despite low gcase activity , argues against a solely loss - of - function mechanism . this theory is supported by the low frequency of parkinson s disease reported in the large gaucher cohort in the international collaborative gaucher group ( icgg ) gaucher registry ( rosenbloom et al . thus , in most cases , the enzymatic deficiency can not predict parkinson s disease . findings from recent studies have further suggested that -syn is involved in the pathogenesis of gba - associated parkinsonism . in the early stages of parkinson s disease , gcase deficits in sporadic parkinson s disease are related to the abnormal accumulation of -syn ( murphy et al . recently , a selective interaction between -syn and gcase was determined ( yap et al . the interaction occurred under lysosomal solution conditions ( ph 5.5 ) , and no significant interaction was noted at ph 7.4 . residue level mapping showed that the interaction site was at the -syn c - terminal residues , 118137 . intriguingly , the n370s mutant form of gcase has a reduced affinity for -syn , so this binding at lysosomal ph might play an important role in facilitating -syn degradation or preventing aggregation . much of the processing of -syn occurs in the cytoplasm , where this interaction was unexpected , but it offers a possibility for lysosomal pathways in -syn degradation . when gba is mutated or absent , the beneficial role of the interaction in -syn processing is disrupted . these findings suggest that the -syn - gcase association is favored in the lysosome and that understanding this noncovalent interaction provides the groundwork to explore molecular mechanisms linking pd with mutant gba alleles . studied the association between -syn pathology and gcase from a different perspective ( mazzulli et al . reduced concentrations of endoglycosidase h - resistant gcase were also reported , ascribed to the depletion of the lysosomal form of the enzyme . gcase knockdown increased the steady - state levels of -syn 1.8-fold relative to controls , while the levels of tau , another disease - associated aggregation - prone protein , did not change . proteins extracted from cerebral cortex samples from patients with synucleinopathies with and without gba mutations , as well as samples from control individuals and patients with gd , were studied by western blotting . patients with gba - associated parkinsonism showed aggregated oligomeric forms of -syn in the insoluble fraction , whereas only monomeric -syn was noted in patients with gba mutations without parkinsonism , including samples from patients with neuropathic gd ( choi et al . used primary cortical neurons to demonstrate that overexpression of either wild - type or a53t - mutant -syn resulted in retention of gcase in the er , associated with reduced lysosomal gcase activity ( mazzulli et al . the proposed gain - of - function or loss - of - function mechanisms do not perfectly explain the association between pd and gd , and there are some clinical exceptions for each of them . therefore , the focus should be on the altered function of -syn in the observed association instead of a single gain or loss of function . a newly identified interaction between -syn and gcase was reported that might influence cellular levels of -syn by either promoting protein degradation and/or preventing aggregation ( sybertz and krainc 2014 ; yap et al . the interaction of -syn and gcase in lysosomes may be beneficial to the degradation of -syn , while intracellular glccer levels control the formation of soluble toxic -syn oligomers and fibrils ( mazzulli et al . in other words , the interactions between -syn and gcase and between -syn and glccer have opposite effects on the aggregation of -syn . some gba mutations result in misfolded gcase , which has no activity but can still interact with -syn in lysosomes and contribute to the degradation . thus , the aggregation of -syn caused by glccer is compromised , which may explain why gd patients with low gcase activity will not develop parkinsonism . when gba mutations affect the interaction site , like n370s , the beneficial role of the interaction in -syn processing is disrupted , and the balance between aggregation and degradation is broken . this may be why carriers of null alleles might have an even higher risk of developing parkinsonism ( neumann et al . , if the aggregation of -syn can not be cleared , it will further inhibit the lysosomal maturation and gcase activity , resulting in additional glccer accumulation and elevated -syn . recent data show that increasing gcase activity with adeno - associated viral vector ( aav ) delivery of the enzyme into the brain of a gd mouse model can reduce the accumulation of snca , tau and ubiquitin ( sardi et al . in addition , neuronal glucosylceramide ( glccer ) accumulates to a certain threshold ; a series of second events is triggered , which includes neuroinflammation and neurodegeneration , then subsequently causes neuronal death ( farfel - becker et al . hence , increasing the gcase activity in the cns represents a potential therapeutic strategy for gba - related and non - gba - associated synucleinopathies , including pd ( schapira and gegg 2013 ) . since a reduction of glucosylceramidase protein levels and activity occurs in the brain of gd and pd patients with gba mutation carriers , gcaes is a potential treatment target for gd therapy . in addition , lysosomal activity of gc is tightly linked to expression of its trafficking receptor , the lysosomal integral membrane protein type-2 ( limp-2 ) , which acts as a chaperone helping to traffic glucosylceramidase from the endoplasmic reticulum to the lysosome ; therefore , manipulating limp-2 expression to increase lysosomal gc activity is also a promising strategy for the treatment of synucleinopathies and gba mutation carriers . additionally , the receptor - interacting protein kinases ( ripks ) , a class of serine / threonine protein kinases , are directly involved in the pathway of pathological events in severe forms of gd . the significant extension of lifespan and improvements of motor coordination in ripk3 knockout mice support the notion that ripk3 is not only a key activator of necrotic cell death , but also the ripk3 pathway might be a molecular target for therapeutic intervention in gd ( vitner et al . further studies using techniques in cell biology , neuropathology and genetics are needed to better reveal the mechanisms that contribute to gba - associated parkinsonism . the role of -syn and gcase will continue to be a source of considerable interest in the field . elucidating the factors underlying this association is necessary to improve genetic counseling for people who carry mutations in this gene . moreover , a better understanding of the role of gcase in the development of parkinsonism will facilitate the development of new therapeutic strategies for gba - associated parkinsonism .

motoneuron loss is the common feature of several neurodegenerative diseases , as well as mechanical injuries affecting the spinal cord ( sc ) . among neurodegenerative diseases , amyotrophic lateral sclerosis ( als ) and spinal muscular atrophy ( sma ) represent the most common diseases affecting spinal and brainstem motoneurons . this disease affects mainly the lower motoneurons within the sc and brainstem , but the pyramidal neurons located in the motor cortex are also frequently damaged . this results in progressive muscle atrophy and spasticity , which ultimately cause death due to respiratory dysfunction [ 1 , 4 ] . als is a heterogeneous disease complex that could be subdivided into two main groups : familial als ( fals ) , which accounts for only 10% of patients , and the more frequent form with no family history , affecting the remaining 90% of als patients , namely , the sporadic als ( sals ) [ 1 , 4 ] . the molecular mechanisms of als pathogenesis remain far to be fully understood and appear extremely heterogeneous . however , a number of gene mutations have been found in fals patients , including a missense mutation in the sod1 gene , encoding for superoxide dismutase 1 protein , which is the most frequent gene mutation found in fals . more recently , aberrant accumulation of either mutant or wild type tar dna - binding protein of 43 kda ( tdp-43 ) has been found in both fals and sals , thus accounting for a common mechanism involving aberrant rna processing and glutamate excitotoxicity [ 1 , 410 ] . sma is the most common inherited motoneuron disease and the main genetic cause of newborn mortality . like als in contrast to the multifactorial origin of als , this disease is unambiguously caused by the recessive mutations or deletion of the survival motor neuron-1 gene ( smn1 ) [ 1113 ] . a number of animal models have been developed attempting to recapitulate at least some of the genetic , anatomical , and functional defects observed in the human als and sma [ 10 , 1417 ] . these models have also been used for testing the efficacy of different repairing strategies such as rehabilitation , pharmacological , genetic , or cell - based approaches [ 10 , 1626 ] . sc injury ( sci ) or nerve damage could also result in severe loss of grey matter neurons , including motoneurons [ 27 , 28 ] . the mechanism of cell loss after contusion injury is complex : the mechanical damage of sc tissue ( primary injury ) destroys many local neurons , but it is followed by a secondary injury that kills a larger neuronal and glial population because of several pathological phenomena , including inflammation or vascular damage . although the described neurodegenerative or traumatic sc diseases are different in their etiology and pathogenesis , they share a common outcome characterized by the death of lower motoneurons . regardless of the pathological reason for motoneuron loss , several studies have investigated the possibility of repairing the motoneuron - depleted sc by using different repairing strategies . these studies have used several animal models of selective motoneuron depletion [ 30 , 31 ] . in the present paper , we performed a comprehensive review of the literature about the use of rodent models of neurotoxic spinal motoneuron degeneration , with a focus on two models obtained by intramuscular injection of volkensin or cholera toxin - b saporin ( ctb - sap ) . in particular , the experimental applications of these models to mimic neurodegenerative diseases , to dissect the molecular mechanisms of neuroplastic changes underlying the functional recovery after motoneuron loss , and to evaluate the effectiveness of several strategies of neural repair are extensively discussed in comparison to the other available preclinical models of disease . the first evidences about the effects of neurotoxins on motoneurons were provided as early as fifty years ago , with some studies showing the effects of tetanus and botulinum toxins on spinal motoneurons [ 3234 ] . afterwards , functional neuroanatomy studies have relied on the effects of lesions to investigate the function of neural systems , and a large variety of neurotoxins has been used to destroy specific cell populations . for instance , excitotoxins such as kainic acids [ 35 , 36 ] or monoamine toxins including 6-hydroxydopamine have been used to produce selective lesions based on the neurotransmitter specificity , but these compounds have shown incomplete anatomical and cell - type specificity . a substantial improvement of these methods of molecular neurosurgery has been provided by the development of axonally transported toxins such as lectins [ 3841 ] , immunotoxins [ 4245 ] , tracer - toxins , and neuropeptide - conjugated toxins . when injected into the target region , these toxins are captured by axon terminals and retrogradely transported towards the cell body , thus causing cell death by ribosome inactivation and apoptosis . plant derived lectins are anatomically but not cell - type selective , being able to kill any neuron projecting to the injection site , by suicide retrograde transport [ 3941 , 46 , 47 ] . this term refers to the uptake and axonal transport of toxins by neurons projecting to the injection site , thus causing a selective lesion based on the specific neural connection rather than cell phenotype [ 31 , 42 , 47 , 48 ] . conversely , immunotoxins as well as tracer- or neuropeptide - conjugated toxins are both anatomically and cell - type selective , since they are internalized by cells after specific chemical binding [ 30 , 42 , 49 ] . a large number of plant derived neurotoxic proteins have been isolated and characterized , thus showing their ability to damage eukaryotic cells by acting on ribosome and catalytically disrupting the elongation step of protein synthesis [ 51 , 52 ] . these ribosome - inactivating proteins ( rips ) include ricin ( from ricinus communis ) , abrin ( from abrus precatorius ) , modeccin ( from adenia digitata ) , and volkensin ( from adenia volkensii ) [ 38 , 39 , 50 , 52 ] . all these rips are axonally transported by peripheral nerves but , among these , modeccin and volkensin are more efficient to kill neurons of the central nervous system ( cns ) by suicide transport [ 4042 , 46 , 53 ] . among the above described rips , volkensin appeared to be the most toxic on cns neurons and it has been the most frequently used to create animal models of spinal motoneuron degeneration . as early as in 1992 , ngrdi and vrbov used volkensin with the aim of creating a reliable model of motoneuron degeneration . similar long - term effects of volkensin on the sc results were shown by leanza and stanzani ( 1998 ) after intramuscular injection of 2.0 ng of this rip in newborn rats . these authors have reported an extensive and long - lasting depletion of spinal motoneurons ( about 90% ) as measured at either two or eight months after the lesion . afterwards , this rodent model was used , also by our research group , either as recipients in experimental approaches of transplant - induced regeneration ( see section 4 ) [ 5557 ] or as models for testing the intrinsic potential for spontaneous regeneration ( see section 3 ) . a substantial improvement of neurotoxic lesion protocols came from the development of targeted rips by conjugation with a specific carrier , such as an antibody , a neuropeptide , or a retrograde tracer [ 30 , 42 , 44 , 45 , 49 , 50 , 59 ] . saporin , an rip from saponaria officinalis , is the most used toxin to prepare targeted neurotoxins . the latter is responsible for the specific binding to the gm1 membrane receptor , internalization , and retrograde transport [ 60 , 61 ] . given these properties , cholera toxin - b subunit could be used either as a retrograde tracer [ 30 , 62 ] or as a targeted neurotoxin after conjugation with saporin . a number of in vivo experiments have used cholera toxin - b saporin ( ctb - sap ) and demonstrated its effectiveness in removing any neuron expressing gm1 ganglioside [ 30 , 49 , 6365 ] . recently , our group has developed a mouse model of lumbar sc motoneuron degeneration by injection of ctb - sap into the gastrocnemius muscle . the toxin has been injected into the medial and lateral gastrocnemius muscles at a dose of 3.0 g / muscle and caused a partial depletion of lumbar motoneuron ( 2530% ) , accompanied by an evident impairment of the hindlimb motor function . given the moderate severity of the lesion , this model is suitable for evaluating the spontaneous recovery of locomotion and the underlying sc plastic changes , such as neurogenesis or synaptic plasticity ( see section 3 ) [ 6669 ] . several evidences have demonstrated that adult mammals could achieve a significant range of spontaneous sensory - motor recovery after injury or disease , by means of various forms of neuroplasticity . this plasticity includes the recruitment of neural precursor cells ( npcs ) and the formation of new pathways as well as synaptic plasticity , within the affected tissue and/or in sensory and supraspinal pathways [ 7073 ] . however , this spontaneous plastic potential is inadequate for allowing complete regeneration and recovery of function , but some therapeutic interventions are able to recruit and potentiate this intrinsic capacity , thus producing a better outcome . since it has been found that sc plasticity is activity - dependent , a number of studies have demonstrated the effectiveness of exercise training and other methods of spinal learning in both animal models and human sci patients [ 70 , 7577 ] . some information is also available about plastic changes occurring in neurodegenerative diseases and , in particular , in motoneuron disease . it is known , for instance , that plastic changes could occur in parkinson 's disease as well as in the respiratory system and brain of als patients [ 7981 ] , but the beneficial effect of exercise training is still controversial [ 82 , 83 ] . given the progressive nature of these diseases , it is obvious that any compensatory change will ultimately be ineffective . despite these limitations , a better understanding of the plastic phenomena occurring in animal models of motoneuron disease would help in elucidating the molecular mechanisms of diseases and finding new putative targets for therapy . anatomical rearrangement and functional compensatory changes in spinal and supraspinal circuitry have been reported in rodent models of neuronal degeneration induced by nerve crushing [ 84 , 85 ] . the previously described murine model of selective ctb - sap induced motoneuron depletion developed in our laboratory has been deeply characterized to evaluate its capacity for spontaneous sensory - motor recovery . noteworthy , a relevant increase of motor performance measured at the grid walk or rotarod test has been observed as early as one month after toxin injection , despite a permanent though moderate motoneuron removal [ 66 , 68 , 69 ] . the cellular and molecular mechanisms underlying this remarkable functional recovery have been studied , including the activation of endogenous npcs , the spontaneous events of synaptic plasticity [ 6669 ] , and the expression and functional roles of neurotrophic factors and/or other molecular factors including cell fate determinants [ 6668 ] and tdp-43 . npcs proliferation and differentiation take place spontaneously in the adult mammals only in the subventricular zone and hippocampus [ 86 , 87 ] . however , multipotent npcs could be isolated from the entire adult cns , including the sc [ 8890 ] . several experiments have demonstrated that these cells could be mobilized after sci but , unfortunately , they only generate migratory cells that differentiate to astrocytes and participate in scar formation [ 89 , 91 , 92 ] . notably , astrocyte activation could also be caused by a selective neurotoxic neuron removal by volkensin suicide transport in either brain or sc [ 31 , 93 ] . moreover , a significant amount of cell proliferation and increase of gfap - positive astrocytes have been found in the sc ventral horn , after selective motoneuron removal by intramuscular injection of ctb - sap . glial reaction is a classical response to cns tissue damage , which generally also involves glial cells themselves and induces a series of events that amplifies and maintains glial activation [ 94 , 95 ] . therefore , the glial reaction observed after selective neuronal loss , with the absence of severe tissue damage and inflammation , could have different origin as well as different consequences on regenerative processes . intrinsic and extrinsic molecular factors regulating adult neurogenesis have been widely explored [ 86 , 97 ] . sonic hedgehog ( shh ) is a secreted glycoprotein promoting npcs proliferation and differentiation to neurons and oligodendrocytes , during both development and adulthood [ 98 , 99 ] . the notch-1 pathway and its inhibitor numb are also involved in the regulation of npcs proliferation , cell fate determination , dendritic morphology , and axon guidance in embryonic and adult cns [ 100103 ] , including sc [ 104 , 105 ] . noggin is a secreted glycoprotein responsible for neural induction during development , by acting as an inhibitor of bone morphogenetic proteins . as shown by chen and colleagues ( 2005 ) , shh , notch-1 , and numb expression are increased in the sc after compression injury . however , unlike their embryonic counterparts , npcs are unable to generate neurons in the adult sc . recently , some experiments have been performed to investigate the expression and the functional role of shh , notch-1 , numb , and noggin on the murine model of ctb - sap induced motoneuron depletion [ 66 , 68 ] . in contrast to those observed in sci models , shh and numb expressions appear transiently decreased after motoneuron removal and then recovered in association with the spontaneous functional recovery , whereas noggin expression progressively increases [ 66 , 68 ] . the reasons for the discrepancy between mechanical and neurotoxic lesion models are elusive but some explanations could be proposed . for instance , mechanical damage affects several neuronal and glial populations , whereas the described neurotoxic lesion selectively kills motoneurons in spatially restricted regions . moreover , ependymal cells undergo a robust proliferation immediately after a mechanical injury [ 89 , 108 ] , whereas they seem unresponsive in the ctb - sap model ( see figure 1 ) . interestingly , a pattern of npcs proliferation and reactive gliosis closely resembling that found in ctb - sap models , with no evidence of neurogenesis , was found in transgenic mouse models of als expressing the mutated human sod1 gene [ 109 , 110 ] . unfortunately , further information concerning these endogenous repairing potentials of als affected sc is still lacking , and the results provided by neurotoxic models are therefore of great importance . however , these processes need to be further clarified because they denote the importance of the role of environmental cues on the behavior of spinal npcs . it is also likely that an experimental approach aimed at artificially modifying shh , numb , and noggin signaling into the sc could stimulate npcs proliferation , reduce glial reaction , and probably drive cell differentiation towards neuronal phenotype . another process promoting the functional restoration consists of the reorganization of spinal , supraspinal , and sensory pathways by mechanisms involving activity - dependent synaptic plasticity [ 71 , 74 , 111 ] . as previously described , a significant amount of spontaneous locomotor recovery is possible in rodent models of both sci and motoneuron disease and could be driven , at least partially , by mechanisms of synaptic plasticity [ 6669 , 112 , 113 ] . the molecular feature of synaptic plasticity has been extensively studied in the hippocampus , as it represents the principal mechanism underlying learning and memory . in fact , it is known that long - term modifications of synaptic efficacy are regulated presynaptically by the expression and phosphorylation of various synaptic vesicle proteins including synapsin - i [ 114116 ] and postsynaptically by changes in the expression and trafficking of glutamate receptors . in particular , alpha - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) ionotropic glutamate receptors are fundamental for cortical and hippocampal synaptic plasticity [ 117120 ] . the emerging role of astrocytes and their expression of connexins in the modulation of synaptic strength are also noteworthy [ 121 , 122 ] . a fundamental role in modulating both pre- and postsynaptic changes is exerted by brain - derived neurotrophic factor ( bdnf ) [ 123125 ] . in fact , synapsin - i is considered as a downstream effector of bdnf [ 123 , 125 ] . moreover , it seems clear that the activity - dependent release of bdnf could regulate the synthesis and synaptic delivery of ampa receptors in different brain areas [ 126 , 127 ] and , conversely , the glutamate receptor activity could modulate bdnf release [ 128 , 129 ] . interestingly , several authors have shown that such mechanisms could take place also in the intact and lesioned sc [ 112 , 113 , 130 ] , as well as in the mouse model of ctb - sap induced motoneuron loss developed in our laboratory . in particular , we have found that the spontaneous recovery of locomotion observed in the motoneuron - depleted mice is linked to the expression levels of both synapsin - i and ampa receptors [ 6668 ] . moreover , this model has confirmed the described role of bdnf and has also provided evidence about novel functional roles of shh , numb , and noggin that , in addition to the traditional role as cell fate determinants , could also participate in modulating synaptic plasticity and functional recovery [ 6669 ] ( see figure 1 ) . information about the occurrence of synaptic plasticity in patients or animal models of als is poor . however , it is noteworthy that the expression of synaptic vesicle proteins is significantly decreased in the sc ventral horn of als patients , thus again confirming that ctb - sap models could be interesting research tools for research in motoneuron disease . tdp-43 is a nuclear dna / rna - binding protein encoded by a highly conserved gene and involved in mrna processing [ 132 , 133 ] . recently , tdp-43 was found in the cytoplasmic protein aggregates observed in some neurons of patients affected by als [ 6 , 133 ] . therefore , increasing attention has been devoted to the toxic effects of mutant tdp-43 on motoneurons but , more recently , it is becoming likely that some of these effects could depend on the loss of function of the normal tdp-43 [ 5 , 7 , 133 , 134 ] . in addition to the described classical role , tdp-43 could be involved in apoptosis , microrna biogenesis , and cell proliferation . notably , tdp-43 has been found in the dendrites , where it could affect local rna translation in an activity - dependent manner [ 135 , 136 ] . moreover , tdp-43 is crucial for synaptic formation and plasticity , as well as for locomotion in drosophila [ 134 , 137 , 138 ] . it has been recently shown in our model of motoneuron loss that synapsin - i expression is linked to that of tdp-43 and that the latter correlates with the expression of ampa receptor subunits glur1 , glur2 , and glur4 . as mentioned above , synaptic plasticity is modulated by ampa receptor trafficking and in particular by the regulation of ca - permeable ampa receptors [ 117119 ] . the ion permeation is linked to the amount of q / r - unedited glur2 subunits included into the ampa channels . therefore , given that tdp-43 is likely involved in the q / r - editing of glur2 subunits , one of the proposed mechanisms of motoneuron death in als is the glutamate toxicity caused by the aberrant increase of unedited glur2 subunits [ 5 , 8 ] . similar processes could take place in the ctb - sap sc lesion model and , interestingly , the same events could affect synaptic plasticity in this model . unlike the functional linkage between ampa receptors and tdp-43 , the association between synapsin - i and tdp-43 is absolutely novel and suggests a model where tdp-43 could affect synaptic strength by modulating the expression of both synapsin - i and ampa receptors ( see figure 1 ) . this hypothesis is supported by other evidences that tdp-43 is present at synapses and controls the local synthesis of synaptic proteins [ 135 , 139 ] . other recent findings have shown that the lack of tdp-43 could affect synapses and cause locomotor deficits in drosophila [ 134 , 137 ] . given the increasing interest in mouse models of tdp-43 gain or loss of function as models of neurodegenerative diseases , including als [ 10 , 16 , 140 ] , is likely that the elucidation of the physiological role of tdp-43 in the sc would provide an important contribution . to date , neurodegenerative disorders such as als and sma do not benefit from any effective therapy . riluzole represents the only approved therapy for als , but its effects consist in prolonging survival and delaying the use of supportive care by a few months . as previously discussed , the adult sc is capable of a significant amount of spontaneous functional restoration , and this is particularly evident in rodent models of sc injury or disease [ 66 , 68 , 71 , 74 , 111 ] . although this capacity is not enough to allow full recovery , it is anyway encouraging because the elucidation of the underlying cellular and molecular mechanisms would provide novel therapeutic tools and targets , thus improving the expected clinical outcomes . as the spontaneous functional recovery could be driven by the recruitment of npcs , regeneration of damaged neurons , and events of synaptic plasticity occurring within the spared circuitries . however , further studies employing representative preclinical models , as well as the design of clinical trials , are mandatory to make this increasing knowledge available for translational applications . the activity - dependent nature of plastic changes within the sc [ 71 , 74 ] has suggested the possibility that the damaged sc could be retrained in an attempt to modify the activity of the spared circuitries and compensate for the partial loss of neurons and connections . locomotor training has proven to be beneficial in spinalized animals [ 76 , 112 , 113 ] , by mechanisms of activity - dependent bdnf - induced synaptic plasticity [ 112 , 113 , 130 , 143 ] . significant clinical improvement could also be achieved by human sci patients as a result of locomotor training [ 70 , 75 ] . the importance of plastic changes in motoneuron diseases needs further investigations and the data provided by neurotoxic models would also be helpful as previously discussed . few studies have investigated the therapeutic value of exercise training in either human patients or animal models of motoneuron disease and produced controversial findings . a couple of studies involving sod1 mouse models demonstrated that a moderate exercise could produce neuroprotective effects on motoneurons , although the impact on the life span is controversial [ 144146 ] . moreover , the beneficial effects seem to be dependent on the type of physical exercise . similar results have been provided by a small number of studies involving human patients [ 82 , 83 ] , thus indicating that further studies are needed to clarify the relationships among neuronal activity , motoneuron vulnerability , and neuroprotection . in this respect , important insights have been provided by the previously described ctb - sap sc lesion model ( see section 3 ) [ 6669 ] , but some of them require further investigation and clinical trials . in particular , the role of neurotrophins and other growth factors has been confirmed in different animal models including the ctb - sap lesioned and the other established animal models of disease [ 17 , 147 ] . however , human trials showed inconsistent or negative effects of growth factors due to different reasons such as bioavailability , poor penetration through the blood - brain barrier , and inadequate or excessive dosing . other studies for effective treatments have focused on the neuromuscular junction and the role of the skeletal muscle as source of chemical and cellular cues sustaining neuronal survival , axonal growth , and synaptic connections , such as trophic support or the role on nogo - a . the ctb - sap model could help in investigating this aspect without unwanted environmental cues , which are normally present in the genetic models of als or sma . cell transplantation was one of the first repairing approaches used in models of sc injury and disease . transplantation of fetal motoneurons was successfully used in models of motoneuron loss induced by nerve crushing , kainic acid , or volkensin [ 5557 , 150 ] and demonstrated that the grafts were able to survive and develop as functionally active mature motoneurons [ 5557 , 150 ] , although their capacity of muscle reinnervation was limited . more recently , cell - based strategies have relied on the potential beneficial effects of stem cells such as embryonic , neural , mesenchymal , and induced pluripotent stem cells [ 1926 , 151153 ] . a number of preclinical studies have proven that stem cell therapy is able to delay the disease progression , rescue motoneuron function , and extend survival in animal models of als or sma . it is obvious , for instance , that replacement of lost motoneurons is an important goal in repairing strategies , but some limitations still occur as previously described , including integration into the host tissue and reinnervation . moreover , resident as well as grafted neurons could be susceptible to degeneration if exposed to a toxic microenvironment like that present within the diseased neural tissue . transplantation of cells including different stem cell types could provide trophic support , remove toxic cues , and exert immunomodulatory effects , which ultimately could result in neuroprotection for motoneurons [ 1926 , 151153 ] . the use of a neurotoxic model , where motoneuron depletion is not accompanied by a chronic disease state or toxic environment , could offer a different point of view for elucidating the beneficial effects of cell - based therapies . a number of stem cell clinical trials [ 19 , 154 ] have shown that some cell - based protocols could be safe and produce promising though modest effects . regarding the cell source , mesenchymal stem cells could be easily obtained from patients and are considered suitable for autologous transplantation . they can be obtained by reprogramming somatic cells without viral methods and differentiated towards multiple phenotypes [ 1926 , 151154 ] . however , to achieve effective cell - based therapies suitable for clinical application , several issues should be addressed , including the optimization of delivery protocols ( route of administration , dose ) and the better elucidation of the graft - host interaction . the ideal route of administration should produce the best therapeutic effects with the minimal invasiveness . intrathecal or intravenous administration could represent effective approaches , because they ensure the widespread distribution of cells , which is ideal when degeneration is not limited to a small area . however , cells must be able to penetrate the blood - brain barrier and migrate correctly towards the affected areas . again , several preclinical studies are needed , by using different animal models , to address these important goals . as previously discussed , npcs proliferation occurs in different animal models of motoneuron loss , including neurotoxic and als models , but external interventions are needed to potentiate this capacity and drive npcs differentiation towards the neuronal phenotype [ 66 , 109 , 110 ] . bambakidis and colleagues ( 2003 ) have treated sc lesioned rats with shh and provided evidence of increased npcs proliferation and their differentiation as oligodendrocytes and neurons [ 73 , 155 ] . in addition , shh promotes survival and exerts neuroprotective effects on cns neurons including motoneurons [ 156 , 157 ] . a recent study showed that g93a mouse model of als produced spontaneous npcs proliferation within sc lamina x , which was increased by lithium administration . moreover , lithium - treated animals showed increased neuronal differentiation and attenuation of disease progression . another growth factor not only stimulating neurogenesis but also promoting neuronal survival , migration , and axon guidance in als models as well as protection of motoneurons against excitotoxicity is vascular endothelial growth factor ( vegf ) [ 158 , 159 ] . beneficial effects of many other growth factors and morphogens , as well as hormones , on sc repair have been published by several authors . axonal growth and other plastic changes could be promoted , for instance , by noggin and bdnf [ 113 , 160162 ] , whereas testosterone treatment has proven to exert neuroprotective effects on motoneurons in ctb - sap lesion models , by preventing dendritic atrophy after removal of surrounding motoneuron [ 63 , 65 ] . further studies are needed to better understand the mechanism of neurodegeneration as well as develop effective methods of therapy and rehabilitation . in this respect , although a large number of studies will be obviously conducted on mouse models of als and sma , the above - described neurotoxic models of motoneuron degeneration will certainly be useful as well . moreover , motoneuron - depleted sc is a simple and powerful tool for cell transplantation and for testing plastic changes and the consequent functional outcome . despite the difference between neurotoxic and genetic rodent models , the described similar effects on neurogenesis and the involvement of tdp-43 and the multiple roles of neurotrophins and morphogens would open a number of novel research pathways aimed at the dissection of pathogenesis and selection of new therapeutic targets and tools for the treatment of motoneuron diseases .

retrogradely transported toxins are widely used to set up protocols for selective lesioning of the nervous system . these methods could be collectively named molecular neurosurgery because they are able to destroy specific types of neurons by using targeted neurotoxins . lectins such as ricin , volkensin , or modeccin and neuropeptide- or antibody - conjugated saporin represent the most effective toxins used for neuronal lesioning . some of these specific neurotoxins could be used to induce selective depletion of spinal motoneurons . in this review , we extensively describe two rodent models of motoneuron degeneration induced by volkensin or cholera toxin - b saporin . in particular , we focus on the possible experimental use of these models to mimic neurodegenerative diseases , to dissect the molecular mechanisms of neuroplastic changes underlying the spontaneous functional recovery after motoneuron death , and finally to test different strategies of neural repair . the potential clinical applications of these approaches are also discussed .

1. Introduction 2. Rodent Neurotoxic Spinal Cord Lesion Models 3. Mechanisms of Spinal Cord Plasticity in Models of Motoneuron Disease 4. Repairing Strategies 5. Concluding Remarks

motoneuron loss is the common feature of several neurodegenerative diseases , as well as mechanical injuries affecting the spinal cord ( sc ) . among neurodegenerative diseases , amyotrophic lateral sclerosis ( als ) and spinal muscular atrophy ( sma ) represent the most common diseases affecting spinal and brainstem motoneurons . this disease affects mainly the lower motoneurons within the sc and brainstem , but the pyramidal neurons located in the motor cortex are also frequently damaged . als is a heterogeneous disease complex that could be subdivided into two main groups : familial als ( fals ) , which accounts for only 10% of patients , and the more frequent form with no family history , affecting the remaining 90% of als patients , namely , the sporadic als ( sals ) [ 1 , 4 ] . the molecular mechanisms of als pathogenesis remain far to be fully understood and appear extremely heterogeneous . however , a number of gene mutations have been found in fals patients , including a missense mutation in the sod1 gene , encoding for superoxide dismutase 1 protein , which is the most frequent gene mutation found in fals . sma is the most common inherited motoneuron disease and the main genetic cause of newborn mortality . a number of animal models have been developed attempting to recapitulate at least some of the genetic , anatomical , and functional defects observed in the human als and sma [ 10 , 1417 ] . these models have also been used for testing the efficacy of different repairing strategies such as rehabilitation , pharmacological , genetic , or cell - based approaches [ 10 , 1626 ] . regardless of the pathological reason for motoneuron loss , several studies have investigated the possibility of repairing the motoneuron - depleted sc by using different repairing strategies . these studies have used several animal models of selective motoneuron depletion [ 30 , 31 ] . in the present paper , we performed a comprehensive review of the literature about the use of rodent models of neurotoxic spinal motoneuron degeneration , with a focus on two models obtained by intramuscular injection of volkensin or cholera toxin - b saporin ( ctb - sap ) . in particular , the experimental applications of these models to mimic neurodegenerative diseases , to dissect the molecular mechanisms of neuroplastic changes underlying the functional recovery after motoneuron loss , and to evaluate the effectiveness of several strategies of neural repair are extensively discussed in comparison to the other available preclinical models of disease . the first evidences about the effects of neurotoxins on motoneurons were provided as early as fifty years ago , with some studies showing the effects of tetanus and botulinum toxins on spinal motoneurons [ 3234 ] . afterwards , functional neuroanatomy studies have relied on the effects of lesions to investigate the function of neural systems , and a large variety of neurotoxins has been used to destroy specific cell populations . for instance , excitotoxins such as kainic acids [ 35 , 36 ] or monoamine toxins including 6-hydroxydopamine have been used to produce selective lesions based on the neurotransmitter specificity , but these compounds have shown incomplete anatomical and cell - type specificity . a substantial improvement of these methods of molecular neurosurgery has been provided by the development of axonally transported toxins such as lectins [ 3841 ] , immunotoxins [ 4245 ] , tracer - toxins , and neuropeptide - conjugated toxins . when injected into the target region , these toxins are captured by axon terminals and retrogradely transported towards the cell body , thus causing cell death by ribosome inactivation and apoptosis . plant derived lectins are anatomically but not cell - type selective , being able to kill any neuron projecting to the injection site , by suicide retrograde transport [ 3941 , 46 , 47 ] . this term refers to the uptake and axonal transport of toxins by neurons projecting to the injection site , thus causing a selective lesion based on the specific neural connection rather than cell phenotype [ 31 , 42 , 47 , 48 ] . conversely , immunotoxins as well as tracer- or neuropeptide - conjugated toxins are both anatomically and cell - type selective , since they are internalized by cells after specific chemical binding [ 30 , 42 , 49 ] . these ribosome - inactivating proteins ( rips ) include ricin ( from ricinus communis ) , abrin ( from abrus precatorius ) , modeccin ( from adenia digitata ) , and volkensin ( from adenia volkensii ) [ 38 , 39 , 50 , 52 ] . all these rips are axonally transported by peripheral nerves but , among these , modeccin and volkensin are more efficient to kill neurons of the central nervous system ( cns ) by suicide transport [ 4042 , 46 , 53 ] . among the above described rips , volkensin appeared to be the most toxic on cns neurons and it has been the most frequently used to create animal models of spinal motoneuron degeneration . as early as in 1992 , ngrdi and vrbov used volkensin with the aim of creating a reliable model of motoneuron degeneration . similar long - term effects of volkensin on the sc results were shown by leanza and stanzani ( 1998 ) after intramuscular injection of 2.0 ng of this rip in newborn rats . these authors have reported an extensive and long - lasting depletion of spinal motoneurons ( about 90% ) as measured at either two or eight months after the lesion . a substantial improvement of neurotoxic lesion protocols came from the development of targeted rips by conjugation with a specific carrier , such as an antibody , a neuropeptide , or a retrograde tracer [ 30 , 42 , 44 , 45 , 49 , 50 , 59 ] . saporin , an rip from saponaria officinalis , is the most used toxin to prepare targeted neurotoxins . the latter is responsible for the specific binding to the gm1 membrane receptor , internalization , and retrograde transport [ 60 , 61 ] . given these properties , cholera toxin - b subunit could be used either as a retrograde tracer [ 30 , 62 ] or as a targeted neurotoxin after conjugation with saporin . a number of in vivo experiments have used cholera toxin - b saporin ( ctb - sap ) and demonstrated its effectiveness in removing any neuron expressing gm1 ganglioside [ 30 , 49 , 6365 ] . the toxin has been injected into the medial and lateral gastrocnemius muscles at a dose of 3.0 g / muscle and caused a partial depletion of lumbar motoneuron ( 2530% ) , accompanied by an evident impairment of the hindlimb motor function . given the moderate severity of the lesion , this model is suitable for evaluating the spontaneous recovery of locomotion and the underlying sc plastic changes , such as neurogenesis or synaptic plasticity ( see section 3 ) [ 6669 ] . however , this spontaneous plastic potential is inadequate for allowing complete regeneration and recovery of function , but some therapeutic interventions are able to recruit and potentiate this intrinsic capacity , thus producing a better outcome . since it has been found that sc plasticity is activity - dependent , a number of studies have demonstrated the effectiveness of exercise training and other methods of spinal learning in both animal models and human sci patients [ 70 , 7577 ] . some information is also available about plastic changes occurring in neurodegenerative diseases and , in particular , in motoneuron disease . given the progressive nature of these diseases , it is obvious that any compensatory change will ultimately be ineffective . despite these limitations , a better understanding of the plastic phenomena occurring in animal models of motoneuron disease would help in elucidating the molecular mechanisms of diseases and finding new putative targets for therapy . anatomical rearrangement and functional compensatory changes in spinal and supraspinal circuitry have been reported in rodent models of neuronal degeneration induced by nerve crushing [ 84 , 85 ] . the cellular and molecular mechanisms underlying this remarkable functional recovery have been studied , including the activation of endogenous npcs , the spontaneous events of synaptic plasticity [ 6669 ] , and the expression and functional roles of neurotrophic factors and/or other molecular factors including cell fate determinants [ 6668 ] and tdp-43 . however , multipotent npcs could be isolated from the entire adult cns , including the sc [ 8890 ] . notably , astrocyte activation could also be caused by a selective neurotoxic neuron removal by volkensin suicide transport in either brain or sc [ 31 , 93 ] . the notch-1 pathway and its inhibitor numb are also involved in the regulation of npcs proliferation , cell fate determination , dendritic morphology , and axon guidance in embryonic and adult cns [ 100103 ] , including sc [ 104 , 105 ] . as shown by chen and colleagues ( 2005 ) , shh , notch-1 , and numb expression are increased in the sc after compression injury . recently , some experiments have been performed to investigate the expression and the functional role of shh , notch-1 , numb , and noggin on the murine model of ctb - sap induced motoneuron depletion [ 66 , 68 ] . in contrast to those observed in sci models , shh and numb expressions appear transiently decreased after motoneuron removal and then recovered in association with the spontaneous functional recovery , whereas noggin expression progressively increases [ 66 , 68 ] . the reasons for the discrepancy between mechanical and neurotoxic lesion models are elusive but some explanations could be proposed . unfortunately , further information concerning these endogenous repairing potentials of als affected sc is still lacking , and the results provided by neurotoxic models are therefore of great importance . however , these processes need to be further clarified because they denote the importance of the role of environmental cues on the behavior of spinal npcs . it is also likely that an experimental approach aimed at artificially modifying shh , numb , and noggin signaling into the sc could stimulate npcs proliferation , reduce glial reaction , and probably drive cell differentiation towards neuronal phenotype . another process promoting the functional restoration consists of the reorganization of spinal , supraspinal , and sensory pathways by mechanisms involving activity - dependent synaptic plasticity [ 71 , 74 , 111 ] . as previously described , a significant amount of spontaneous locomotor recovery is possible in rodent models of both sci and motoneuron disease and could be driven , at least partially , by mechanisms of synaptic plasticity [ 6669 , 112 , 113 ] . the molecular feature of synaptic plasticity has been extensively studied in the hippocampus , as it represents the principal mechanism underlying learning and memory . in particular , alpha - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) ionotropic glutamate receptors are fundamental for cortical and hippocampal synaptic plasticity [ 117120 ] . in particular , we have found that the spontaneous recovery of locomotion observed in the motoneuron - depleted mice is linked to the expression levels of both synapsin - i and ampa receptors [ 6668 ] . moreover , this model has confirmed the described role of bdnf and has also provided evidence about novel functional roles of shh , numb , and noggin that , in addition to the traditional role as cell fate determinants , could also participate in modulating synaptic plasticity and functional recovery [ 6669 ] ( see figure 1 ) . information about the occurrence of synaptic plasticity in patients or animal models of als is poor . however , it is noteworthy that the expression of synaptic vesicle proteins is significantly decreased in the sc ventral horn of als patients , thus again confirming that ctb - sap models could be interesting research tools for research in motoneuron disease . therefore , increasing attention has been devoted to the toxic effects of mutant tdp-43 on motoneurons but , more recently , it is becoming likely that some of these effects could depend on the loss of function of the normal tdp-43 [ 5 , 7 , 133 , 134 ] . in addition to the described classical role , tdp-43 could be involved in apoptosis , microrna biogenesis , and cell proliferation . it has been recently shown in our model of motoneuron loss that synapsin - i expression is linked to that of tdp-43 and that the latter correlates with the expression of ampa receptor subunits glur1 , glur2 , and glur4 . as mentioned above , synaptic plasticity is modulated by ampa receptor trafficking and in particular by the regulation of ca - permeable ampa receptors [ 117119 ] . therefore , given that tdp-43 is likely involved in the q / r - editing of glur2 subunits , one of the proposed mechanisms of motoneuron death in als is the glutamate toxicity caused by the aberrant increase of unedited glur2 subunits [ 5 , 8 ] . similar processes could take place in the ctb - sap sc lesion model and , interestingly , the same events could affect synaptic plasticity in this model . given the increasing interest in mouse models of tdp-43 gain or loss of function as models of neurodegenerative diseases , including als [ 10 , 16 , 140 ] , is likely that the elucidation of the physiological role of tdp-43 in the sc would provide an important contribution . to date , neurodegenerative disorders such as als and sma do not benefit from any effective therapy . riluzole represents the only approved therapy for als , but its effects consist in prolonging survival and delaying the use of supportive care by a few months . as previously discussed , the adult sc is capable of a significant amount of spontaneous functional restoration , and this is particularly evident in rodent models of sc injury or disease [ 66 , 68 , 71 , 74 , 111 ] . although this capacity is not enough to allow full recovery , it is anyway encouraging because the elucidation of the underlying cellular and molecular mechanisms would provide novel therapeutic tools and targets , thus improving the expected clinical outcomes . as the spontaneous functional recovery could be driven by the recruitment of npcs , regeneration of damaged neurons , and events of synaptic plasticity occurring within the spared circuitries . the activity - dependent nature of plastic changes within the sc [ 71 , 74 ] has suggested the possibility that the damaged sc could be retrained in an attempt to modify the activity of the spared circuitries and compensate for the partial loss of neurons and connections . locomotor training has proven to be beneficial in spinalized animals [ 76 , 112 , 113 ] , by mechanisms of activity - dependent bdnf - induced synaptic plasticity [ 112 , 113 , 130 , 143 ] . few studies have investigated the therapeutic value of exercise training in either human patients or animal models of motoneuron disease and produced controversial findings . a couple of studies involving sod1 mouse models demonstrated that a moderate exercise could produce neuroprotective effects on motoneurons , although the impact on the life span is controversial [ 144146 ] . moreover , the beneficial effects seem to be dependent on the type of physical exercise . similar results have been provided by a small number of studies involving human patients [ 82 , 83 ] , thus indicating that further studies are needed to clarify the relationships among neuronal activity , motoneuron vulnerability , and neuroprotection . in this respect , important insights have been provided by the previously described ctb - sap sc lesion model ( see section 3 ) [ 6669 ] , but some of them require further investigation and clinical trials . in particular , the role of neurotrophins and other growth factors has been confirmed in different animal models including the ctb - sap lesioned and the other established animal models of disease [ 17 , 147 ] . however , human trials showed inconsistent or negative effects of growth factors due to different reasons such as bioavailability , poor penetration through the blood - brain barrier , and inadequate or excessive dosing . other studies for effective treatments have focused on the neuromuscular junction and the role of the skeletal muscle as source of chemical and cellular cues sustaining neuronal survival , axonal growth , and synaptic connections , such as trophic support or the role on nogo - a . the ctb - sap model could help in investigating this aspect without unwanted environmental cues , which are normally present in the genetic models of als or sma . cell transplantation was one of the first repairing approaches used in models of sc injury and disease . transplantation of fetal motoneurons was successfully used in models of motoneuron loss induced by nerve crushing , kainic acid , or volkensin [ 5557 , 150 ] and demonstrated that the grafts were able to survive and develop as functionally active mature motoneurons [ 5557 , 150 ] , although their capacity of muscle reinnervation was limited . more recently , cell - based strategies have relied on the potential beneficial effects of stem cells such as embryonic , neural , mesenchymal , and induced pluripotent stem cells [ 1926 , 151153 ] . a number of preclinical studies have proven that stem cell therapy is able to delay the disease progression , rescue motoneuron function , and extend survival in animal models of als or sma . moreover , resident as well as grafted neurons could be susceptible to degeneration if exposed to a toxic microenvironment like that present within the diseased neural tissue . transplantation of cells including different stem cell types could provide trophic support , remove toxic cues , and exert immunomodulatory effects , which ultimately could result in neuroprotection for motoneurons [ 1926 , 151153 ] . regarding the cell source , mesenchymal stem cells could be easily obtained from patients and are considered suitable for autologous transplantation . however , to achieve effective cell - based therapies suitable for clinical application , several issues should be addressed , including the optimization of delivery protocols ( route of administration , dose ) and the better elucidation of the graft - host interaction . intrathecal or intravenous administration could represent effective approaches , because they ensure the widespread distribution of cells , which is ideal when degeneration is not limited to a small area . again , several preclinical studies are needed , by using different animal models , to address these important goals . as previously discussed , npcs proliferation occurs in different animal models of motoneuron loss , including neurotoxic and als models , but external interventions are needed to potentiate this capacity and drive npcs differentiation towards the neuronal phenotype [ 66 , 109 , 110 ] . another growth factor not only stimulating neurogenesis but also promoting neuronal survival , migration , and axon guidance in als models as well as protection of motoneurons against excitotoxicity is vascular endothelial growth factor ( vegf ) [ 158 , 159 ] . axonal growth and other plastic changes could be promoted , for instance , by noggin and bdnf [ 113 , 160162 ] , whereas testosterone treatment has proven to exert neuroprotective effects on motoneurons in ctb - sap lesion models , by preventing dendritic atrophy after removal of surrounding motoneuron [ 63 , 65 ] . in this respect , although a large number of studies will be obviously conducted on mouse models of als and sma , the above - described neurotoxic models of motoneuron degeneration will certainly be useful as well . despite the difference between neurotoxic and genetic rodent models , the described similar effects on neurogenesis and the involvement of tdp-43 and the multiple roles of neurotrophins and morphogens would open a number of novel research pathways aimed at the dissection of pathogenesis and selection of new therapeutic targets and tools for the treatment of motoneuron diseases .

the editors of the london hospital gazette either agreed with e.m.p . or were oblivious to his blatant scorn for immigrant and indigenous working - class patients . indeed , the gazette routinely sneered at the local residents who made up the bulk of their patients , poking fun at their quaint expressions of pain and minimising the degree of distress they might be experiencing . peoples regularly focussed on their loathsome bodies : the problem was not so much that these patients writhed in pain , but that they were incapable of screwing up the courage to be brave in the face of misfortune . disparaged was the inability of jews , turks , and heretics to endure suffering with the reserved intrepidness of stalwart britons . failure of willpower was portrayed as particularly despicable since many of these outsiders were believed to possess dulled sensibilities in the first place . slaves , savages , and dark - skinned people generally were depicted as possessing a limited capacity to truly feel , a biological fact that conveniently diminished any culpability amongst their so - called superiors for any acts of abuse inflicted on them . writing in 1811 , for instance , a professional planter was determined not to let the evidence of anatomy dissuade him of his prejudices about the bodies of black slaves . although the knife of the anatomist has never been able to detect anatomical differences between slaves and their white masters , he admitted , it was obvious that slaves possessed less exquisite bodies and minds . because of their dulled sensitivities , slaves were better able to endure , with few expressions of pain , the accidents of nature ( 201 ) . this was providential indeed , since they were subjected to so many accidents of nature while labouring in slave plantations . the need to insist on the physical insensitivity of slaves did not diminish with the end of slavery . quite the contrary , if hierarchies of labour and citizenship were to be retained , belief in the insensitivity of black bodies was more necessary than ever . a year after abraham lincoln s emancipation proclamation , anthropologist karl christoph vogt provided a physiological justification for their continued abuse . vogt s lectures on man ( 1864 ) informed readers that the negro stands far below the white race in terms of the acuteness of the senses . admittedly , in hospitals that had sprung up during the civil war , we see negroes suffering from the gravest diseases cowering on their couches without taking any notice of the attending physicians . but their wretched endurance was certainly more from disposition than from habit or education ( 188 ) . in other words , african - americans cowered in silent tenacity , not because of any enlightened custom or educated sensibility but simply because of a physiological disposition . it was a myth that a generation of african - american physicians writing in the early years of the twentieth century both struggled to come to grips with and attempted to debunk . one of the main forums for this generation of doctors was the journal of the national medical association , a journal dedicated to promoting african - american interests in medicine . in the 1914 edition , the surgeon - in - chief to st . agnes hospital admitted that there was a major debate about the ability of african - americans to endure pain and take anaesthetics . as a generalisation , he was prepared to accept thatthe negro submits to pain with resignation , his sensibilities being less acute than those of a more highly - wrought nervous nature ; that , as a rule , he is a favourable subject for anesthesia , provided his emotional spirit be not aroused and provided he have confidence in his advisers . the negro submits to pain with resignation , his sensibilities being less acute than those of a more highly - wrought nervous nature ; that , as a rule , he is a favourable subject for anesthesia , provided his emotional spirit be not aroused and provided he have confidence in his advisers . after this concession to those who believed that african - americans had less sensitive nervous systems and were easily swayed emotionally , he went on to warn against translating these generalisations into more casual attitudes to providing pain - relief for african - american patients . if you think , he continued , that , because the negro is hardy and resistant , he will on that account always survive great risks at tremendous odds , regardless of circumstances , you will at some time be sorely surprised . but what was it about the non - european body that rendered it less susceptible to painful stimuli ? racial sciences placed great emphasis on the development and complexity of peoples brains . since the existence of feeling depended on the activity of the brain , observed a writer signing himself philanthropos in the early 1880s , it was logical that the more perfect development of that organ , the greater the perception of sensations such as pain . for him , the rough proportion between sensibility and intellectual development explained why savages will undergo [ with ] equanimity tortures which no civilized man ( except perhaps under great excitement ) could endure ( 1883 , 11 ) . or , as the author of pain and sympathy ( 1907 ) concluded when attempting to explain why the savage could bear physical torture without shrinking : the higher the life , the keener is the sense of pain silas weir mitchell s famous statement of 1892 , during the process of being civilized we have won intensified capacity to suffer . after all , the savage does not feel pain as we do : nor as we examine the descending scale of life do animals seem to have the acuteness of pain - sense at which we have arrived ( 108 ) . the savage does not feel pain as we do : nor as we examine the descending scale of life do animals seem to have the acuteness of pain - sense at which we have arrived ( 108 ) . non - european peoples could be denigrated as possessing lesser bodies : their position at the lower echelons of the great chain of feeling was due to their physiological insensibility . on the other hand , certain peoples could also be designated as inferior on precisely the opposite grounds : excessive sensitivity or , at the very least , exaggerated responses to pain . as an author writing in the british journal of nursing in 1906 asked , why does the hebrew race manifest such feeble resistance to pain compared to all other nations ? just a few years earlier , the author of the highly respectable textbook entitled the diagnostics of internal medicine ( 1901 ) also accused the semitic stock , and the celtic and italic [ sic ] groups of appearing to possess an average greater sensibility to pain than the teutonic and slavonic groups ( butler 1901 , 35 ) . or , as essayist louis bertrand pontificated in the art of suffering ( 1936 ) , people from the southern or eastern parts of europe lacked the capacity to control themselves when experiencing pain . he also criticised the jews , an ancient race with a refined or decadent sensibility for being explanations for their acute sensitivities lay as much in their physiological degeneracy as it did in their moral inferiority ( or their inability to restrain their emotions ) . a degenerate physiology was certainly one explanation for such peculiar sensitivity to painful stimuli , but , in addition , these groups were accused of possessing immature psyches . irishmen and jews made the most noise on the operating table , according to an author in the british medical journal in 1929 . he claimed to have observed thatthe hebrew cried out through fear that if he failed to attract full attention he might miss some of the benefits of hospital care ; while the irishmen called loudly upon god and the saints , and wept and groaned because he was an emotional being to whose nature the repression of feeling , whether pleasant or painful , was foreign . the hebrew cried out through fear that if he failed to attract full attention he might miss some of the benefits of hospital care ; while the irishmen called loudly upon god and the saints , and wept and groaned because he was an emotional being to whose nature the repression of feeling , whether pleasant or painful , was foreign . rather , irish patients lacked adequate psychological inhibitions and jews had learnt the bitter lesson of persecution so were keen to ensure that they were not overlooked ( pain 1929 , 164 ) . either way , their lack of inhibition stamped them as inferior . whether generalising according to race or religion or drawing meticulous regional distinctions , ascriptions of pain - sensitivity registered fears and desires linked to cultural alliances and affinities rather than physiological facts nevertheless , these alleged physiological traits served as useful indicators for making broader social generalisations . hair and eye colour , for instance , were convenient stand - ins for racial groups . often , the racial references were implicit , as in a 1899 article in the american journal of psychology that concluded that male schoolchildren in michigan who had light eyes and hair were less sensitive to pain than those with ( carman 1899 , 396 ) . lurking behind such pseudo - surveys were assumptions that peoples from western and northern european stock were more stoical when compared to newer immigrants from more southern parts . in 1959 , this type of pseudoscientific research excited an almost feverish debate in the letters to the editor pages of the highly esteemed british medical journal . the question that ignited the debate was simple : could pain thresholds ( that is , the point at which a person subjected to a noxious stimuli complained of pain ) be correlated with eye colour ? the editors started things off by reporting on a study of 403 patients whose teeth had been filled at the melbourne university dental school . they noted that the researchers had found thatthe more blue the eyes the less [ pain ] reaction . as the colour went through blue - grey , green , hazel , light brown , and dark brown so the reaction to pain increased on the average . the more blue the eyes the less [ pain ] reaction . as the colour went through blue - grey , green , hazel , light brown , and dark brown so the reaction to pain increased on the average . this was no freak coincidence , the editors continued , speculating that patients with blue eyes were likely to come from north european stock , traditionally a phlegmatic race , unlike brown - eyed patients who were more likely to have descended from more excitable mediterranean peoples a doctor from hove ( sussex ) maintained that amongst his patients there was a positive correlation not only between dark brown eyes and a low pain threshold , but also between this eye - colour and over - reaction to pain . yet another doctor in hove pursed the argument , introducing an anti - semitic twist . for him , the positive association between brown eyes and excitable reactions to pain was due to the fact that members of the jewish race , in whom these physical features was present were notorious for their lowered [ pain ] threshold . bizarrely , he petitioned readers to investigate whether red - haired jews also had brown eyes , implying that this might be significant in evaluating their degree of pain - sensitivity ( wauchope 1959 , 1098 ) . as one doctor admitted , when he was a medical student working in a casualty department , i eased my burden considerably by always selecting blue - eyed and fair - haired children as my share of painful dressings . nordic children either have a higher pain threshold than other children or greater self - control . i eased my burden considerably by always selecting blue - eyed and fair - haired children as my share of painful dressings . he believed that gender also exerted an influence , with the nordic girl being better at bearing pain than her male counterpart ( hawksley 1959 , 958 ) . in such ways , beliefs about pain thresholds and pain responses translated directly into differential treatment . ironically , in this case , patients who were perceived to be less sensitive ( blue - eyed patients ) won better treatment than those who were believed to be suffering the most . gender was equally dominant in debates about differential pain sensitivity . were women the weaker sex or the more stoical one ? in a letter to his friend margaret king on 26 january 1792 , poet william cowper came out strongly on the side of female strength . king had written to him describing the patience with which a friend of hers had endured the terrible operation of having her breast laid open , that is , having undergone a mastectomy without anaesthetics . cowper commented that such patience was strong proof that your sex surpasses ours in heroic fortitude . indeed , there was more true heroism in suffering his [ god s ] will with meek submission than there was in heroism in a field of battle . in war , there were a great many incitements to disregard pain : renown and glory being two important ones . in contrast , no laurels are to be won by sitting patiently under the knife of a surgeon , so the virtue is of a less suspicious character , the principle of it more simple , and the practice more difficult ( cowper 1792 ) . this juxtaposition of patience and heroism the first being passive and female , in contrast to the active and masculine character of the second was not always judged to be a cause for celebration . a century after cowper s letter , brunless observed that , despite the fact that a man might be a hopeless coward in bearing bodily pain ( even to the extent of being fearfully depressed even by a toothache or headache ) , he would still prove himself capable of dying on a battlefield as few woman could have done . unlike cowper , she disparaged women for being capable only of patience , while men possessed that higher virtue of endurance brunless s point was that the grand heroics involved in wounding and being wounded in combat were beyond a woman s competency , but even she conceded that women might be better at bearing everyday afflictions ( such as toothaches ) . as the author of passages from the diary ( 1834 ) put it , the female sex showed great firmness in enduring a degree of physical pain , which would utterly break down the stubborn strength of man women s patience also impressed physician edward henry sieveking . writing in the 1860s , he believed in the greater sensitiveness of the female . women s more delicate organisation unfortunately meant that she was more likely to suffer a however , by way of balance , women were endowed with more placid and patient endurance than generally characterises the members of the ruder sex it was a theme that was still being repeated over 70 years later , when a survey showed that 70 % of physicians and dentists believed that women were superior to men in withstanding pain ( josey and miller 1932 , 375 ) . even at the end of the 1980s , a british study commissioned by the drug company that made nurofen found that 75 % of people agreed that women were better able to tolerate pain than men . interestingly , the generalisation was held to be correct by 86 % of women compared with only 64 % of men ( nurofen 1989 , 1 ) . particularly pessimistic account in 1910 , women s resilience was simply ascribed to their long practice in suffering the blows of the male it was common to hear it said that nature , when she gave the woman that proud and exclusive duty [ of childbearing ] , without doubt also gave her the means of discharging it , as a physician writing in the british medical journal in 1949 put it . i am sure that woman bears pain better and more patiently than man , he concluded ( cook 1949 , 781 ) . as other historians have shown , this argument was highly racialised , with distinctions being made between the hardy primitive woman giving birth and her more sensitive civilized counterpart ( hoberman 2005 , 8695 ) . was women s for instance , the medical superintendent of the virol pathological research laboratories , writing in the british journal of nursing in 1913 and 1914 , initially seemed to be making an argument for socialisation as the chief mechanism by which women learnt to bear their tribulations . a woman who had been trained to live for others , he wrote , will only complain when the pain is so bad as to interfere with her duties . in contrast , a woman who had been taught to think much of her own case , and to use words loosely would make a great fuss over slight pain , and describe it in inflated and incorrect language . he spoke scornfully of women whose vapourisings were a nuisance to everybody : however , he also seemed to adhere to the view that women were naturally stoical . after all , he admitted , very many men were also addicted to making a great fuss the natural tendency of a woman is to unselfishness while , with men this often has to be acquired ( gordon 1913 , 22 and gordon 1914 , 27 ) . occurring time and again throughout these debates , commentators frequently attempted to ensure that pre - existing prejudices about a particular group were upheld , even if it meant embracing contradictory arguments . thus , commentators argued both that women were innately stoical ( and therefore could be given less pain relief than men ) and were profoundly weak ( and thus liable to hysterical or exaggerated pains ) . this seeming contradiction is illustrated in the views of edward henry sieveking ( the inventor of the aesthesiometer ) and francis galton ( the founder of eugenics ) . as we saw earlier , sieveking believed that women were endowed with more placid and patient endurance than generally characterises the members of the ruder sex . this did not mean that women were superior to men in the way they comported themselves in pain . sieveking was equally convinced that men were superior to women because the nature of their respective pains differed . men s pains had a much more definite , local character , while those of women were less discriminating . as a result , when treating male patients , the wise physician would direct his attention to the seat of the lesion itself or the conducting nerves . baffle[d ] because women s pains were commonly due to reflex or reflected irritation . if a physician failed to remember or recognise the sensitive organisation of the female nervous system , sieveking argued , they would find themselves dealing blows at random , and in the dark , not always to the destruction of the malady or the benefit of the patient . her proclivity to emotional influences , and the greater motality [ sic ] and excitability of her imagination . doctors needed to adopt a roundabout way of examining all the organs of the female body , in order to determine the real source of any particular pain complained of ( 1867 , 13133 ) . sievekind was attempting to reconcile women s commendable fortitude with their physiological inferiority and emotional unpredictability . galton s dilemma was different : he needed to reconcile his belief that a delicate power of sense discrimination was an indication of superiority ( in which case , women were ranked above men ) with his need to insist on the inferiority of ( european ) women in comparison with ( european ) men . in his inquiries into human faculty and its development ( 1883 ) , galton argued that all information about natural events passed through the avenue of our senses . as a result , the more perceptive the senses are of difference , the larger is the field upon which our judgment and intelligence can act . it followed that a chief attribute of a high race was acuteness of the senses . of course , galton elaborated , this did not imply that european women ( noted for their exquisite sensitivity ) were more advanced than european men . nervous irritability , while their menfolk possessed more delicate powers of discrimination ( 1920 ) . like sieveking , he could admit to women s acute senses while denying that their sensitivity was evidence of high rank . such attitudes affected the level of relief given to women in pain . even in giving birth , certain types of women were deemed not to require analgesics . in the words of obstetrician g. ernest herman writing in 1901 , the doctor s job at a labour was to tie and cut the cord , press the placenta out of the vagina , and provide reassurance . he went on to say that among the higher classes he also acts as an anaesthetist , implying that this was not the case for lower classes ( 13 and 16 ) . indeed , it was only from the 1940s that mainstream medical personnel began questioning assumptions that european , white - american , african - american , and primitive women experienced labour pains in different ways . not surprisingly , perhaps , rebuttals were especially prominent within the african - american medical community . in 1966 , for instance , william f. mengert ( professor of obstetrics and gynaecology at the university of illinois medical center in chicago ) published his thoughts on labour pains in an article in the journal of the national medical association . as a medical student , he recalled , he had been taught by john whitridge williams , the founder of academic obstetrics in the united states . williams had believed that negro babies at birth had soft heads and that these , therefore , molded easily through pelves that might otherwise cause trouble , for example , with a white baby whose head would not so readily alter its shape . instead , the attitude of the white doctor undoubtedly was responsible for most of this belief . at dallas , mengert and his team had undertaken a clinical study in whichwe decided not to interfere with any patient in labor until it became obvious to all [ that ] she could not give birth by herself . the end - point chosen was a 2 h arrest of labor after the membranes were ruptured [ and ] the cervix fully dilated and retracted behind the head . we decided not to interfere with any patient in labor until it became obvious to all [ that ] she could not give birth by herself . the end - point chosen was a 2 h arrest of labor after the membranes were ruptured [ and ] the cervix fully dilated and retracted behind the head . by strictly adhering to these criteria , it became obvious that caucasian women also would give birth vaginally if allowed to labor . indeed , contrary to the belief that non - white women had flexible pelves , they found that southern negroes were six to eight times more likely to suffer from contracted pelves than northern whites the implication was clear : under the mistaken belief that african - american infants heads were softer and their mothers pelves more flexible ( when , in fact , the opposite was the case ) , it had been routine to allow southern negro women to labour unaided , while northern white women in labour were given assistance . when white women were also refused assistance , they proved just as capable of producing the infant as their darker - skinned counterparts . it is interesting that these researchers chose to withhold assistance to white women rather than intervening more in the birthing practices involving african - american women . ethnic , religious , and gender variations were important markers of sensitivity to pain , but they were not the only ones . numerous commentators speculated about whether the civilising process itself had increased people s sensitivity to painful stimuli . civilized man has of will ceased to torture , argued neurologist silas weir mitchell , butin our process of being civilized we have won , i suspect , intensified capacity to suffer . the savage does not feel pain as we do : nor as we examine the descending scale of life do animals seem to have the acuteness of pain - sense at which we have arrived ( 1892 , 108 ) . in our process of being civilized the savage does not feel pain as we do : nor as we examine the descending scale of life do animals seem to have the acuteness of pain - sense at which we have arrived ( 1892 , 108 ) . perhaps one reason for the heightened sensitivities of civilized man , anaesthetics and analgesics had an effect on people s ability ( as well as willingness ) to cope with acute afflictions . physicians increasingly observed that , as civilisation progressed , their patients were less capable of bearing the afflictions of their flesh . it required them to treat people in their care differently , as a dentist writing in 1935 british dentistry journal noted . there can be no doubt , he admitted , that our patients are now very different from the pre - war days ; they are not so ready to bear pain , and are more frightened of being hurt . as a consequence , the old idea of the manipulation in the mouth almost regardless of the feelings of the patient has gone , and rightly so , for ever [ sic ] and we are at the dawn of a new era of sympathetic dentistry writing in the same decade , pioneering pain surgeon ren leriche fervently believed in the truth of this argument , illustrating it with an account of a young patient whose elbow joints had fused together after an injury and needed re - sectioning . the young man s grandfather had undergone an identical operation after being wounded at the battle of sedan during the franco - prussian war in 1870 . the grandfather had refused any ansthetic because he was afraid that the limb might be amputated while he was unconscious , his grandson could not even contemplate making such a decision . despite being a brave , stout - hearted , energetic youth , he would not have allowed us to cut even a centimetre of his skin without administering an ansthetic . leriche hastened to add : rather , it was a sign of a nervous system differently developed , and more sensitive . in other words , increased sensitivity to pain was a consequence of the enhanced refinement of senses , which has advanced so rapidly during the century . of course , people in all times anxiously sought to shield themselves from discomfort , leriche acknowledged , but until recent times , they met with little success . they continued to suffer in silence , and , becoming more hardened to pain , they came to feel it less . this meant , of course , that people in the twentieth century were bound to suffer more readily than their predecessors . even the slightest sensory disturbances , he argued , seem to have exaggerated importance . far more than our ancestors , we try to avoid the slightest pain , however fleeting it is , because we know that we have the means of doing so . and , by this very fact , we make ourselves more readily susceptible to pain and we suffer more . every time we fix our attention on anything , we become more conscious of it . , we try to avoid the slightest pain , however fleeting it is , because we know that we have the means of doing so . and , by this very fact , we make ourselves more readily susceptible to pain and we suffer more . every time we fix our attention on anything , we become more conscious of it . he argued that by furnishing us with the means of so easily relieving pain , , antipyrine ( also known as phenazone ) and aspirin rendered us more sensitive to it . as he astutely pointed out , this change in the sensory mechanism of mankind had occurred at the level of real physiology , for physiology means neither more nor less than the observation of what is occurring in ourselves we have already seen examples of this with regards ethnicity , religion , and gender . however , other markers tended to be categorised under two broad headings : first , personal characteristics and , second , traits shared by individuals grouped according to class or occupation . in the first category , individuals possessed subtly different physiologies and personalities . sensitivity to painful stimuli was often linked to an individual s balance of the four humours , for instance . well into the late nineteenth century , physicians argued that melancholics and those with phlegmatic ( sluggish and fat ) temperaments were not especially receptive to pain , in comparison to thin , excitable choleric people ( collier 1889 , 624 ) . as physiological models gradually gave way to more psychological ones , temperament was increasingly judged to be decisive . in an address at the london school of tropical medicine in 1908 , for example , sir william bennett advised physicians to pay attention to their patients temperaments , comparing the reactions of a hospital porter with that of an officer whose bravery on the field was beyond dispute . the porter underwent his grave operation without anaesthetics and without a murmur of complaint , even thanking the surgeon afterwards . in contrast , the officer howled loudly despite the fact that his procedure involved simply trying to bend a partially stiff joint . bennett claimed that it would be ridiculous to attribute cowardice to an officer who had conducted himself honourably in battle . his screams could only be explained by the fact that he possessed a highly - strung nervous temperament which seems to be quite unable to control itself under pain inflicted in what is commonly called in other words , the officer s nervous sensitivity could be over - ridden in the excitement of battle but was irrepressible in the stark setting of a hospital surgery . for another group of thinkers , pain - sensitivity was , literally , embodied in the brain and skull . phrenologists , for instance , speculated that the organ of destructiveness and the organ of fighting were crucial in predisposing men and women to physical stoicism . according to their head - map , the organ of destructiveness was located above the ear , extending backwards from about an inch and a half in front and top of the ears ( wells 1885 , 154 ) . people who possessed a large organ of destructiveness could not only inflict [ pain ] upon others without compunction if not with positive pleasure , but they could also endure pain heroically ( wells 1891 , 165 ) . ( sizer and drayton 1886 , 127 ) , and would even submit one of [ their ] own limbs unflinching to the surgeon , if necessary ( wells 1891 , 166 ) . phrenologists also located the organ for insensitivity to pain in the organ of fighting , which lay on both sides of the skull , near the organ of friendship , but somewhat lower , or behind , and a little above the ear ( hufeland 1807 , 92 ) . this organ , also called the organ of courage , denoted bodily courage , that disregard and inattention to bodily pain . the second category of individuals who were categorised by their degree of sensitivity to pain that is , those grouped according to class , occupation , or education was even more common than humoral , temperamental , and phrenological ones . who could doubt that there were crucial differences in pain sensitivity between nervous scholars and muscular agricultural labourers of people , the president of the british medical association asked in 1889 ? ( collier , 1889 624 ) . the author of sensibility to pain ( 1900 ) concurred , drawing a positive correlation between acute sensitivity to pain and excellent intellectual abilities . the nervous system of brighter people react [ ] quicker in response to the actions of the outside world upon them ( swift , 1900 31517 ) . unlike the physiologists and phrenologists , however , these commentators believed that the correlation between pain sensitivity and class ( or education ) was social rather than innate : the circumstances of a person s life dictated whether he toughened - up or remained fragile . in all these debates , acts of apportioning levels of sensitivity were profoundly prejudicial . outsider groups that is , categories of people who were different to those passing judgment were in a catch-22 situation : the alleged insensitivity of workers , immigrants , hysterics , and chronically - ill patients was proof of their humble status , yet the profound sensitivity of these same people was also proffered as evidence of their inferiority . in the context of class , middle - class commentators believed both that labouring men were insensate ( because they possessed rudimentary nervous systems ) and that they were oversensitive ( because they lacked strength of will ) . conversely , it was taken as obvious that the sensitivity of the educated , wealthy classes showed that they were highly ranked in the chain of civilisation ; yet the insensitivity of the same people confirmed the fact that they possessed superior levels of self - control . for instance , john finney was the first president of the american college of surgeons . in his influential book the significance and effect of pain ( 1914 ) , he amiably claimed thatit does not always follow that because a patient bears what appears to be a great amount of pain with remarkable fortitude , that individual is more deserving of credit or shows greater self - control than the one who does not ; for it is a well - established fact that pain is not felt to the same degree by all individuals alike . it does not always follow that because a patient bears what appears to be a great amount of pain with remarkable fortitude , that individual is more deserving of credit or shows greater self - control than the one who does not ; for it is a well - established fact that pain is not felt to the same degree by all individuals alike . however , in the same section , he made pejorative statements about people with low pain thresholds ( they possessed a yellow streak ) and insisted that patients capable of bearing pain showed wonderful fortitude . is there or can there be anything more sublime or more inspiring in its effect upon others than such an exhibition of self - control ? ( 14 ) . the civilised , white , professional man might be exquisitely sensitive to pain but , through acts of willpower , was capable of masking his reaction , which was the in contrast , the savage , the uneducated , and the dark - skinned might bear a great amount of pain with remarkable fortitude but was not necessarily deserving of credit because it was a well - established fact that pain is not felt to the same degree by all individuals alike . this also helps explain why both highly civilised people as well as degenerates and neurotics were said to be sensitive to pain : the civilized man had cultivated a sensitivity , which was under the control of a highly complex mind , while the degenerate was nothing more than a body - in - pain , out of control . so far , i have explored beliefs from the last two centuries about the pain sensitivities of individuals grouped according to ethnicity , religion , gender , alleged level of civilization , class , education , and temperament . however , the salience of these beliefs has changed over time , often with surprising rapidity , indicating that clinical notions of sensitivity to pain have been highly unstable . this is illustrated in the works of eminent surgeon john eric erichsen , whose the science and art of surgery . being a treatise on surgical injuries , diseases , and operations went through ten different editions between 1853 and 1895 . notably , in the first and second editions of his classic text in 1853 and 1857 , erichsen was relatively uninterested in surgical pain , confining himself to very general statements about pain susceptibility . he was also indifferent to questions about the sensitivity of specific groups to painful stimuli although he did call attention to the great difference in the mental fortitude of individuals and to the fact that in the heat of action ( 1853 , 79 and 1857 , 88 ) . in the editions from the 1860s , erichsen made two significant additions . first , this was the decade in which he first singled out women and children as being of great nervous susceptibility . second , by the 1860s , he was emphasising the degree of pain , as opposed to the severity of injury , important because it represents a shift in erichsen s thinking , including an increased acknowledgement that emotional responses to bad events could have significant effects on physiological sensations . after all , this was the period in which erichsen invented the concept of trauma in the sense we use it today , that is , as a psychological state as opposed to a or bodily injury , as it meant in the original greek . it was in the 1860s that erichsen had observed , in the context of people who had experienced railway accidents , that there was not necessarily a direct and commensurate relationship between the degree of physical injury and nervous disarrangement ( 1866 , 9 ) . in surgery as well , he noted , there was no necessary correlation between the severity of the wound and the degree of pain complained of ( 1861 , 945 ; 1864 , 1012 ; 1869 , 1078 ) . in 1872 ( the first edition which had an extended section on anaesthetics ) and again in 1877 , erichsen elaborated on the importance of constitution as well as sex , disparaging those with an irritable and anxious mind . furthermore , it was no longer simply women who were less capable of bearing pain but nervous and hysterical women in particular . furthermore , the moral condition of the patient , although briefly mentioned in earlier editions , took centre stage in the 1870s . unlike earlier editions , the dangers of indulging in excesses were clearly spelt out in 1872 and 1877 with particular censor reserved for people whose habits could not be called temperate and sober , whose diet was not sufficient and of good quality , and whose minds had been over - strained by the anxieties of business or the labours of a professional life . erichsen was most disparaging of the pain - coping capacities ofthe poor inhabitant of a large and densely peopled town , who has from earliest childhood inhaled an impure and fetid atmosphere , whose scanty diet has consisted of the refuse of the shops , or the semi - decomposed offal of the stalls , and whose nervous system has been irritated and at the same time exhausted in the daily struggle for a precarious livelihood , or over - stimulated by habitual excesses in strong drinks , by which he has hoped to purchase temporary forgetfulness in the cares of a sordid life ( 1872 , 3 and 136 and 1877 , 56 and 180 ) . the poor inhabitant of a large and densely peopled town , who has from earliest childhood inhaled an impure and fetid atmosphere , whose scanty diet has consisted of the refuse of the shops , or the semi - decomposed offal of the stalls , and whose nervous system has been irritated and at the same time exhausted in the daily struggle for a precarious livelihood , or over - stimulated by habitual excesses in strong drinks , by which he has hoped to purchase temporary forgetfulness in the cares of a sordid life ( 1872 , 3 and 136 and 1877 , 56 and 180 ) . in other words , by the 1870s , erichsen s invocation of fears associated with fetid environments or disease - carrying miasma ( a scientific theory that was in steep and terminal decline in this period ) as well as his anxiety about the strains placed on modern middle - class men reflected commonly held fears of his times . by the time the eighth edition was published in 1884 , erichsen had been appointed president of the royal college of surgeons and his devoted student , marcus beck , edited his textbook . perhaps for these reasons , dramatic changes can be identified . the ability to bear surgical pain was still linked to an individual s constitution , occupation , place of residence , and moral status ( particularly the sordid imbibing of alcohol ) , but surgeons were also told thatmen who live hardy out - door lives are less sensitive to pain than those who follow occupations of an opposite kind . the skin , which is the main seat of sensibility in wounds , when hardened by exposure and work , is less sensitive than when it has been habitually protected from such influences . men who live hardy out - door lives are less sensitive to pain than those who follow occupations of an opposite kind . the skin , which is the main seat of sensibility in wounds , when hardened by exposure and work , is less sensitive than when it has been habitually protected from such influences . this was a new passage , but its basic theme was consistent with earlier editions . however , a completely new figure suddenly appeared : that of the savage . interestingly , erichsen and beck s savage took his place in the textbook alongside invocations of european femininity . the relevant paragraph began by stating that the higher man rises in the scale of civilisation , the more acute does his sensibility to pain appear to become , or possibly the less well able is he to bear it . as a consequence , asavage probably suffers less than a civilised man from any given injury , and hence may display more fortitude . an hysterical woman probably does not suffer more than one with a more healthy nervous system , but she complains more loudly , for she has her feelings in all things less under control . race appears to exercise an influence in pain ; some of the native races of india appear to suffer far less than europeans under surgical operations of a similar kind ( 1884 , 56 and 28586 . savage probably suffers less than a civilised man from any given injury , and hence may display more fortitude . an hysterical woman probably does not suffer more than one with a more healthy nervous system , but she complains more loudly , for she has her feelings in all things less under control . race appears to exercise an influence in pain ; some of the native races of india appear to suffer far less than europeans under surgical operations of a similar kind ( 1884 , 56 and 28586 . in other words , erichmen and beck s great chain of feeling positioned civilised man at one end and the savage at the other , but placed between these two extremes were hysterical women . the savage felt less pain , which enabled him to display more fortitude . it was a misleading fortitude because it was really simply due to physiological insensitivity . in contrast , the acute sensibilities of the hysterical woman were similar to the rest of her sex , but her lack of self - control meant that she possessed a lesser capacity for endurance . finally , the tenth edition of 1895 withdrew even token sympathy for the morally-sordid patient who was barely capable of bearing pain . while editions in the 1870s and 1880s warned against patients who had been over - stimulated by habitual excesses in strong drinks , by which [ they had ] hoped to purchase temporary forgetfulness in the cares of a sordid life , but in 1895 , the last phrase was excised ( 1895 , 5 and 303 ) . those who imbibed alcohol were possessed of weak , degenerate bodies , which were incapable of fortitude in times of suffering , and even poverty and deprivation could no longer be proffered as excuses . these debates about different people s propensity to actually feel were complicated by the awareness that irrespective of the innate sensibilities of a particular individual or group emotional states dramatically affected levels of pain awareness and tolerance . this was what erichsen was referring to when , in the 1860s , for the first time he included edward deacon girdlestone in the 1880s was also interested in the influence of mental factors on pain sensation . he recalled being informed of a butcher who had slipped while attempting to hook up a large piece of meat : the hook had penetrated his arm . on being examined , the butcher was described aspale , almost pulseless , and expressed himself as suffering acute agony . the arm could not be moved without causing excessive pain ; and in cutting off the sleeve he frequently cried out . pale , almost pulseless , and expressed himself as suffering acute agony . the arm could not be moved without causing excessive pain ; and in cutting off the sleeve he frequently cried out . yet , when the arm was exposed , it was discovered that the hook had simply pierced the sleeve of his coat . for girdlestone , the patient , he explained , was not a hysterical female , nor yet a poet ; but only a butcher ! if a man s imagination is able to create acute pain out of nothing , is it not reasonable to credit man with the power and the habit of magnifying already existing little pains ? ( 1884 , 22 ) . was not a hysterical female , nor yet a poet ; but only a butcher ! query : - if a man s imagination is able to create acute pain out of nothing , is it not reasonable to credit man with the power and the habit of magnifying already existing little pains ? ( 1884 , 22 ) . mental factors were generally seen as arising out of particular environmental contexts . it became a clich to observe that the high excitement of combat lessened the pain of being wounded . ren leriche s exploration of surgical pain became an influential exposition about the way people in extreme situations might fail to register pain despite being severely wounded . in the context of the 191418 war , leriche asserted that there was all the difference in the world between the reactions [ to wounding and surgery ] of a european and those of an asiatic or an african . he was profoundly impressed by the almost complete indifference to pain shown by russian allies and claimed that his russian colleagues advised him that it was useless to give an ansthetic to certain cossacks before operating on them because they felt nothing , one day , hedisarticulated , without any ansthetic , though with considerable repugnance on my part , three fingers and their metacarpals of one wounded russian , and the whole foot of his comrade . neither one man nor the other showed the least tremor , but turned the hand or raised the leg when asked to do so , and not showing even the slightest sign of momentary weakness , just as if under the most perfect local ansthetic . disarticulated , without any ansthetic , though with considerable repugnance on my part , three fingers and their metacarpals of one wounded russian , and the whole foot of his comrade . neither one man nor the other showed the least tremor , but turned the hand or raised the leg when asked to do so , and not showing even the slightest sign of momentary weakness , just as if under the most perfect local ansthetic . leriche was not making an argument about the propensity of various nationalities or races to ignore pain . in attempting to explain this strange phenomenon , leriche turned neither to the racial sciences nor to other ideas about innate physiological differences : rather , he insisted , a mental factor had to be acknowledged . he noted that russian soldiers possessed the same physiology ( or , to use his mannered language , the same appropriate apparatus ) as other people . consequently , a psychological dimension must have intervened by either suppressing the expression of pain or diminishing its acuteness : we all know that , in certain circumstances , we do not suffer pain , when we ought , in fact , to be acutely conscious of it . many wounded men , in the heat of action , have had their flesh lacerated and torn , without being conscious of anything . when our attention is intensely fixed on something , we may be quite unconscious of pain , and may be prevented from feeling , as we otherwise would , the lacerations of our nerve endings and of our nerves . we all know that , in certain circumstances , we do not suffer pain , when we ought , in fact , to be acutely conscious of it . many wounded men , in the heat of action , have had their flesh lacerated and torn , without being conscious of anything . when our attention is intensely fixed on something , we may be quite unconscious of pain , and may be prevented from feeling , as we otherwise would , the lacerations of our nerve endings and of our nerves . willpower had certainly nothing to do with it . rather , the explanation had to be in certain movements of our hormones , or of the blood , which were diverted into directions other than normal , as the result of fixed attention or of emotion and have the effect of displacing the area ( or altering the atmosphere ) of pain . in addition , he noted , the appreciation of pain was affected bydiet , vitamins , atmospheric conditions , and everything that is capable of bringing conditional reflexes into action ; for certainly the mechanism of sensibility can not escape the effects of association which are produced in us by actions regularly repeated ( 1938 , 23 and 7 ) . diet , vitamins , atmospheric conditions , and everything that is capable of bringing conditional reflexes into action ; for certainly the mechanism of sensibility can not escape the effects of association which are produced in us by actions regularly repeated ( 1938 , 23 and 7 ) . leriche s observations , which were drawn from his experiences during the world war i , were perceptive but anecdotal . they were confirmed by a more systematic study based on the second world war of the twentieth century . henry k. beecher , who served in combat zones on the venafro and cassino fronts , was struck by the fact that many severely wounded men did not complain of pain . medical officers found that there was no necessary correlation between the size and depth of any specific wound and men s expressions of suffering . rather than anecdote , beecher decided to explore this paradox systematically , questioning 215 seriously wounded men . to his surprise , three - quarters did not report experiencing significant pain . one third claimed to be feeling no pain at all , while another quarter said they were experiencing only slight pain . penetrating abdominal wounds , for instance , were more painful ( nearly half of men with such wounds admitted that their pain was bad ) than penetrating wounds of the thorax ( 12 % ) or cerebral wounds ( 7 % ) . remarkably , three quarters of all seriously wounded men did not even ask for pain relief , despite the fact that being asked the question would have served as a reminder that relief was available . what was happening ? it was relatively easy to explain the severity of suffering for men with abdominal wounds : such wounds caused blood and intestinal contents to spill into the peritoneal cavity , spreading infection . however , even significant numbers of these men did not complain of serious pain . perhaps , beecher speculated , men who had been wounded were simply less sensitive generally . but this explanation failed to account for the fact that a badly wounded patient who says he is having no wound pain will protest as vigorously as a normal individual at an inept venipuncture . instead , beecher argued , there must be a difference between wounds caused in civilian contexts ( a car accident , for example ) and those caused during combat . perhaps the strong emotions aroused in combat were responsible for the absence of acute pain . pain might also be alleviated by the fact that wartime wounding would release a soldierfrom an exceedingly dangerous environment , one filled with fatigue , discomfort , anxiety , fear and real danger of death , and gives him a ticket to the safely of the hospital . his troubles are about over , or he thinks they are . from an exceedingly dangerous environment , one filled with fatigue , discomfort , anxiety , fear and real danger of death , and gives him a ticket to the safely of the hospital . this was in contrast to civilian accidents , which only heralded in the beginning of disaster beecher s findings were profoundly influential in post - war reworking of notions of pain . as pain researchers harold wolff and stewart wolf found in the 1950s , most people perceived pain at around similar intensities , but their threshold for reaction varied widely . this did not surprise them since the ability to perceive pain depends upon the intactness of relatively simple and primitive nerve connections . while reacting to pain was modified by the highest cognitive functions and depends in part upon what the sensation means to the individual in the light of his past experiences it was a classic statement that became the dominant way of thinking about pain in the second half of the twentieth century . this distinction between perceiving and reacting to pain received a significant boost from 1943 when physicians began operating on the brains of people suffering agonizing and intractable pain . to everyone s astonishment , lobotomies ( and its numerous surgical variations , such as prefrontal leucotomies and topectomies ) had an unexpected effect : after the operation , patients were still aware that they were experiencing something they identified as pain but were utterly undisturbed by it ( ostenasek 1948 , 229 ) . as leading psychosurgeons walter freeman and james w. watts wrote in their influential textbook psychosurgery . in the treatment of mental disorders and intractable pain ( 1950),the individual might use the same terms to describe the pain after the operations as he used before . [ but ] the attitude was different . the pain was present , but it was a sensation , rather than a threat ( 353 ) . the individual might use the same terms to describe the pain after the operations as he used before . [ but ] the attitude was different . the pain was present , but it was a sensation , rather than a threat ( 353 ) . these patients could still perceive when they were being pricked with pins , scratched , or subjected to extremes of heat or cold , and their threshold for identifying the level of stimulation was unaffected : they simply were not emotionally affected by it . physiologists , psychologists , and sociologists enthusiastically sought to confirm this distinction between perception and reaction , instigating a huge number of experiments seeking to document the two very distinctive thresholds . like their predecessors , they shared a curiosity about racial and ethnic difference , contrasting ( for instance ) the pain thresholds of northern and southern europeans , mikmaq indians , native alaskan indians , eskimos , or african - americans ( for instance , see chapman and jones 1944 ; jewsbury 1951 , 336 ; sherman 943 , 441 ; meehan , stoll , and hardy 1954 , 397400 ; zborowski 1969 ; zborowski 1958 , 25668 ) . some of the most interesting of these experiments involved testing the effect of group cohesion and identification on pain tolerance . for instance , in the late-1950s researchers at mcgill university set out to manipulate an ethnocentric prestige motive or inter - group rivalry . for instance , when jewish women were casually informed that , compared to non - jews , jews were inferior in their capacity to withstand pain , the jewish women s tolerance levels soared . this result was not replicated when protestant women were told the same thing about non - protestants generally . however , when protestant women were told that christians tolerated less pain than jews , the pain tolerance level of these protestant women increased . by specifying the rivalrous comparison group ( jews ) as opposed to the vague non - protestant category , the researchers increased the salience of these women s identification . in other words , pride in one s group identification ( as jews for the jewish women and as christians as opposed to jews for the protestant women ) made these women willing to endure discomfort for the sake of their group s reputation . for all participants , the ability to tolerate pain was assumed to confer a high status of their group ( lambert , libman , and poser 1960 , 35057 ) . research into the way the acuteness , salience , duration , and affective qualities of pain varied according to the meanings attached to the noxious stimuli bolstered the arguments of many scholars that the pain of the laboratory and the pain - malady could not be considered as one and the same thing , according to rne leriche in 1938 . experimental pain was the result of a short excitation repeated from time to time , leriche observed : in contrast , the pain people experiencing in their everyday lives were encountering a continuous phenomenon , with special paroxysms certainly , but with a background which remains unchanged over months or even years . this was a very different experience to the transient disagreeable sensation of pricking or pinching that can be provoked in a healthy individual . he castigated physiologists for adhering to a concept of pain that was too mechanical , too purely artificial , to be capable of reconciliation with which we doctors see in the human patient , and pleaded with them to pay attention to the affective or mental quality of pathological pain syndromes ( 48183 ) . not surprisingly , that other great pain - researcher beecher agreed with leriche . he opposed studies that sought to understand the mechanisms of pain in experimental settings , artificially producing pain through pricking people s skin , giving them electric shocks , applying heat to their foreheads or teeth , or tightening a tourniquet around their limbs . in experimental pharmacology and measurement of the subjective response ( 1952 ) , beecher maintained that no one who has worked with problems of pathological pain can doubt the importance in their field of the environment , of emotional factors , or the reaction to pain . it requires little imagination , he continued , to suppose that the sickbed of the patient in pain , with its ominous threat against his happiness , his security , his very life , provides an entirely different milieu ( and reaction ) than the laboratory , with its dispassionate and unemotional atmosphere . to suppose that the sickbed of the patient in pain , with its ominous threat against his happiness , his security , his very life , provides an entirely different milieu ( and reaction ) than the laboratory , with its dispassionate and unemotional atmosphere . in other words , pain experiences consist of both perceptive aspects and reactive ones , and the only way to truly understand human suffering was through observing and listening to the man himself in real pain of pathological origin ( 15960 ) . these debates reached their height in 1965 when ronald melzack and patrick wall effectively overturned commonly - understand mechanism of pain with their gate control theory , introducing the idea of a gating mechanism in the dorsal horns of the spinal cord that allowed the perception of pain to be modified . crucially , the gate control theory insisted that sensory , cognitive , and affective processes influence people s experience of pain . in effect , mind and the body became fully integrated ( 97179 ) . he worked in the surgical - dressing room of a hospital in an area of london with a large immigrant population , but his dismissal of the pain of fellow - humans is alive and well in clinical settings today . it was not until the 1980s that the under - treatment for pain in minority groups and women began to be addressed , and even today these patients continue to be strongly affected by prejudices about their high threshold for perceiving pain ( green , anderson , baker , et al . debates about the relative sensitivity of different people to noxious stimuli were not merely academic . the seriousness of people s sufferings was calibrated according to such characterisations and sympathy was unevenly rationed . myths about the lower susceptibility of certain patients to painful stimuli justified physicians and other care - givers prescribing fewer and less effective analgesics and anaesthetics , which affected all the groups discussed here . the belief that not every person - in - pain suffered to the same degree was systemic to hierarchical systems generally , shifting in line with other social changes including slave emancipation , anti - imperialism , unionism , and suffrage . after all , it was the colonialist s voice declaring that indigenous peoples were insensible to pain ; the slave - owner professing the extraordinary hardiness of africans ; the professor informing us that the miner s back was sturdy enough to bear the weight of coal ; and the male anthropologist failing to recognise suffering in the birthing - hut in kenya while his medical colleague in london recognised the exquisite sensitivities of european women when their infant s head emerged through the maternal parts like a veritable stone ( knaggs 1931 , 6 and 9 ) . in each case , commentators retained contradictory ideas simultaneously : the humble status of workers and immigrants meant that they were likely to be insensitive to noxious stimuli ; the profound inferiority of these same patients meant that they were especially likely to respond with exaggerated sensitivity . pain - assignation claimed to be based on natural hierarchical schemas , but the great chain of feeling was more fluid than it seemed . the question of whose pain is heard was not only correlated with power differentials between different groups in society ( in which case , the solution was to improve access to resources ) ; patients considered to be

who was truly capable of experiencing pain ? in this article , i explore ideas about the distribution of bodily sensitivity in patients from the early nineteenth century to 1965 in anglo - american societies . while certain patients were regarded as truly hurting , other patients distress could be disparaged or not even registered as being real pain . such judgments had major effects on regimes of pain - alleviation . indeed , it took until the late twentieth century for the routine underestimation of the sufferings of certain groups of people to be deemed scandalous . often the categorizations were contradictory . for instance , the humble status of workers and immigrants meant that they were said to be insensitive to noxious stimuli ; the profound inferiority of these same patients meant that they were especially likely to respond with exaggerated sensitivity . how did physicians hold such positions simultaneously ? pain - assignation claimed to be based on natural hierarchical schemas , but the great chain of feeling was more fluid than it seemed .

Pain sensibilities The gendering of sensitivities The civilizing process A case-study in change: Erichsens textbook Complications due to mental factors Conclusion

indeed , the gazette routinely sneered at the local residents who made up the bulk of their patients , poking fun at their quaint expressions of pain and minimising the degree of distress they might be experiencing . peoples regularly focussed on their loathsome bodies : the problem was not so much that these patients writhed in pain , but that they were incapable of screwing up the courage to be brave in the face of misfortune . writing in 1811 , for instance , a professional planter was determined not to let the evidence of anatomy dissuade him of his prejudices about the bodies of black slaves . because of their dulled sensitivities , slaves were better able to endure , with few expressions of pain , the accidents of nature ( 201 ) . this was providential indeed , since they were subjected to so many accidents of nature while labouring in slave plantations . admittedly , in hospitals that had sprung up during the civil war , we see negroes suffering from the gravest diseases cowering on their couches without taking any notice of the attending physicians . it was a myth that a generation of african - american physicians writing in the early years of the twentieth century both struggled to come to grips with and attempted to debunk . one of the main forums for this generation of doctors was the journal of the national medical association , a journal dedicated to promoting african - american interests in medicine . after this concession to those who believed that african - americans had less sensitive nervous systems and were easily swayed emotionally , he went on to warn against translating these generalisations into more casual attitudes to providing pain - relief for african - american patients . since the existence of feeling depended on the activity of the brain , observed a writer signing himself philanthropos in the early 1880s , it was logical that the more perfect development of that organ , the greater the perception of sensations such as pain . or , as the author of pain and sympathy ( 1907 ) concluded when attempting to explain why the savage could bear physical torture without shrinking : the higher the life , the keener is the sense of pain silas weir mitchell s famous statement of 1892 , during the process of being civilized we have won intensified capacity to suffer . after all , the savage does not feel pain as we do : nor as we examine the descending scale of life do animals seem to have the acuteness of pain - sense at which we have arrived ( 108 ) . the savage does not feel pain as we do : nor as we examine the descending scale of life do animals seem to have the acuteness of pain - sense at which we have arrived ( 108 ) . non - european peoples could be denigrated as possessing lesser bodies : their position at the lower echelons of the great chain of feeling was due to their physiological insensibility . just a few years earlier , the author of the highly respectable textbook entitled the diagnostics of internal medicine ( 1901 ) also accused the semitic stock , and the celtic and italic [ sic ] groups of appearing to possess an average greater sensibility to pain than the teutonic and slavonic groups ( butler 1901 , 35 ) . or , as essayist louis bertrand pontificated in the art of suffering ( 1936 ) , people from the southern or eastern parts of europe lacked the capacity to control themselves when experiencing pain . he claimed to have observed thatthe hebrew cried out through fear that if he failed to attract full attention he might miss some of the benefits of hospital care ; while the irishmen called loudly upon god and the saints , and wept and groaned because he was an emotional being to whose nature the repression of feeling , whether pleasant or painful , was foreign . the hebrew cried out through fear that if he failed to attract full attention he might miss some of the benefits of hospital care ; while the irishmen called loudly upon god and the saints , and wept and groaned because he was an emotional being to whose nature the repression of feeling , whether pleasant or painful , was foreign . rather , irish patients lacked adequate psychological inhibitions and jews had learnt the bitter lesson of persecution so were keen to ensure that they were not overlooked ( pain 1929 , 164 ) . the question that ignited the debate was simple : could pain thresholds ( that is , the point at which a person subjected to a noxious stimuli complained of pain ) be correlated with eye colour ? this was no freak coincidence , the editors continued , speculating that patients with blue eyes were likely to come from north european stock , traditionally a phlegmatic race , unlike brown - eyed patients who were more likely to have descended from more excitable mediterranean peoples a doctor from hove ( sussex ) maintained that amongst his patients there was a positive correlation not only between dark brown eyes and a low pain threshold , but also between this eye - colour and over - reaction to pain . ironically , in this case , patients who were perceived to be less sensitive ( blue - eyed patients ) won better treatment than those who were believed to be suffering the most . in contrast , no laurels are to be won by sitting patiently under the knife of a surgeon , so the virtue is of a less suspicious character , the principle of it more simple , and the practice more difficult ( cowper 1792 ) . women s more delicate organisation unfortunately meant that she was more likely to suffer a however , by way of balance , women were endowed with more placid and patient endurance than generally characterises the members of the ruder sex it was a theme that was still being repeated over 70 years later , when a survey showed that 70 % of physicians and dentists believed that women were superior to men in withstanding pain ( josey and miller 1932 , 375 ) . was women s for instance , the medical superintendent of the virol pathological research laboratories , writing in the british journal of nursing in 1913 and 1914 , initially seemed to be making an argument for socialisation as the chief mechanism by which women learnt to bear their tribulations . as a result , when treating male patients , the wise physician would direct his attention to the seat of the lesion itself or the conducting nerves . in the words of obstetrician g. ernest herman writing in 1901 , the doctor s job at a labour was to tie and cut the cord , press the placenta out of the vagina , and provide reassurance . indeed , it was only from the 1940s that mainstream medical personnel began questioning assumptions that european , white - american , african - american , and primitive women experienced labour pains in different ways . not surprisingly , perhaps , rebuttals were especially prominent within the african - american medical community . in 1966 , for instance , william f. mengert ( professor of obstetrics and gynaecology at the university of illinois medical center in chicago ) published his thoughts on labour pains in an article in the journal of the national medical association . indeed , contrary to the belief that non - white women had flexible pelves , they found that southern negroes were six to eight times more likely to suffer from contracted pelves than northern whites the implication was clear : under the mistaken belief that african - american infants heads were softer and their mothers pelves more flexible ( when , in fact , the opposite was the case ) , it had been routine to allow southern negro women to labour unaided , while northern white women in labour were given assistance . in our process of being civilized the savage does not feel pain as we do : nor as we examine the descending scale of life do animals seem to have the acuteness of pain - sense at which we have arrived ( 1892 , 108 ) . as a consequence , the old idea of the manipulation in the mouth almost regardless of the feelings of the patient has gone , and rightly so , for ever [ sic ] and we are at the dawn of a new era of sympathetic dentistry writing in the same decade , pioneering pain surgeon ren leriche fervently believed in the truth of this argument , illustrating it with an account of a young patient whose elbow joints had fused together after an injury and needed re - sectioning . sensitivity to painful stimuli was often linked to an individual s balance of the four humours , for instance . well into the late nineteenth century , physicians argued that melancholics and those with phlegmatic ( sluggish and fat ) temperaments were not especially receptive to pain , in comparison to thin , excitable choleric people ( collier 1889 , 624 ) . his screams could only be explained by the fact that he possessed a highly - strung nervous temperament which seems to be quite unable to control itself under pain inflicted in what is commonly called in other words , the officer s nervous sensitivity could be over - ridden in the excitement of battle but was irrepressible in the stark setting of a hospital surgery . according to their head - map , the organ of destructiveness was located above the ear , extending backwards from about an inch and a half in front and top of the ears ( wells 1885 , 154 ) . phrenologists also located the organ for insensitivity to pain in the organ of fighting , which lay on both sides of the skull , near the organ of friendship , but somewhat lower , or behind , and a little above the ear ( hufeland 1807 , 92 ) . who could doubt that there were crucial differences in pain sensitivity between nervous scholars and muscular agricultural labourers of people , the president of the british medical association asked in 1889 ? outsider groups that is , categories of people who were different to those passing judgment were in a catch-22 situation : the alleged insensitivity of workers , immigrants , hysterics , and chronically - ill patients was proof of their humble status , yet the profound sensitivity of these same people was also proffered as evidence of their inferiority . conversely , it was taken as obvious that the sensitivity of the educated , wealthy classes showed that they were highly ranked in the chain of civilisation ; yet the insensitivity of the same people confirmed the fact that they possessed superior levels of self - control . for instance , john finney was the first president of the american college of surgeons . in his influential book the significance and effect of pain ( 1914 ) , he amiably claimed thatit does not always follow that because a patient bears what appears to be a great amount of pain with remarkable fortitude , that individual is more deserving of credit or shows greater self - control than the one who does not ; for it is a well - established fact that pain is not felt to the same degree by all individuals alike . it does not always follow that because a patient bears what appears to be a great amount of pain with remarkable fortitude , that individual is more deserving of credit or shows greater self - control than the one who does not ; for it is a well - established fact that pain is not felt to the same degree by all individuals alike . the civilised , white , professional man might be exquisitely sensitive to pain but , through acts of willpower , was capable of masking his reaction , which was the in contrast , the savage , the uneducated , and the dark - skinned might bear a great amount of pain with remarkable fortitude but was not necessarily deserving of credit because it was a well - established fact that pain is not felt to the same degree by all individuals alike . this also helps explain why both highly civilised people as well as degenerates and neurotics were said to be sensitive to pain : the civilized man had cultivated a sensitivity , which was under the control of a highly complex mind , while the degenerate was nothing more than a body - in - pain , out of control . so far , i have explored beliefs from the last two centuries about the pain sensitivities of individuals grouped according to ethnicity , religion , gender , alleged level of civilization , class , education , and temperament . however , the salience of these beliefs has changed over time , often with surprising rapidity , indicating that clinical notions of sensitivity to pain have been highly unstable . second , by the 1860s , he was emphasising the degree of pain , as opposed to the severity of injury , important because it represents a shift in erichsen s thinking , including an increased acknowledgement that emotional responses to bad events could have significant effects on physiological sensations . erichsen was most disparaging of the pain - coping capacities ofthe poor inhabitant of a large and densely peopled town , who has from earliest childhood inhaled an impure and fetid atmosphere , whose scanty diet has consisted of the refuse of the shops , or the semi - decomposed offal of the stalls , and whose nervous system has been irritated and at the same time exhausted in the daily struggle for a precarious livelihood , or over - stimulated by habitual excesses in strong drinks , by which he has hoped to purchase temporary forgetfulness in the cares of a sordid life ( 1872 , 3 and 136 and 1877 , 56 and 180 ) . in other words , erichmen and beck s great chain of feeling positioned civilised man at one end and the savage at the other , but placed between these two extremes were hysterical women . in contrast , the acute sensibilities of the hysterical woman were similar to the rest of her sex , but her lack of self - control meant that she possessed a lesser capacity for endurance . finally , the tenth edition of 1895 withdrew even token sympathy for the morally-sordid patient who was barely capable of bearing pain . while editions in the 1870s and 1880s warned against patients who had been over - stimulated by habitual excesses in strong drinks , by which [ they had ] hoped to purchase temporary forgetfulness in the cares of a sordid life , but in 1895 , the last phrase was excised ( 1895 , 5 and 303 ) . these debates about different people s propensity to actually feel were complicated by the awareness that irrespective of the innate sensibilities of a particular individual or group emotional states dramatically affected levels of pain awareness and tolerance . when our attention is intensely fixed on something , we may be quite unconscious of pain , and may be prevented from feeling , as we otherwise would , the lacerations of our nerve endings and of our nerves . when our attention is intensely fixed on something , we may be quite unconscious of pain , and may be prevented from feeling , as we otherwise would , the lacerations of our nerve endings and of our nerves . rather , the explanation had to be in certain movements of our hormones , or of the blood , which were diverted into directions other than normal , as the result of fixed attention or of emotion and have the effect of displacing the area ( or altering the atmosphere ) of pain . in addition , he noted , the appreciation of pain was affected bydiet , vitamins , atmospheric conditions , and everything that is capable of bringing conditional reflexes into action ; for certainly the mechanism of sensibility can not escape the effects of association which are produced in us by actions regularly repeated ( 1938 , 23 and 7 ) . they were confirmed by a more systematic study based on the second world war of the twentieth century . one third claimed to be feeling no pain at all , while another quarter said they were experiencing only slight pain . penetrating abdominal wounds , for instance , were more painful ( nearly half of men with such wounds admitted that their pain was bad ) than penetrating wounds of the thorax ( 12 % ) or cerebral wounds ( 7 % ) . to everyone s astonishment , lobotomies ( and its numerous surgical variations , such as prefrontal leucotomies and topectomies ) had an unexpected effect : after the operation , patients were still aware that they were experiencing something they identified as pain but were utterly undisturbed by it ( ostenasek 1948 , 229 ) . like their predecessors , they shared a curiosity about racial and ethnic difference , contrasting ( for instance ) the pain thresholds of northern and southern europeans , mikmaq indians , native alaskan indians , eskimos , or african - americans ( for instance , see chapman and jones 1944 ; jewsbury 1951 , 336 ; sherman 943 , 441 ; meehan , stoll , and hardy 1954 , 397400 ; zborowski 1969 ; zborowski 1958 , 25668 ) . for instance , in the late-1950s researchers at mcgill university set out to manipulate an ethnocentric prestige motive or inter - group rivalry . for instance , when jewish women were casually informed that , compared to non - jews , jews were inferior in their capacity to withstand pain , the jewish women s tolerance levels soared . however , when protestant women were told that christians tolerated less pain than jews , the pain tolerance level of these protestant women increased . for all participants , the ability to tolerate pain was assumed to confer a high status of their group ( lambert , libman , and poser 1960 , 35057 ) . research into the way the acuteness , salience , duration , and affective qualities of pain varied according to the meanings attached to the noxious stimuli bolstered the arguments of many scholars that the pain of the laboratory and the pain - malady could not be considered as one and the same thing , according to rne leriche in 1938 . experimental pain was the result of a short excitation repeated from time to time , leriche observed : in contrast , the pain people experiencing in their everyday lives were encountering a continuous phenomenon , with special paroxysms certainly , but with a background which remains unchanged over months or even years . he castigated physiologists for adhering to a concept of pain that was too mechanical , too purely artificial , to be capable of reconciliation with which we doctors see in the human patient , and pleaded with them to pay attention to the affective or mental quality of pathological pain syndromes ( 48183 ) . these debates reached their height in 1965 when ronald melzack and patrick wall effectively overturned commonly - understand mechanism of pain with their gate control theory , introducing the idea of a gating mechanism in the dorsal horns of the spinal cord that allowed the perception of pain to be modified . crucially , the gate control theory insisted that sensory , cognitive , and affective processes influence people s experience of pain . he worked in the surgical - dressing room of a hospital in an area of london with a large immigrant population , but his dismissal of the pain of fellow - humans is alive and well in clinical settings today . debates about the relative sensitivity of different people to noxious stimuli were not merely academic . myths about the lower susceptibility of certain patients to painful stimuli justified physicians and other care - givers prescribing fewer and less effective analgesics and anaesthetics , which affected all the groups discussed here . in each case , commentators retained contradictory ideas simultaneously : the humble status of workers and immigrants meant that they were likely to be insensitive to noxious stimuli ; the profound inferiority of these same patients meant that they were especially likely to respond with exaggerated sensitivity . pain - assignation claimed to be based on natural hierarchical schemas , but the great chain of feeling was more fluid than it seemed . the question of whose pain is heard was not only correlated with power differentials between different groups in society ( in which case , the solution was to improve access to resources ) ; patients considered to be

nurses are the largest group of professionals within the global health care system , with a total of 19.3 million nursing and midwifery personnel in the world . the current and growing shortage of registered nurses ( rns ) in health care systems is thus a global concern [ 2 , 3 ] . in fact , the european commission has estimated that there will be a shortage of 590,000 nurses by the year 2020 . in the united states , employment of rns most countries within the organization for economic cooperation and development ( oecd ) have reported a nursing shortage , which is predicted to get worse because the current nursing population is aging . this shortage of rns influences the delivery of health care and negatively affects patient outcomes ; an insufficient nurse staffing level is associated with negative patient outcomes [ 8 , 9 ] and decreased nurse job satisfaction . at the same time of this global nursing shortage , many nurses are considering leaving their job , profession or are out of the nursing workforce . according to flinkman et al . literature review , nurses ' intention to leave the profession varied from 4% up to 54% across the studies internationally . in a next ( nurses early exit ) study , conducted in ten european countries ( n = 30,330 ) , 13% of nurses had thought about leaving the profession frequently . according to an rn4cast ( nurse forecasting in europe ) study ( n = 33,659 nurses / europe ; 27,509 nurses / united states ) , the proportion of nurses planning to leave their current job ranged from 49% ( finland , greece ) to 14% ( united states ) . in european sample of this study , every tenth ( 9% ) of nurses was having intention to leave the profession . furthermore , in salminen study , nearly half ( 37% , n = 343 ) of young rns ( under 35 years ) working in hospitals have reported frequent intention to leave the profession in finland . in sweden , , it was reported that the percentage of rns intending to leave nursing within 3 years was low , at 3% . yet , of the total licensed rn population in 2008 , it was estimated that more than 15% ( n = 466,564 ) were not actually employed within nursing . turnover intention appears to be a multistage process consisting of psychological , cognitive , and behavioral components and has been found to predict the actual decision to leave the profession [ 12 , 19 , 20 ] . according to a study by hasselhorn et al . , the majority of leavers began the process with serious consideration in the final year preceding leaving , and the actual decision to leave was then made within the 6 months prior to determination . in carless and arnup 's study , one year prior to changing careers , actual career changers were actively looking for a new career and had a high intention to leave their current job . furthermore , another study revealed that nurses left nursing within 6 months of their decision to leave . the final decision to leave the profession is likely to be the result of an individual reflection process [ 21 , 23 ] with multiple underlying causes [ 11 , 12 ] . from the society 's and healthcare 's points of view , professional turnover is a more significant form of work transitions than organizational turnover [ 24 , 25 ] . those rns , who are leaving the profession , are reducing the total number of nurses in the manpower , which has an impact on the present nursing shortage and is leading to a permanent loss of productivity . nurses leaving the profession take their tacit knowledge , experience , and contribution from the organizations and also from the nursing workforce . the financial investments used on nurse 's education , orientation , and continuing education are lost . moreover , nurse turnover is also costly to organizations : first , because it results in the direct and indirect costs of filling the positions , and second , because of the loss of organizational productivity and knowledge . the youngest generation of nurses are the most willing to leave the job and the nursing profession [ 12 , 15 , 16 , 27 , 28 ] . kovner and djukic have reported that in the united states more rn graduates left their first nursing job ( 26% ) than nursing profession ( 2% ) during the first two years in career . according to next study , in most european countries the intent to leave the profession was highest in the age groups between 25 and 35 years of age . in robinson study , diploma - qualified nurses ' movement into other activities was highest around the age of 28 and declined thereafter . according to barron and west study , rate of leaving the nursing profession for a better job was highest at the age of 32 . several factors are related to young rns ' intentions to leave the profession , including an imbalance of effort and reward , high psychological demands , and higher job strain , which all influence young nurses ' intention to resign from their nursing careers . according to a swedish study of rudman et al . , graduates of a younger age are more vulnerable to early career burnout , which is associated with the intention to leave the profession . in a study by flinkman et al . , both the survey questionnaire and the open - ended questions showed that young nurses ' intentions to leave the profession were connected with the highly demanding work , burnout and dissatisfaction with salary levels . experiences of resent graduates have been widely examined in the nursing research [ 3436 ] . however , less research is available concerning graduate nurses ' intention to leave the profession . in a study by scott et al . , graduates ( mean age 29 ) who did not like being a nurse reported a higher desire to leave the profession . in a study by lavoie - tremblay et al . , those young graduates ( 24 or younger ) who reported having poor work environment , were more likely to state they would leave the profession . most of the earlier turnover research is concerned with nurses leaving the job or the organization ; less research is conducted on what motivates nurses to leave their profession [ 11 , 25 ] . this is understandable , because nurse turnover is expensive and organizations are usually more concerned with their own nursing manpower than workforce in a whole . studies concerning nurses ' turnover and their intent to leave the profession have been mainly executed using quantitative methods and survey questionnaires [ 11 , 27 , 39 ] , revealing the need to explore this phenomenon using qualitative methods and to produce studies with more in - depth understanding . accordingly , qualitative research and research from generational perspective has been recommended in order to get a more comprehensive picture of turnover [ 27 , 40 ] . knowing that young nurses ' transition from a student to nursing career is a demanding and stressful process and thus could lead to nurse turnover from the profession [ 37 , 38 ] , it is important to explore views and perceptions regarding early career experiences from the young nurses point of view . therefore , the aim of this study was to gain a deeper understanding of young nurses ' career transition processes , as experienced by young registered nurse , aged between 29 and 32 years . young registered nurse ( rn ) in this study refers to a person who has completed bachelor of health care degree in the university of applied sciences and whose intention to leave the profession has started before the age of 30 years . this inquiry has been designed as a longitudinal qualitative case study using interpretive narrative method . a case is being defined as a longitudinal career story interpreted by a young registered nurse . this is a qualitative case study , in which an interpretative narrative approach [ 42 , 43 ] is adopted . qualitative case study has been defined as a research methodology grounded in an interpretive , constructivist paradigm . case studies often contain narratives that approach the complexities and contradictions of real life and can be used for exploring , describing , and understanding a phenomenon in its context [ 44 , 45 ] . an intensive investigation of cases can provide contextual knowledge of real - life phenomena that would be unobtainable with a population - based approach . case - centred research can add knowledge of how people experience career transitions and construct new career identities . according to sharp case - centred approaches are an excellent and relevant way of doing nursing research , because nursing practice is centred on cases ( patients and in broader sense organizations and social context ) . even though nurses have not fully embraced case study as a comprehensive approach for research [ 45 , page 103 ] , case studies can be seen as an option for many nursing studies , because it allows to use various sources of evidence and it has a potential to uncover multiple realities . both the interview material and data analysis method in this study can be defined as narrative . the underlying assumption of narrative inquiry is the idea that individuals make sense of their world by telling stories . narrative approach does not assume objectivity , but rather it privileges positionality , pluralism , relativism , and subjectivity [ 50 , 51 ] . there is an underlying assumption in a narrative approach that there is neither a single , absolute truth in human reality nor one correct reading or interpretation of a text [ 50 , page 2 ] . narrative turn is a part of a paradigmatic shift from realism toward constructivism in science , which then further refers to fundamental changes in beliefs concerning reality and knowledge . events in a narrative are selected , organized , connected , and evaluated in accordance with the audience to which the narrative is told ( e.g. , the interviewer ) . interpretation is inevitable because stories are representations , and a researcher does not have direct access to another 's experiences . stories are usually composed and received in context , telling much about the society , culture , and the social reality the teller is living in . stories are a way for a person to make sense of disruptive events in their lives or when there has been a breach between the ideal and real , self and society . cochran suggests that narrative study is a paradigm in career research , because the topic of the career is not ultimately focused on the distinct parts of the story but instead on how the different elements of a professional history are interrelated and brought together . transitions from one profession to another involve always narrative reflection . by narrating their professional history and transitions , a successful transition to a new workplace or occupation could be considered as completed once a person is able to resolve the conflicts and contradictions in his or her career narrative . key informants or critical cases that were knowledgeable about the phenomenon of interest . in the year 2006 , we invited young nurses to write the narrative stories of their nursing careers and why they had an intention to leave nursing . we advertised in tehy the magazine of the union of health and social care professionals and in an open discussion forum for nurses via the internet ( http://www.hoitajat.net/ ) . to be eligible for the study , we required the participants to be a registered nurse , have work experience in nursing , have an intention to leave a nursing profession that had started before the age of 30 , have a willingness to articulate , and reflect upon , their experiences in their nursing career . be a registered nurse , have work experience in nursing , have an intention to leave a nursing profession that had started before the age of 30 , have a willingness to articulate , and reflect upon , their experiences in their nursing career . we requested that they add their contact information if they wanted participate the interviews concerning the intention of leaving the nursing profession . three young female registered nurses were willing to tell their story to the interviewer and met the inclusion criteria . we decided to accomplish longitudinal career story interviews with these three nurses in order to get an in - depth understanding of the phenomenon of interest . we considered that three young nurses was a sufficient number , because in case study method sample sizes are nonrepresentative and narrative interview data usually accumulate with a large quantity of rich data . the nurses were living in different parts of finland , one in a large city and the remaining two in smaller cities . nursing education in finland is arranged in universities of applied sciences , and study consists of 210 credit points . the first author conducted all of the interviews : a female researcher who was at the time in her 30s , a registered nurse , and a phd student in nursing science . the process of gathering the initial data was conducted between december 2006 and may 2007 . in the beginning of the first interview the interviewees were asked to draw on a3-sized paper their pathway as a nurse and put into this drawing the significant events in , and experiences of , their nursing career . narratives of personal experiences or life stories are often told in response to open - ended questions , which do not limit or specify the type and the form of the narrative . in the first interview i would like you to tell me , freely and with your own words , your story as a nurse . you can start your story at any point of your life that you have felt to be significant for you becoming a nurse . you can end your story at any point in your life that you want . after this open - ended question , the interviewer acted as an active listener and avoided interrupting the participant 's narrative . when the complete story had been obtained , some detailed relevant questions were presented to encourage further elaboration on the narrative ( e.g. , could you tell more about why you applied to nursing school ? ) . further questioning helps participants to recall details , turning points , and other meaningful events in their narrative story . in the last phase of the interview , the interviewer asked the participant whether she wanted to say anything else ; the interviews ended when the participant did not want to add anything further . the second interviews took place four years later , between january and march 2011 , when all of these young nurses had made the transition to a new career . in the beginning of the second interview , the participants were asked to continue their earlier career - pathway drawing and were asked to continue their story of being a nurse from any point in their life that they chose . the lengths of the interviews varied from 113 minutes ( longest first interview ) to 25 minutes ( shortest second interview ) . one nurse chose to be interviewed at her workplace , and the other participants were interviewed in their homes . interviews were recorded on a digital recorder and were transcribed verbatim by the first author , because analysis and interpretation of such data depends on the adequacy of a carefully prepared manuscript . firstly , we followed the holistic - content method developed by lieblich et al . , where the life story of a person is taken as a whole ; researchers studying life histories and case studies also use this approach . after the first readings of the data , the first author formed three career story narratives on the basis of the interviews , career - pathway drawings and written stories . in these narratives , the first author selected and organized the separate events from the nurses ' careers into complete narratives with a chronological order . polkinghorne describes this kind of method as narrative analysis , where the researcher configures a new narrative on the basis of the narrative material . we found in the preliminary readings that while these narratives were unique , certain main themes also appeared to be reflected both within and between these stories . a thematic approach to narratives is useful when trying to find common thematic elements across research participants and the events they report . as sandelowski and leeman [ 60 , page 1407 ] conclude , the identification of themes is foundational to qualitative research of all kinds . the first author followed these particular themes throughout the stories and compared , contrasted , and interpreted these themes in the context of the overall career histories . these emerging interpretations and themes were discussed , reviewed , and refined with the coauthors . the results of the analysis are reported in accordance with these themes . by presenting cases and themes simultaneously it was possible to reach a more holistic and multidimensional perspective to these stories . to evaluate the validity of narrative studies , riessman argues that it is trustworthiness , rather than truth , which should be evaluated in narrative studies . first , our data analysis is described in detail , and direct citations from the narratives are provided in relation to each story to reveal the basis from which the analysis was conducted . according to polkinghorne [ 61 , page 6 ] , readers should be able to follow the presented evidence and argument enough to make their own judgment as to the relative validity of the claim . second , we used data triangulation ( narrative interviews , career - pathway drawings , and written stories ) to enhance the trustworthiness of the analysis . third , the narrative stories and themes were reviewed in research seminars in a doctoral school of nursing science and in a narrative summer school to ensure that the chosen themes resonated with the narrative stories . fourth , the accuracy was reviewed by sending the final paper draft to the participants , to allow them to verify the authenticity of the data and ensure that they agreed with the interpretations . as the original transcripts were in finnish , the citations used in this paper have been translated into english by a professional translator in order to reproduce the citations as accurately as possible and maintain the authenticity of the participant 's responses . we informed them that they had the right to withdraw at any stage , and that the collected data would be treated with confidentiality . we removed the following details from the career stories : places of residence , names of the health care organization , names of family members , and names of colleagues . we also changed some minor details in these career stories , which we considered to be insignificant , in order to protect participants ' identity . furthermore , we used pseudonyms to protect participant anonymity . because nurses participated voluntarily and in their leisure time , at the time of the first interview , anna ( a pseudonym ) was a young woman at a crossroad in her nursing career . she showed a strong intention for a career change , yet at the same time she was tired and distressed because she was not sure how such a career change would be possible . her narrative was detailed , complex , and often included cynical humor and sarcasm ; she also often vocalized direct quotations from other people 's speech to make parts of her narrative more dramatic . anna used a great amount of time in the first interview analyzing what she wanted for her future career and what she might be capable of doing if she were to leave nursing work . she had talked about this career change with her relatives and friends and had thought deeply about the advantages and disadvantages of leaving the nursing profession . betty ( a pseudonym ) already had a new university education at the time of her first interview . she told her story in a good - humored way , often laughing . during her story , there were some more dramatic stories for example , an unsuccessful resuscitation of a small baby she had , however , stayed in her nursing career for quite a short time . her narratives were well structured , and she used a flowing narrative style to tell her stories . she had a strong sense of treating other people in a fair and just way . during her time at the nursing school confidential tasks were already being given to cecilia , and teachers were saying that she would become a good teacher . she had already begun to be interested in management when she was in the nursing school and was willing to develop herself professionally from the beginning of her nursing career . when anna was in both grammar school and high school , she had no intention of becoming a nurse . she considered many careers , for example , a veterinarian or journalist , but was not sure , or at least confident , of what she wanted to do in her forthcoming career . after high school she applied to university to read nonnursing discipline but was not accepted . she was at that time in practical training in a hospital and liked the work , despite the fact that it was not exactly the work she was considering as a career choice . not knowing what else to do , she however applied to a nursing school and was accepted . anna related that she did not know anything about nursing when she was applying to nursing school , but she was confident that she was never going to work as a bedside nurse . in the admission test she achieved a very high overall score but a low motivation score . in her retrospective analysis she reflected that the test had probably quite accurately measured the reality of the situation : she was not especially eager to get into the nursing school . at that time her mother and friends had said that a career in nursing might not be right for anna 's personality . well , it was some sort of drifting , that i had never any kind of calling . and then i thought that i really did not know where to apply , so then it was the nursing school , because perhaps nursing was something kind of similar to psychology , although i did not really want to admit to myself that it was n't really like that . well , it was some sort of drifting , that i had never any kind of calling . and then i thought that i really did not know where to apply , so then it was the nursing school , because perhaps nursing was something kind of similar to psychology , although i did not really want to admit to myself that it was n't really like that . betty was also not interested in a nursing career when she was in high school . after high school betty applied to university but , disappointingly for her , was not accepted . she was doing voluntary work during a gap year and through this became interested in nursing work . she decided to apply for a nursing education because she thought that nursing could be a stepping stone for working in developing countries or in becoming a physician . at the very beginning of her nursing studies she had already started to hesitate : was she choosing the right career ? in the beginning of her story she recalledi do not even know how to say why i ended up becoming a nurse , but i feel that i have sort of drifted or something . i do not even know how to say why i ended up becoming a nurse , but i feel that i have sort of drifted or something . cecilia too believed that she became a nurse through serendipity , and a nursing career had not been a childhood dream for her either . after high school cecilia decided to move abroad and to start university studies there . before leaving , cecilia was working for a few months in a hospital and started to consider what it would be like to be a nurse . coincidentally she noticed an advertisement for the nursing studies in a newspaper , decided to apply , and was accepted . one of her relatives had worked in a hospital when cecilia was a small child . she thought that there was a certain attraction to nursing work , although at the same time the hospital was quite a frightening place , with white working clothes and with peculiar smells . it 's not as if i had thought that i would like to work in a hospital . it 's not as if i had thought that i would like to work in a hospital . even though these young nurses did not have nursing as a first career option , they all graduated from nursing school with good grades . they also all received positive feedback from the school and from clinical practices , and their memories from the nursing school were mainly positive in their narratives . since the beginning of her nursing career , anna had been working in a ward where many patient were alcoholic , drug abusers , or elderly patients with no hope of recovery . she said that she did not like the routine tasks related to patient hygiene and housekeeping within the ward . anna did not like working close to the patients ; she highlighted nursing tasks that were menial , unpleasant , and intimate . perhaps because of the work - related burnout , she recalledi'm just so irritated by our patients to be honest . when we think of the picture that the so - called normal people have of a hospital , that there nurses take care of car - crash patients and make them healthy and heal them and all kinds of things . and the reality is some are intoxicated , some alcoholic , and then there are old people with many diseases whose nursing feels more like torturing them . when we think of the picture that the so - called normal people have of a hospital , that there nurses take care of car - crash patients and make them healthy and heal them and all kinds of things . and the reality is some are intoxicated , some alcoholic , and then there are old people with many diseases whose nursing feels more like torturing them . betty also had to take on a great deal of responsibility as a graduate nurse : in some wards she was the only rn during a shift , as all the others were nursing aids . when she was employed in her last summer job as a nurse she thought that the nurses where she was working had an increased workload , and that they were extremely busy . she was no longer enjoying nursing work , and she felt that being productive was seen as more important than taking good care of the patients . she told a story about one nightshift when the workload was almost overwhelmingthat hospital , it was a place like , it was really something like eight people in the same room and some of those people were screaming all night long . that 's also when i thought that this is not making any sense ! and there 's two of us nurses and over forty patients on the wards . that hospital , it was a place like , it was really something like eight people in the same room and some of those people were screaming all night long . that 's also when i thought that this is not making any sense ! and there 's two of us nurses and over forty patients on the wards . cecilia liked nursing work related to the patients and their care and enjoyed both the atmosphere of the workplace and her coworkers . how much she liked her job , cecilia also verbalized feelings of guilt and of being burdened because she thought that she was unable to provide the level of care that she considered adequate . she told a story of her last summer in the hospital when there were many inexperienced doctors on the ward . she recalls we had had some really difficult patient cases and that summer for some reason there happened to be some really , really inexperienced doctors at the beginning of their careers , candidates of medical science , and all the time you had to be kind of mentoring their actions . it felt like you had to watch out all the time , and that besides your own work you kind of had to also take care of how everything else runs there . we had had some really difficult patient cases and that summer for some reason there happened to be some really , really inexperienced doctors at the beginning of their careers , candidates of medical science , and all the time you had to be kind of mentoring their actions . it felt like you had to watch out all the time , and that besides your own work you kind of had to also take care of how everything else runs there . there was a strong hierarchy in the hospitals , and the lowest in the hierarchy were nursing students in some workplaces they were not even permitted to go into the coffee room yet graduate nurses and new nurses in the ward were also low down . betty noticed during her nursing education that medical students were even lower in the hierarchy than nursing students ; after this realisation she decided against pursuing a medical career . strong hierarchy was a disappointment for these young nurses ; however , they felt that they could not change the situation . during her interviews , cecilia often recounted her experiences of poor management in the hospital . for example , she had supervised students , often using her own leisure time to do so , but these efforts were not compensated ; furthermore , she had lacked feedback about what she was good at , and what she needed to do in order to develop . she had wanted to participate in further education , but as a temporary worker she was not offered this possibility . cecilia felt that managers in the hospital did not actually understand how demanding the nurses ' work was , and that they had very unrealistic proposals about how to develop patient care . she also confronted ethical problems in the ward ; she felt that nurses in the ward were not able to provide humane and proper care . she recalledand then , like i told you , my husband came to pick me up from work and then i started to cry that i can not take this anymore , this is terrible , i can not . and then , like i told you , my husband came to pick me up from work and then i started to cry that i can not take this anymore , this is terrible , i can not . anna felt that nursing would not provide her with intellectual challenges after a few years of work , and that nurses had no career development possibilities . similarly , betty had sought career development opportunities but was not able to see the potential for pursuing them in her nursing career ; she had hoped that nurses would have some kind of career ladder , to allow for such progress . thus , this lack of professional development was a common concern , as expressed by bettyi find it kind of terrible to think that you graduate as a nurse and then you will be a nurse for the rest of your life . that maybe you move from one ward to another ward or from place to place but the work is always the same everywhere . i find it kind of terrible to think that you graduate as a nurse and then you will be a nurse for the rest of your life . that maybe you move from one ward to another ward or from place to place but the work is always the same everywhere . anna thought that even a much higher salary would not compensate for work that was otherwise unsatisfactory . betty said that the salary was too low when compared to the responsibilities of nursing work ; she was getting a better salary in her new job outside nursing , even though there she was not able to kill anyone . anna , betty , and cecilia all did shift work during their nursing career . combining private life with shift work was demanding for them , because they found planning their lives around it challenging . recovering from night shifts was also difficult , because it was hard to be awake at nights and sleep during the day . betty , furthermore , said that if she did not get enough sleep , she was prone to suffer from migraines . all of these nurses were working under fixed - term contracts at the beginning of their career , because during the economic depression , graduate nurses were mainly offered only fixed - term employments . anna felt that the charge - nurse in the ward was using fixed - term contracts as a way to exert power . this power relation changed when there started to be a shortage of nurses in the ward , which provided anna with a bargaining opportunity . after telling the ward sister that she was looking for a new job she thought that getting a permanent job was a good thing for gaining control over one 's own life , although at the same time she also thought that she was then somewhat trapped.there were these fixed - term contracts which could be from two weeks to three months or then for six months . they could n't give me their word that there would be longer periods which made me feel really insecure when i thought about my future . there were these fixed - term contracts which could be from two weeks to three months or then for six months . they could n't give me their word that there would be longer periods which made me feel really insecure when i thought about my future . betty did not work in nursing for long and only with temporary work contracts . while studying for a new career she worked for short periods in numerous hospitals . in some places there was no proper orientation for the job , and there was no support for a recently graduated , inexperienced nurse . she had the possibility to apply for a permanent contract but never did so ; she did not want a permanent job because she was sure that she was not going to work for the next forty years as a nurse . within the narratives , all these young nurses described what kind of people nurses are : nurturing , altruistic , and willing to serve . they did not , however , place themselves within these expected images of nurses . in their stories , these young nurses narrate nursing as a profession , not as a vocational calling . they construct themselves as talented and ambitious and striving for career advancement . throughout anna ' story , she described herself as intellectual , creative , and systematic , but that she was not able to use any of these qualities in nursing . she thought that many other nurses were hoping to change career but found it impossible because of the demands of their families . her boyfriend concurred , as he had said that she was too bright and talented for a nursing work my husband used to tell me well with that mind , with those talents , with that brain , it is obvious you 're not going to enjoy it there , that he has always been saying to me that you 're in the wrong profession . well with that mind , with those talents , with that brain , it is obvious you 're not going to enjoy it there , that he has always been saying to me that you 're in the wrong profession . betty also related that she had not been a nurturer when she was a child . when betty applied to the nursing school , her parents said that she was too ambitious for a nursing career , and that she would not stay in nursing for long . however , betty was a strong - minded woman and decided at that time to continue , despite her parents ' worries . after graduation , stuck in that ward , where she was not capable of using her knowledge and talents as a nurse . cecilia also produced stereotypical images of nurses in her stories : she described that nurses had a role within the hospital of the physicians ' handmaids . she thought that nurses had a lower professional status in the hospital than physicians and that a nurse 's role was not autonomous.nurses are physicians ' handmaids in the hospital . nurses are physicians ' handmaids in the hospital . so developing your own thinking skills , development , was kind of incomplete . she also thought that the average person did not know how demanding the nursing job is or how many skills nursing work requires , and that she did not like the stereotypical picture of nurses given in the media and on television i 'm disgusted by the word just the typical sort of little blond scrubber that just fools around with the doctors and giggles and puts her cool hand to the feverish patient 's forehead wearing a short skirt with her bottom up . just the typical sort of little blond scrubber that just fools around with the doctors and giggles and puts her cool hand to the feverish patient 's forehead wearing a short skirt with her bottom up . in her search for a new career anna tried to discover one that would better suit her personality as an intellectual , creative , and systematic person . her decision for changing careers had started as a sinking feeling , although the interviews did not reveal a single instance where this intention would have started . this transition process had lasted for many years and was difficult for anna ; indeed a number of times during the interview she remembered that she had often cried and had occasionally felt very tired . however , she presented herself as an active actor who had the power to change a situation that was not satisfactory to her . after the first interview anna applied to university for a nonnursing discipline , and was accepted . she graduated , and she found a new workplace outside nursing , and also resigned from her permanent nursing job . anna was the only one of these three nurses who thought that she would never work as a registered nurse again . as anna sharedi guess it was pretty much the whole thing and many people have asked me why because this is such a dramatic career change . that it is a bunch of various things . i guess it was pretty much the whole thing and many people have asked me why because this is such a dramatic career change . that it is a bunch of various things . according to betty 's story , how she ended up in a different career was as serendipitous as her initial choice to become a nurse . a friend had given her an extra application form for the entrance exam to study nonnursing discipline in the university ; after sending in the application she decided to prepare carefully for the exam , studied hard , and was accepted . she managed her financial problems while studying by working as a nurse and by living frugally , which required both self - discipline and sacrificing her own comfort . after graduating from a university , she received a demanding work outside the nursing field . at the time of the second interview betty had some thoughts of continuing her education in university to doctoral level and also doing some nursing work occasionally under temporary contracts . in finland reregistration is not required , so betty would have the possibility to return to nursing.at the moment i can not even imagine what i would be if i had not gone through the nursing school . although i 've thought many times that me being a nurse has been a total waste , that i 've been squandering the resources of society or something like that . i think that i would be missing something if i would've not done that . it has given me a lot . at the moment i can not even imagine what i would be if i had not gone through the nursing school . although i 've thought many times that me being a nurse has been a total waste , that i 've been squandering the resources of society or something like that . i think that i would be missing something if i would've not done that . it has given me a lot . during the transition process many changes were happening in cecilia 's private life . her family was very supportive of her career change , because they saw that she was not satisfied with nursing . she graduated , and because she was having a university degree , she got a new demanding job in human resource management in a large organization . she enjoyed her new work , even though it was demanding and the workdays were long . i do not know if it has been a bad choice , cause it has made many things possible , but the choice that i 've clearly made intentionally has been to leave cause i 've been thinking that it is possible to do things better . but maybe the path that has leaded me away from nursing to here , where i am now , could 've been more direct . i do not know if it has been a bad choice , cause it has made many things possible , but the choice that i 've clearly made intentionally has been to leave cause i 've been thinking that it is possible to do things better . but maybe the path that has leaded me away from nursing to here , where i am now , could 've been more direct . in this inquiry , we used a qualitative case - study approach to form an in - depth investigation into the reasons why young registered nurses may leave nursing profession . this study produced three rich , detailed accounts of career transition from a perspective of a young generation . the results of this exploratory qualitative study reflect a shift toward insights into understanding professional turnover as a complex and long - lasting process . this could be one reason why earlier quantitative studies do not provide a clear picture of the reasons why nurses leave nursing . all of these career stories are unique but also have similarities ( see figure 1 ) . none of these young nurses had nursing as a childhood dream ; it was more serendipitous that they had ended up as nurses . the work content of nursing was demanding , and the practice environment was not ideal in terms of nurse - patient ratios , rush , shift hours , working contracts , salary , and general appreciation . furthermore , these young women felt that they did not fit to the stereotypical picture and image of nurses . and finally , nursing was not able to provide the career development possibilities and intellectual challenges that these women were able to gain by applying to university studies and by starting a new career . although these nurses narrated their career changes as having happened serendipitously , they also constructed themselves as active and competent actors . throughout their career transition process , these nurses had the possibility to make financial and personal sacrifices for a new education , and they were supported by their families . findings of this inquiry support the work of others ; for example , in takase 's literature review turnover intention was described as a multistage process , with a range of factors considered as the antecedents . additionally , according to cheung 's qualitative interview study of former nurses the decision to leave the profession was a difficult one for nurses , and represents a complex psychological process . the case studies here present three phases of a career transition : nurses reassessed their work ; they began the transition process ( e.g. , by getting a new education ) ; and finally there was a socialization phase for the new job . trice and morand described similar phases based on the literature : separation from the old role , transition , and then integration into the new role . as typical for voluntary career transitions these nurses had both time and the possibility to consider options for new education and for a new career . there was no single particular experience or crisis that made these nurses leave the profession . this is contrary to the findings of cheung and morrell studies , and where also a single , jarring event or shock initiated nurse 's thoughts of quitting . as seen in this inquiry , the long - lasting process of career transition can actually start even before young nurses enter the nursing education . none of these nurses who shared their stories held nursing as a childhood dream ; they described their career choices as a second career choice , or that they had drifted into nursing , rather than as a purposefully selected occupation . after being a while in a nursing career all of these nurses applied to their primary educational choice , to study at the university . nursing career chosen as a default choice or not having nursing as a childhood career dream has been associated with a shorter tenure in nursing . also a study conducted in sweden gave similar results ; of graduates ( n = 672 ) who were negative or uncertain about their career choice ( 26% ) , a majority had considered leaving the profession . in an earlier study by santamki et al . , approximately one - third of finnish nurses ( 35% , n = 3,352 ) reported that they had chosen nursing coincidentally , or as a second - best alternative . by investigating nurses ' longitudinal career stories there is an opportunity to gain understanding of the complex dynamics of why nurses choose their career and how leaving considerations and behaviors evolve over time . however , future research is needed into whether the motives for choosing nursing career affect the length of the career in nursing . according to these young nurses stories , the work content of nursing was demanding , and the nursing practice environment was not ideal in terms of nurse - patient ratios , rush , shift hours , and general appreciation . nurses felt tired because they could not provide as adequate nursing care as they were willing . nurses ' narratives bought up ethical dilemmas associated with not being able to provide humane and proper care . not being able to do one 's best and not being able to influence working conditions were in contradiction with the talent and ambitions of these young women . similar themes came up in mcgillis hall and kiesners study , where nurses related narratives about guilt and over commitment when the working environment prevented them from providing complete and high - quality care . we believe that narrative stories can provide a window into people 's beliefs and experience [ 72 , page 209 ] and can provide richer understanding of how young nurses experienced the reality of nursing and how these lived experiences affected their career decisions . they described what kind of people nurses are supposed to be : nurturing , altruistic , and willing to serve . similar traditional stereotypes of good women have been presented on media and in public when nursing has been presented as a virtue script . nursing profession has been associated with femininity and powerlessness [ 76 , 77 ] , and stereotypical public images such as angels with pretty faces and empty heads , physicians ' handmaids , or naughty nurses still exist in western countries . these young nurses did not , however , place themselves within these expected stereotypic images of nurses . this could be one reason why these nurses left profession talented and bright women did not fit the stereotypes of nurses as nurturing and serving and of being lower in the hospital hierarchy . as seen in this inquiry , public stereotypes can have negative impact on nurses ' self - esteem and could lead nurses to leave their profession . although there is considerable amount of research concerning stereotypes and images of nurses [ 77 , 80 ] , a search of the literature revealed only few earlier studies where the low status of the profession was connected to nurse turnover [ 78 , 81 ] . more quantitative and also qualitative research is needed on how public stereotypes and image of nursing possibly affect young nurse 's turnover . stories can reveal deeply hidden assumptions that influence the actions of people ; through stories we can better understand how nurses explain and make sense of the stereotypes and images associated with the profession . by investigating in - depth stories there is a possibility to gain deeper understanding of the historical , sociocultural , and gendered perceptions which partly could lead younger generation of nurses to leave their profession . in these stories , young nurses described themselves as talented and determined to go ahead in their career , attributes not historically attached to nurses [ 77 , 80 ] . for example , in the uk [ 82 , page 305 ] , media tend to view nursing profession was not able to provide the career development possibilities and intellectual challenges that these women were able to gain by applying to university studies and by starting a new career . for example , in a study by salminen , rns who judged that they had the potential to carry out more challenging tasks had a higher intention to leave the profession . additionally , hasselhorn et al . found that former nurses had indicated too low demands of nursing work as one reason to leave the profession , and former nurses in a swedish study by fochsen et al . stated that a lack of professional opportunities and restricted professional autonomy were central reasons for leaving . most of this earlier literature was focused on turnover from the perspectives of organizations or professions , with the researchers viewing it as a preventable or negative result [ 11 , 27 ] . however , as seen in this inquiry , turnover can also be beneficial for an individual nurse [ 12 , 23 ] . changing workplace , and even career , can provide nurses with the opportunity to move to positions better suited to their motives , ambitions , skills , and career goals . according to hasselhorn et al . , a proportion of nurses with an intention to leave the profession are young , highly qualified , and seeking a new challenge . in light of this study , we think that it would be beneficial to health care systems to retain this group of nurses within the profession by opening up to them new possibilities and challenges within nursing . on the contrary , nurses who display a low level of work ability might benefit from leaving the nursing profession . in - depth investigations of career stories could give a broader perspective to the research of nurse 's turnover , by focusing on career transition from a perspective of an individual nurse and by enlightening how a single nurse can benefit from a change of career . what kind of new knowledge of nurses ' professional turnover can we expect to get based on qualitative case - study approach ? the purpose of this inquiry was not to seek universal explanations of why young nurses leave nursing profession . the results are not meant to be generalized to a certain population as case study research commonly needs to be generalized theoretically [ 47 , 53 ] . it is clear that no one methodology by itself will be able to address nurse turnover as a multifaceted phenomenon effectively . there is still a need to statistically measure and model variables connected with turnover ; also interventions to reduce turnover intention need to be developed and tested . the advantage of large sample sized research is the breath and the empirical generalizability of the evidence . however , as flyvbjerg remains , the disadvantage of this kind of knowledge is one of depth ; therefore , qualitative case studies are needed as well . first , nurses were given an opportunity to tell their own story of why they had initially intended to leave , and why they eventually left , nursing . the voices of nurses who have intended to leave nursing have rarely been studied from their own perspective . second , in contrast to the majority of previous studies that have mainly focused on turnover intentions and not actual turnover , a unique feature of this study is its longitudinal nature , which allows the reader to follow nurses through the process of their career transition . third , nurses here provided stories of negative images and stereotypes in relation to their intention to leave nursing ; in earlier studies of professional turnover , nurses ' own negative stereotypes and images have not been significant factors in causing an intention to leave . furthermore , it provides unique real - life contextual knowledge of young nurses ' experiences of leaving a nursing career . this knowledge can be used as a basis for further studies and in discussions concerning nurses ' intentions to leave nursing from a generational perspective . more in - depth research is needed in order to better understand why nurses leave , and even more significantly , what could motive the younger generation of nurses to stay in nursing . in addition , there is also a need for longitudinal research that tracks nurses throughout the career change processes . the findings reported in this study may be limited due to the sampling methods which were used . it is possible that the nurses who volunteered , and thus were prepared to tell their career stories , were different from other nurses who had also considered leaving the profession . these nurses were interested in the subject under investigation , they had all thought about the motives for why they had an intention to leave and were willing to narrate their experiences . all of the nurses who participated were open to the experience , sociable , and talkative . these are all also characteristics that are associated with persons who are more likely to change their career . all of these nurses had the personal and financial opportunities to gain a new education at university , something that is not possible for all nurses with an intention to leave . furthermore , the participants were all aware of the interviewer 's occupation as a registered nurse ; thus it is possible that this knowledge may have influenced the way in which they recounted their career stories . they were also aware that the topic of the research was the intention to leave nursing ; this in turn might have brought up more negative issues from their career , in order to justify their reasons for leaving it . the use of the qualitative case study method made it possible to examine nurses ' career turnover as an ongoing and multidimensional process , in which several causes consequently impacted on the final decision to leave the nursing profession . we demonstrated that narrative career interviews are a feasible way of collecting data regarding career transition processes . we also claim that in - depth narrative stories can provide unique real - life contextual knowledge of nurses ' experiences when living through these kinds of career transitions . we conclude that the qualitative case - study approach is a potential and novel way of doing turnover research in nursing science . given the complex nature of nurse turnover , such a qualitative , in - depth approach is essential , alongside with quantitative studies , for a sound development of the nurse turnover research .

in a time of global nursing shortages an alarming number of young registered nurses have expressed a willingness to leave the profession . in this qualitative case study we investigate in depth why young nurses leave nursing profession and reeducate themselves for a new career . the study is based on longitudinal interviews of three young registered nurses in finland . these nurses were first interviewed between december 2006 and may 2007 , when they were 2932 years old and having an intention to leave the profession . the second interview took place four years later , from january 2011 to march 2011 when all of them had made the transition to a new career . data were analyzed in two stages . in the first stage , comprehensive career story narratives were formed on the basis of the interviews . in the second stage , emerging themes in these stories were compared , contrasted , and interpreted in the context of the overall career histories . nursing as a second career choice and demanding work content as well as poor practice environment and the inability to identify with the stereotypical images of nurses were main themes that emerged from these career stories . the results of this interpretative qualitative study reflect a shift toward insights into understanding professional turnover as a complex and long - lasting process .

1. Introduction 2. Material and Methods 3. Results and Discussion 4. Conclusions

at the same time of this global nursing shortage , many nurses are considering leaving their job , profession or are out of the nursing workforce . literature review , nurses ' intention to leave the profession varied from 4% up to 54% across the studies internationally . in a next ( nurses early exit ) study , conducted in ten european countries ( n = 30,330 ) , 13% of nurses had thought about leaving the profession frequently . according to an rn4cast ( nurse forecasting in europe ) study ( n = 33,659 nurses / europe ; 27,509 nurses / united states ) , the proportion of nurses planning to leave their current job ranged from 49% ( finland , greece ) to 14% ( united states ) . in european sample of this study , every tenth ( 9% ) of nurses was having intention to leave the profession . furthermore , in salminen study , nearly half ( 37% , n = 343 ) of young rns ( under 35 years ) working in hospitals have reported frequent intention to leave the profession in finland . turnover intention appears to be a multistage process consisting of psychological , cognitive , and behavioral components and has been found to predict the actual decision to leave the profession [ 12 , 19 , 20 ] . , the majority of leavers began the process with serious consideration in the final year preceding leaving , and the actual decision to leave was then made within the 6 months prior to determination . in carless and arnup 's study , one year prior to changing careers , actual career changers were actively looking for a new career and had a high intention to leave their current job . the final decision to leave the profession is likely to be the result of an individual reflection process [ 21 , 23 ] with multiple underlying causes [ 11 , 12 ] . those rns , who are leaving the profession , are reducing the total number of nurses in the manpower , which has an impact on the present nursing shortage and is leading to a permanent loss of productivity . moreover , nurse turnover is also costly to organizations : first , because it results in the direct and indirect costs of filling the positions , and second , because of the loss of organizational productivity and knowledge . the youngest generation of nurses are the most willing to leave the job and the nursing profession [ 12 , 15 , 16 , 27 , 28 ] . kovner and djukic have reported that in the united states more rn graduates left their first nursing job ( 26% ) than nursing profession ( 2% ) during the first two years in career . according to next study , in most european countries the intent to leave the profession was highest in the age groups between 25 and 35 years of age . according to barron and west study , rate of leaving the nursing profession for a better job was highest at the age of 32 . several factors are related to young rns ' intentions to leave the profession , including an imbalance of effort and reward , high psychological demands , and higher job strain , which all influence young nurses ' intention to resign from their nursing careers . , graduates of a younger age are more vulnerable to early career burnout , which is associated with the intention to leave the profession . , both the survey questionnaire and the open - ended questions showed that young nurses ' intentions to leave the profession were connected with the highly demanding work , burnout and dissatisfaction with salary levels . however , less research is available concerning graduate nurses ' intention to leave the profession . , graduates ( mean age 29 ) who did not like being a nurse reported a higher desire to leave the profession . studies concerning nurses ' turnover and their intent to leave the profession have been mainly executed using quantitative methods and survey questionnaires [ 11 , 27 , 39 ] , revealing the need to explore this phenomenon using qualitative methods and to produce studies with more in - depth understanding . therefore , the aim of this study was to gain a deeper understanding of young nurses ' career transition processes , as experienced by young registered nurse , aged between 29 and 32 years . young registered nurse ( rn ) in this study refers to a person who has completed bachelor of health care degree in the university of applied sciences and whose intention to leave the profession has started before the age of 30 years . this inquiry has been designed as a longitudinal qualitative case study using interpretive narrative method . a case is being defined as a longitudinal career story interpreted by a young registered nurse . qualitative case study has been defined as a research methodology grounded in an interpretive , constructivist paradigm . even though nurses have not fully embraced case study as a comprehensive approach for research [ 45 , page 103 ] , case studies can be seen as an option for many nursing studies , because it allows to use various sources of evidence and it has a potential to uncover multiple realities . events in a narrative are selected , organized , connected , and evaluated in accordance with the audience to which the narrative is told ( e.g. cochran suggests that narrative study is a paradigm in career research , because the topic of the career is not ultimately focused on the distinct parts of the story but instead on how the different elements of a professional history are interrelated and brought together . by narrating their professional history and transitions , a successful transition to a new workplace or occupation could be considered as completed once a person is able to resolve the conflicts and contradictions in his or her career narrative . in the year 2006 , we invited young nurses to write the narrative stories of their nursing careers and why they had an intention to leave nursing . to be eligible for the study , we required the participants to be a registered nurse , have work experience in nursing , have an intention to leave a nursing profession that had started before the age of 30 , have a willingness to articulate , and reflect upon , their experiences in their nursing career . be a registered nurse , have work experience in nursing , have an intention to leave a nursing profession that had started before the age of 30 , have a willingness to articulate , and reflect upon , their experiences in their nursing career . three young female registered nurses were willing to tell their story to the interviewer and met the inclusion criteria . we decided to accomplish longitudinal career story interviews with these three nurses in order to get an in - depth understanding of the phenomenon of interest . we considered that three young nurses was a sufficient number , because in case study method sample sizes are nonrepresentative and narrative interview data usually accumulate with a large quantity of rich data . the nurses were living in different parts of finland , one in a large city and the remaining two in smaller cities . the first author conducted all of the interviews : a female researcher who was at the time in her 30s , a registered nurse , and a phd student in nursing science . the process of gathering the initial data was conducted between december 2006 and may 2007 . in the beginning of the first interview the interviewees were asked to draw on a3-sized paper their pathway as a nurse and put into this drawing the significant events in , and experiences of , their nursing career . in the first interview i would like you to tell me , freely and with your own words , your story as a nurse . in the last phase of the interview , the interviewer asked the participant whether she wanted to say anything else ; the interviews ended when the participant did not want to add anything further . the second interviews took place four years later , between january and march 2011 , when all of these young nurses had made the transition to a new career . in the beginning of the second interview , the participants were asked to continue their earlier career - pathway drawing and were asked to continue their story of being a nurse from any point in their life that they chose . the lengths of the interviews varied from 113 minutes ( longest first interview ) to 25 minutes ( shortest second interview ) . after the first readings of the data , the first author formed three career story narratives on the basis of the interviews , career - pathway drawings and written stories . polkinghorne describes this kind of method as narrative analysis , where the researcher configures a new narrative on the basis of the narrative material . we found in the preliminary readings that while these narratives were unique , certain main themes also appeared to be reflected both within and between these stories . the first author followed these particular themes throughout the stories and compared , contrasted , and interpreted these themes in the context of the overall career histories . the results of the analysis are reported in accordance with these themes . first , our data analysis is described in detail , and direct citations from the narratives are provided in relation to each story to reveal the basis from which the analysis was conducted . second , we used data triangulation ( narrative interviews , career - pathway drawings , and written stories ) to enhance the trustworthiness of the analysis . third , the narrative stories and themes were reviewed in research seminars in a doctoral school of nursing science and in a narrative summer school to ensure that the chosen themes resonated with the narrative stories . fourth , the accuracy was reviewed by sending the final paper draft to the participants , to allow them to verify the authenticity of the data and ensure that they agreed with the interpretations . as the original transcripts were in finnish , the citations used in this paper have been translated into english by a professional translator in order to reproduce the citations as accurately as possible and maintain the authenticity of the participant 's responses . we removed the following details from the career stories : places of residence , names of the health care organization , names of family members , and names of colleagues . we also changed some minor details in these career stories , which we considered to be insignificant , in order to protect participants ' identity . because nurses participated voluntarily and in their leisure time , at the time of the first interview , anna ( a pseudonym ) was a young woman at a crossroad in her nursing career . anna used a great amount of time in the first interview analyzing what she wanted for her future career and what she might be capable of doing if she were to leave nursing work . she was at that time in practical training in a hospital and liked the work , despite the fact that it was not exactly the work she was considering as a career choice . even though these young nurses did not have nursing as a first career option , they all graduated from nursing school with good grades . when she was employed in her last summer job as a nurse she thought that the nurses where she was working had an increased workload , and that they were extremely busy . she was no longer enjoying nursing work , and she felt that being productive was seen as more important than taking good care of the patients . there was a strong hierarchy in the hospitals , and the lowest in the hierarchy were nursing students in some workplaces they were not even permitted to go into the coffee room yet graduate nurses and new nurses in the ward were also low down . all of these nurses were working under fixed - term contracts at the beginning of their career , because during the economic depression , graduate nurses were mainly offered only fixed - term employments . anna felt that the charge - nurse in the ward was using fixed - term contracts as a way to exert power . this power relation changed when there started to be a shortage of nurses in the ward , which provided anna with a bargaining opportunity . while studying for a new career she worked for short periods in numerous hospitals . in their stories , these young nurses narrate nursing as a profession , not as a vocational calling . she thought that many other nurses were hoping to change career but found it impossible because of the demands of their families . her boyfriend concurred , as he had said that she was too bright and talented for a nursing work my husband used to tell me well with that mind , with those talents , with that brain , it is obvious you 're not going to enjoy it there , that he has always been saying to me that you 're in the wrong profession . cecilia also produced stereotypical images of nurses in her stories : she described that nurses had a role within the hospital of the physicians ' handmaids . she also thought that the average person did not know how demanding the nursing job is or how many skills nursing work requires , and that she did not like the stereotypical picture of nurses given in the media and on television i 'm disgusted by the word just the typical sort of little blond scrubber that just fools around with the doctors and giggles and puts her cool hand to the feverish patient 's forehead wearing a short skirt with her bottom up . in her search for a new career anna tried to discover one that would better suit her personality as an intellectual , creative , and systematic person . after the first interview anna applied to university for a nonnursing discipline , and was accepted . at the time of the second interview betty had some thoughts of continuing her education in university to doctoral level and also doing some nursing work occasionally under temporary contracts . she graduated , and because she was having a university degree , she got a new demanding job in human resource management in a large organization . in this inquiry , we used a qualitative case - study approach to form an in - depth investigation into the reasons why young registered nurses may leave nursing profession . the results of this exploratory qualitative study reflect a shift toward insights into understanding professional turnover as a complex and long - lasting process . this could be one reason why earlier quantitative studies do not provide a clear picture of the reasons why nurses leave nursing . all of these career stories are unique but also have similarities ( see figure 1 ) . none of these young nurses had nursing as a childhood dream ; it was more serendipitous that they had ended up as nurses . the work content of nursing was demanding , and the practice environment was not ideal in terms of nurse - patient ratios , rush , shift hours , working contracts , salary , and general appreciation . furthermore , these young women felt that they did not fit to the stereotypical picture and image of nurses . throughout their career transition process , these nurses had the possibility to make financial and personal sacrifices for a new education , and they were supported by their families . additionally , according to cheung 's qualitative interview study of former nurses the decision to leave the profession was a difficult one for nurses , and represents a complex psychological process . trice and morand described similar phases based on the literature : separation from the old role , transition , and then integration into the new role . as typical for voluntary career transitions these nurses had both time and the possibility to consider options for new education and for a new career . there was no single particular experience or crisis that made these nurses leave the profession . as seen in this inquiry , the long - lasting process of career transition can actually start even before young nurses enter the nursing education . none of these nurses who shared their stories held nursing as a childhood dream ; they described their career choices as a second career choice , or that they had drifted into nursing , rather than as a purposefully selected occupation . after being a while in a nursing career all of these nurses applied to their primary educational choice , to study at the university . , approximately one - third of finnish nurses ( 35% , n = 3,352 ) reported that they had chosen nursing coincidentally , or as a second - best alternative . according to these young nurses stories , the work content of nursing was demanding , and the nursing practice environment was not ideal in terms of nurse - patient ratios , rush , shift hours , and general appreciation . these young nurses did not , however , place themselves within these expected stereotypic images of nurses . this could be one reason why these nurses left profession talented and bright women did not fit the stereotypes of nurses as nurturing and serving and of being lower in the hospital hierarchy . as seen in this inquiry , public stereotypes can have negative impact on nurses ' self - esteem and could lead nurses to leave their profession . although there is considerable amount of research concerning stereotypes and images of nurses [ 77 , 80 ] , a search of the literature revealed only few earlier studies where the low status of the profession was connected to nurse turnover [ 78 , 81 ] . stories can reveal deeply hidden assumptions that influence the actions of people ; through stories we can better understand how nurses explain and make sense of the stereotypes and images associated with the profession . by investigating in - depth stories there is a possibility to gain deeper understanding of the historical , sociocultural , and gendered perceptions which partly could lead younger generation of nurses to leave their profession . in these stories , young nurses described themselves as talented and determined to go ahead in their career , attributes not historically attached to nurses [ 77 , 80 ] . for example , in the uk [ 82 , page 305 ] , media tend to view nursing profession was not able to provide the career development possibilities and intellectual challenges that these women were able to gain by applying to university studies and by starting a new career . for example , in a study by salminen , rns who judged that they had the potential to carry out more challenging tasks had a higher intention to leave the profession . found that former nurses had indicated too low demands of nursing work as one reason to leave the profession , and former nurses in a swedish study by fochsen et al . most of this earlier literature was focused on turnover from the perspectives of organizations or professions , with the researchers viewing it as a preventable or negative result [ 11 , 27 ] . , a proportion of nurses with an intention to leave the profession are young , highly qualified , and seeking a new challenge . in light of this study , we think that it would be beneficial to health care systems to retain this group of nurses within the profession by opening up to them new possibilities and challenges within nursing . what kind of new knowledge of nurses ' professional turnover can we expect to get based on qualitative case - study approach ? the purpose of this inquiry was not to seek universal explanations of why young nurses leave nursing profession . the results are not meant to be generalized to a certain population as case study research commonly needs to be generalized theoretically [ 47 , 53 ] . however , as flyvbjerg remains , the disadvantage of this kind of knowledge is one of depth ; therefore , qualitative case studies are needed as well . first , nurses were given an opportunity to tell their own story of why they had initially intended to leave , and why they eventually left , nursing . the voices of nurses who have intended to leave nursing have rarely been studied from their own perspective . third , nurses here provided stories of negative images and stereotypes in relation to their intention to leave nursing ; in earlier studies of professional turnover , nurses ' own negative stereotypes and images have not been significant factors in causing an intention to leave . this knowledge can be used as a basis for further studies and in discussions concerning nurses ' intentions to leave nursing from a generational perspective . more in - depth research is needed in order to better understand why nurses leave , and even more significantly , what could motive the younger generation of nurses to stay in nursing . it is possible that the nurses who volunteered , and thus were prepared to tell their career stories , were different from other nurses who had also considered leaving the profession . these nurses were interested in the subject under investigation , they had all thought about the motives for why they had an intention to leave and were willing to narrate their experiences . all of the nurses who participated were open to the experience , sociable , and talkative . all of these nurses had the personal and financial opportunities to gain a new education at university , something that is not possible for all nurses with an intention to leave . furthermore , the participants were all aware of the interviewer 's occupation as a registered nurse ; thus it is possible that this knowledge may have influenced the way in which they recounted their career stories . they were also aware that the topic of the research was the intention to leave nursing ; this in turn might have brought up more negative issues from their career , in order to justify their reasons for leaving it . the use of the qualitative case study method made it possible to examine nurses ' career turnover as an ongoing and multidimensional process , in which several causes consequently impacted on the final decision to leave the nursing profession .

trauma is one of the most common causes of hospital admission and in - hospital mortality [ 1 - 3 ] . recently , the mortality rate in severe trauma patients has decreased [ 4 - 6 ] due to emphasis on damage control resuscitation ( dcr ) . blood product transfusion plays an important role in early dcr and improved survival [ 7 , 8 ] . in a randomized trial , del junco et al ( 2013 ) reported that early blood product transfusions during prehospital transportation improved patient outcomes including improved patient acid - base status , decreased blood product usage , and reduced early in - hospital mortality . other observational studies showed similar benefits among patients who received early blood transfusions either during prehospital or initial emergency department ( ed ) resuscitation phases [ 10 - 12 ] . these studies documented improved oxygen carrying capacity , more efficient intravascular volume expansion , and reduced risk of early trauma induced coagulopathy [ 10 - 12 ] among patients receiving early blood transfusions . thus , early prehospital blood product transfusion is an appropriate intervention and improves patient outcomes in the setting of severe trauma patients with high risk of massive hemorrhage . however , criteria available to guide decisions regarding early blood product transfusions among trauma patients vary in published reports [ 12 , 13 ] . variables that were used in some studies ( such as imaging , blood gas , lactate , etc . ) may not be feasible on scene or reliably adapted by ems personnel without specific training including the use of focused assessment with sonography for trauma ( fast ) [ 13 , 14 ] . in recent years , several blood transfusion scoring systems were derived to predict the need for massive blood transfusion in trauma patients . these tools perform at different levels of sensitivity and specificity [ 15 - 19 ] . some variables such as blood pressure , heart rate , mechanisms of injury , severity of injuries , and shock index were commonly used for blood transfusion prediction [ 18 , 20 ] . other factors such as fast results , the occurrence of pelvic trauma , lactate level , and mental status were also used as valuable predictors [ 14 , 17 ] . the majority of these blood transfusion scores were derived as prerequisites for massive blood transfusion protocols which may not apply universally to patients requiring blood transfusions in general . some scores require more detailed information ( e.g. , fast results , presence of pelvic trauma , etc . ) that are not available or noted at the scene [ 14 , 15 ] . several previous studies attempted to develop a blood transfusion score to determine appropriate prehospital blood transfusion needs in severe trauma patients , but there is no validated study to use as a reference . to fill this gap , this study derived and validated a simple and pragmatic prehospital blood transfusion scoring system designed to determine blood transfusion needs in trauma patients . ems personnel can easily use this quick scoring system at the scene with limited information on hand . a retrospective review of local trauma registry for the period january 2004 through december 2013 was performed . analysis included data associated with trauma patients transported to the study center ed ( level 1 trauma center ) . this study focused on the indication of time - sensitive blood product transfusions either at the scene , en route to ed , or during early ed ( defined as the initial 4 h of ed stay ) resuscitations . patients who received any blood products before final ed dispositions were included in the experimental group while those who did not receive any blood products before final ed dispositions were included in the control group . to minimize patient selection bias , all trauma patients were included in this study regardless of mode of transportation ( e.g. , ground , helicopter , private vehicles , ambulatory , etc . ) because some patients received blood products emergently ( within 4 h upon arrival ) in the ed although they were not transported by ems . since this study enrolled all intend - to - treat trauma patients in the local registry , no patients were excluded from the study . since this study intended to identify trauma patients who may require earlier blood transfusion , patients who received any blood products during prehospital transportation or within the first 4 h of ed arrival were screened as positive for early transfusion . therefore , two groups were studied ( early blood transfusion versus non - early blood transfusion groups ) . those who did not receive any blood products , or received blood products later in their hospital stay ( after 4 h of arrival ) were included in the non - early transfusion group . early transfusions were defined as those occurring within 4 h of arrival because the majority of our severe trauma patients remained less than 4 h at ed and the primary end point of this study was final ed disposition . blood products included fresh frozen plasma , platelets , whole blood , or packed red blood cells ( prbc ) . variables not applicable during the prehospital phase were excluded from this study ( e.g. fast , pelvic trauma sustained ) . basic patient demographics ( age , sex , and race / ethnicity ) and clinical variables ( initial ems / ed vital signs , ems / ed glasgow coma scale ( gcs ) , and mechanisms of injury ) were analyzed and compared between these two groups . given that a number of patients receiving blood transfusions were not transferred by ems , excluding these patients would cause selection bias . the ems and ed vital signs were compared and an analysis determined whether initial ed vital signs could be used instead of ems vital signs . this allowed for expansion of our patient population to all severe trauma patients regardless of mode of transportation . to determine the independent risks predictive of early blood product transfusions in the study population , a multivariate logistic regression model was used . finally , a scoring system was derived and validated internally for identifying severe trauma patients in the field who qualified for early blood product transfusions . the local institutional review board ( irb ) approved this study . to identify trauma patients who qualified for early blood transfusion , retrospective derivation and validation of a blood transfusion scoring system was implemented . variables chosen for model building were reviewed by clinicians experienced in acute trauma care to ensure consistent clinical significance while protecting against over - fitting . to avoid redundant variables in the final model , spearman correlation and a regression with variance inflation ( vif ) option including all predictive variables was used . the model s discrimination was summarized using the area under the receiver operational characteristic curve ( auc ) and the model goodness of fit was measured using the hosmer and lemeshow test . accuracy of the prediction was reported with sensitivity , specificity , positive and negative likelihood ratios . finally , the sensitivity , specificity , and auc were compared between derivation versus validation data . repeated analyses were done using different randomized data to determine the stability of the scoring system . student s t - test was used to compare continuous variables , while analysis of variance with bonferroni correction was used to analyze differences among groups . cronbach s alpha ( ) coefficient was measured to determine reliability and consistency of interchanging ed vital signs with ems vital signs . in general , 0.7 was considered reliable . independent clinical variables were initially entered into a univariate logistic regression analysis followed by multivariate logistic regression analysis . all descriptive and statistical analyses were conducted using sas / stat 9.2 ( cary , nc ) and stata 12.0 ( college station , tx ) . a retrospective review of local trauma registry for the period january 2004 through december 2013 was performed . analysis included data associated with trauma patients transported to the study center ed ( level 1 trauma center ) . this study focused on the indication of time - sensitive blood product transfusions either at the scene , en route to ed , or during early ed ( defined as the initial 4 h of ed stay ) resuscitations . patients who received any blood products before final ed dispositions were included in the experimental group while those who did not receive any blood products before final ed dispositions were included in the control group . to minimize patient selection bias , all trauma patients were included in this study regardless of mode of transportation ( e.g. , ground , helicopter , private vehicles , ambulatory , etc . ) because some patients received blood products emergently ( within 4 h upon arrival ) in the ed although they were not transported by ems . since this study enrolled all intend - to - treat trauma patients in the local registry , no patients were excluded from the study . since this study intended to identify trauma patients who may require earlier blood transfusion , patients who received any blood products during prehospital transportation or within the first 4 h of ed arrival were screened as positive for early transfusion . therefore , two groups were studied ( early blood transfusion versus non - early blood transfusion groups ) . those who did not receive any blood products , or received blood products later in their hospital stay ( after 4 h of arrival ) were included in the non - early transfusion group . early transfusions were defined as those occurring within 4 h of arrival because the majority of our severe trauma patients remained less than 4 h at ed and the primary end point of this study was final ed disposition . blood products included fresh frozen plasma , platelets , whole blood , or packed red blood cells ( prbc ) . variables not applicable during the prehospital phase were excluded from this study ( e.g. fast , pelvic trauma sustained ) . basic patient demographics ( age , sex , and race / ethnicity ) and clinical variables ( initial ems / ed vital signs , ems / ed glasgow coma scale ( gcs ) , and mechanisms of injury ) were analyzed and compared between these two groups . given that a number of patients receiving blood transfusions were not transferred by ems , excluding these patients would cause selection bias . the ems and ed vital signs were compared and an analysis determined whether initial ed vital signs could be used instead of ems vital signs . this allowed for expansion of our patient population to all severe trauma patients regardless of mode of transportation . to determine the independent risks predictive of early blood product transfusions in the study population , a multivariate logistic regression model was used . finally , a scoring system was derived and validated internally for identifying severe trauma patients in the field who qualified for early blood product transfusions . to identify trauma patients who qualified for early blood transfusion , retrospective derivation and validation of a blood transfusion scoring system was implemented . variables chosen for model building were reviewed by clinicians experienced in acute trauma care to ensure consistent clinical significance while protecting against over - fitting . to avoid redundant variables in the final model , spearman correlation and a regression with variance inflation ( vif ) option including all predictive variables was used . the model s discrimination was summarized using the area under the receiver operational characteristic curve ( auc ) and the model goodness of fit was measured using the hosmer and lemeshow test . accuracy of the prediction was reported with sensitivity , specificity , positive and negative likelihood ratios . finally , the sensitivity , specificity , and auc were compared between derivation versus validation data . repeated analyses were done using different randomized data to determine the stability of the scoring system . student s t - test was used to compare continuous variables , while analysis of variance with bonferroni correction was used to analyze differences among groups . cronbach s alpha ( ) coefficient was measured to determine reliability and consistency of interchanging ed vital signs with ems vital signs . in general , 0.7 was considered reliable . independent clinical variables were initially entered into a univariate logistic regression analysis followed by multivariate logistic regression analysis . all descriptive and statistical analyses were conducted using sas / stat 9.2 ( cary , nc ) and stata 12.0 ( college station , tx ) . a total of 24,303 patients were listed in the trauma registry during the study period . a subgroup of 784 patients received blood product transfusions either during the prehospital period or within 4 h of arrival to ed resulting in a transfusion rate of 3.23% . the missing data rate ranged 0 - 9.4% among main variables and 1.8 - 11.7% among other variables ( supplementary table 1 , www.jocmr.org ) . since this study focused on the prediction of blood transfusion among trauma patients , 25 patients with unknown information on blood transfusion were excluded from the final analysis . table 1 describes the general information among patients who received blood product transfusions versus those not receiving transfusions . patients who received blood transfusion were predominately male , arrived via ambulance / helicopter , sustained more penetrating injuries , had more severe injuries , and were noted to have less stable vital signs . as compared to patients who did not receive blood products , the majority of these variables demonstrated significant differences except for age and race / ethnicity ( table 1 ) . n : number ; ed : emergency department ; sd : standard deviation ; iqr : interquartile range . in order to determine whether prehospital vital signs ( e.g. pulse , systolic blood pressure ( sbp ) , and gcs ) could be replaced by ed vital signs , cronbach s alpha ( ) coefficient was measured to determine the reliability and consistency of these exchanges . reliability coefficients ( ) measured were gcs = 0.99 , pulse = 0.70 , and sbp = 0.69 , indicating strong reliability between prehospital and ed vital signs . to derive and validate a blood transfusion needs scoring system , registry data were randomly split into two equal parts ( supplementary table 2 , www.jocmr.org ) . our findings demonstrated no significant differences between derivation and validation data , indicating no significant patient selection bias between the two datasets . when derivation data were used for identifying independent risk factors , patient age , gcs , penetrating injury , initial ed pulse , and sbp odds ratios of each risk factor ( table 2 ) were determined using either univariate or multivariate logistic regression analysis with the final model of c - statistic ( auc ) equal to 0.90 ( 95% confidence interval : 0.89 - 0.92 ) . results of the hosmer - lemeshow goodness of fit test for logistic regression confirmed that the distribution fit the data well and the risk prediction was well calibrated ( ( 8) = 11.57 , p = 0.172 ) . these five risk factors ( patient age , gcs , penetrating injury , initial ed pulse , and sbp ) were then incorporated into the blood transfusion score with an associated integer point value ( table 3 ) resulting in a total score ranging from -4 to 17 . in order to confirm the accuracy of using the study transfusion score , this score was applied back to the derivation data yielding a similar auc ( 0.87 , 95% confidence interval : 0.85 - 0.89 ) when compared to the auc determined by the logistic regression model . ci : confidence interval ; ed : emergency department ; sbp : systolic blood pressure ; gcs : glasgow coma scale . a validation dataset was used for internal validation yielding an auc of 0.86 ( 95% confidence interval : 0.84 - 0.88 ) . 1 , p > 0.05 ) . using a score > 5 as the predictive cutoff value for early blood transfusion needs , the sensitivity and specificity of the derivation data were 83% and 80% , respectively . similar results were obtained when analyzing the validation data ( supplementary table 3 , www.jocmr.org ) . moreover , if a score > 8 was considered as the predictive cutoff value for early blood transfusion needs , derivation data set analysis specificity was about 90% , whereas sensitivity dropped to near 70% . our studies suggest in general that a score of > 5 be considered for patients with potential need for early blood transfusion and a score of > 8 be considered for high risk patients . repeat analyses were performed using different randomized data in the derivation and validation sets yielding similar results indicating adequate stability of the study scoring system ( data not shown ) . receiver operating characteristic curve comparisons between deviation and validation data using blood transfusion scoring system . trauma patients with higher scores had higher correlation of receiving blood transfusions earlier in their care timelines . our study recommends strong consideration of ems initiated prehospital blood transfusion for patients with high blood transfusion needs scores . variables used in this scoring system are easy to harvest thereby facilitating a pragmatic protocol which may improve patient outcomes . the significance of this study is that the early blood transfusion needs score will provide appropriate guidance to ems personnel implementing dcr in the field . evidence from previous studies supported blood product transfusions being one of the most effective interventions during initial dcr and proving to be particularly beneficial in severe trauma patients [ 10 , 13 , 24 ] . this strategy can shorten length of stay in hospital , protect against acute traumatic coagulopathy , and reduce in - hospital and 30-day mortality [ 12 , 13 ] . early blood transfusions may subsequently decrease the total amount of blood transfusion requirements per patient . when considering poor outcomes observed among trauma patients with delayed blood transfusions during the initial resuscitation and among trauma patients receiving delayed excessive and unnecessary or futile blood transfusions [ 10 , 11 , 25 , 26 ] , it is apparent that a strategy that balances the timely delivery of blood products produces improved patient outcomes . given limited on scene information , the intricate challenge for ems personnel and physicians is to determine which specific patients require early blood transfusions in the prehospital setting . in general , time sensitive decisions are best supported by simple , easy - to - use tools as compared to those requiring sophisticated calculations . in this study , we show unequivocally that all variables required for blood transfusion needs score calculations can be obtained within a few minutes in the field . the variables used in the calculation of blood transfusion needs scores in this study have been reported previously in the literature [ 15 , 17 - 19 ] . among these variables , sbp and heart rate ( hr ) are two variables commonly used to determine the need for blood transfusion in the literature [ 12 , 28 ] . isolated hypotension was found to be less reliable unless combined with additional variables ( such as age , penetrating injury , hr , etc . ) . some studies reported hr can be masked by medication effects especially among geriatric patients with chronic medical conditions [ 30 , 31 ] . of interest a recent report on geriatric trauma patients with pre - injury cardiac medications ( e.g. beta - blocker ) in some studies , shock index ( e.g. hr / sbp ) was determined to be more accurate and a better predictor of early blood transfusion needs [ 33 - 35 ] . results from an analysis of the national trauma data bank also showed a combination of si and age is a better predictor of life threatening shock in older trauma patients . generally speaking , gcs is less reliable in determining the need for blood transfusion on its own , but has clinical value when considered along with other variables . taken together , it is ideal to derive a tool with a combination of all available information while also addressing the individual weight of each variable to increase the probability of predicting time - sensitive interventions . in this study however , concern regarding outcomes of patients receiving blood transfusions based on pseudo negative / positive score results can not be ruled out . a few studies reported unnecessary blood transfusions with no significant harmful outcomes [ 39 , 40 ] . if indicated , emergency physicians may decide to stop the prehospital initiated blood transfusion upon patient arrival or when further detailed information is obtained ( e.g. , imaging studies ) . future research is needed to further delineate the risks of initiating limited unnecessary blood transfusions . this is a retrospective study with limited information , missing data and potential for patient population selection bias . however , the overall missing data rate among the major variables was less than 10% ( supplementary table 1 , www.jocmr.org ) . data imputed in this study posed less significant bias with respect to incomplete data ( supplementary table 1 , www.jocmr.org ) . we were not able to include all possible variables that may predict blood transfusions needs . the simplest variables that were easily approachable were used , therefore , vital signs , age , and mental status were chosen . ed vital signs as opposed to vital signs at the scene were used for blood transfusion needs prediction which may be a source of bias . this was done to include patients who were not transported by ems but still required early blood transfusion during ed resuscitation . our ems vital signs correlated well with the ed vital signs within the same categorical range due to limited ems transportation time in this study ( usually less than 10 - 15 min ) . the use of initial ed vital signs is acceptable although future studies with external validation of a larger sample size are recommended . an early blood transfusion needs scoring system was derived and internally validated to predict severe trauma patients requiring blood transfusion during prehospital or initial ed resuscitation . a score > 5 indicated risk of receiving early blood transfusion with about 80% sensitivity and specificity in both the derivation and validation cohorts . this is a retrospective study with limited information , missing data and potential for patient population selection bias . however , the overall missing data rate among the major variables was less than 10% ( supplementary table 1 , www.jocmr.org ) . data imputed in this study posed less significant bias with respect to incomplete data ( supplementary table 1 , www.jocmr.org ) . we were not able to include all possible variables that may predict blood transfusions needs . the simplest variables that were easily approachable were used , therefore , vital signs , age , and mental status were chosen . ed vital signs as opposed to vital signs at the scene were used for blood transfusion needs prediction which may be a source of bias . this was done to include patients who were not transported by ems but still required early blood transfusion during ed resuscitation . our ems vital signs correlated well with the ed vital signs within the same categorical range due to limited ems transportation time in this study ( usually less than 10 - 15 min ) . the use of initial ed vital signs is acceptable although future studies with external validation of a larger sample size are recommended . an early blood transfusion needs scoring system was derived and internally validated to predict severe trauma patients requiring blood transfusion during prehospital or initial ed resuscitation . a score > 5 indicated risk of receiving early blood transfusion with about 80% sensitivity and specificity in both the derivation and validation cohorts . hw , rdr , and cds conceived the study and developed the design in consultation with all of the authors . hw , rdr , jl , sb , as , and nrz drafted the articles .

backgroundthere is no existing adequate blood transfusion needs determination tool that emergency medical services ( ems ) personnel can use for prehospital blood transfusion initiation . in this study , a simple and pragmatic prehospital blood transfusion needs scoring system was derived and validated.methodslocal trauma registry data were reviewed retrospectively from 2004 through 2013 . patients were randomly assigned to derivation and validation cohorts . multivariate logistic regression was used to identify the independent approachable risks associated with early blood transfusion needs in the derivation cohort in which a scoring system was derived . sensitivity , specificity , and area under the receiver operational characteristic ( auc ) were calculated and compared using both the derivation and validation data.resultsa total of 24,303 patients were included with 12,151 patients in the derivation and 12,152 patients in the validation cohorts . age , penetrating injury , heart rate , systolic blood pressure , and glasgow coma scale ( gcs ) were risks predictive of early blood transfusion needs . an early blood transfusion needs score was derived . a score > 5 indicated risk of early blood transfusion need with a sensitivity of 83% and a specificity of 80% . a sensitivity of 82% and a specificity of 80% were also found in the validation study and their auc showed no statistically significant difference ( auc of the derivation = 0.87 versus auc of the validation = 0.86 , p > 0.05).conclusionsan early blood transfusion scoring system was derived and internally validated to predict severe trauma patients requiring blood transfusion during prehospital or initial emergency department resuscitation .

Introduction Methods Participant selection Inclusion criteria Exclusion criteria Study design and protocol Deriving and validating a scoring system for early blood transfusion Other statistical analyses Results Discussion Limitation Conclusion Conflicts of Interest Author Contributions

trauma is one of the most common causes of hospital admission and in - hospital mortality [ 1 - 3 ] . recently , the mortality rate in severe trauma patients has decreased [ 4 - 6 ] due to emphasis on damage control resuscitation ( dcr ) . blood product transfusion plays an important role in early dcr and improved survival [ 7 , 8 ] . in a randomized trial , del junco et al ( 2013 ) reported that early blood product transfusions during prehospital transportation improved patient outcomes including improved patient acid - base status , decreased blood product usage , and reduced early in - hospital mortality . other observational studies showed similar benefits among patients who received early blood transfusions either during prehospital or initial emergency department ( ed ) resuscitation phases [ 10 - 12 ] . these studies documented improved oxygen carrying capacity , more efficient intravascular volume expansion , and reduced risk of early trauma induced coagulopathy [ 10 - 12 ] among patients receiving early blood transfusions . thus , early prehospital blood product transfusion is an appropriate intervention and improves patient outcomes in the setting of severe trauma patients with high risk of massive hemorrhage . however , criteria available to guide decisions regarding early blood product transfusions among trauma patients vary in published reports [ 12 , 13 ] . variables that were used in some studies ( such as imaging , blood gas , lactate , etc . ) may not be feasible on scene or reliably adapted by ems personnel without specific training including the use of focused assessment with sonography for trauma ( fast ) [ 13 , 14 ] . in recent years , several blood transfusion scoring systems were derived to predict the need for massive blood transfusion in trauma patients . some variables such as blood pressure , heart rate , mechanisms of injury , severity of injuries , and shock index were commonly used for blood transfusion prediction [ 18 , 20 ] . other factors such as fast results , the occurrence of pelvic trauma , lactate level , and mental status were also used as valuable predictors [ 14 , 17 ] . the majority of these blood transfusion scores were derived as prerequisites for massive blood transfusion protocols which may not apply universally to patients requiring blood transfusions in general . , fast results , presence of pelvic trauma , etc . ) several previous studies attempted to develop a blood transfusion score to determine appropriate prehospital blood transfusion needs in severe trauma patients , but there is no validated study to use as a reference . to fill this gap , this study derived and validated a simple and pragmatic prehospital blood transfusion scoring system designed to determine blood transfusion needs in trauma patients . ems personnel can easily use this quick scoring system at the scene with limited information on hand . a retrospective review of local trauma registry for the period january 2004 through december 2013 was performed . analysis included data associated with trauma patients transported to the study center ed ( level 1 trauma center ) . this study focused on the indication of time - sensitive blood product transfusions either at the scene , en route to ed , or during early ed ( defined as the initial 4 h of ed stay ) resuscitations . patients who received any blood products before final ed dispositions were included in the experimental group while those who did not receive any blood products before final ed dispositions were included in the control group . to minimize patient selection bias , all trauma patients were included in this study regardless of mode of transportation ( e.g. because some patients received blood products emergently ( within 4 h upon arrival ) in the ed although they were not transported by ems . since this study enrolled all intend - to - treat trauma patients in the local registry , no patients were excluded from the study . since this study intended to identify trauma patients who may require earlier blood transfusion , patients who received any blood products during prehospital transportation or within the first 4 h of ed arrival were screened as positive for early transfusion . therefore , two groups were studied ( early blood transfusion versus non - early blood transfusion groups ) . those who did not receive any blood products , or received blood products later in their hospital stay ( after 4 h of arrival ) were included in the non - early transfusion group . early transfusions were defined as those occurring within 4 h of arrival because the majority of our severe trauma patients remained less than 4 h at ed and the primary end point of this study was final ed disposition . variables not applicable during the prehospital phase were excluded from this study ( e.g. basic patient demographics ( age , sex , and race / ethnicity ) and clinical variables ( initial ems / ed vital signs , ems / ed glasgow coma scale ( gcs ) , and mechanisms of injury ) were analyzed and compared between these two groups . this allowed for expansion of our patient population to all severe trauma patients regardless of mode of transportation . to determine the independent risks predictive of early blood product transfusions in the study population , a multivariate logistic regression model was used . finally , a scoring system was derived and validated internally for identifying severe trauma patients in the field who qualified for early blood product transfusions . the local institutional review board ( irb ) approved this study . to identify trauma patients who qualified for early blood transfusion , retrospective derivation and validation of a blood transfusion scoring system was implemented . variables chosen for model building were reviewed by clinicians experienced in acute trauma care to ensure consistent clinical significance while protecting against over - fitting . to avoid redundant variables in the final model , spearman correlation and a regression with variance inflation ( vif ) option including all predictive variables was used . the model s discrimination was summarized using the area under the receiver operational characteristic curve ( auc ) and the model goodness of fit was measured using the hosmer and lemeshow test . accuracy of the prediction was reported with sensitivity , specificity , positive and negative likelihood ratios . finally , the sensitivity , specificity , and auc were compared between derivation versus validation data . repeated analyses were done using different randomized data to determine the stability of the scoring system . student s t - test was used to compare continuous variables , while analysis of variance with bonferroni correction was used to analyze differences among groups . independent clinical variables were initially entered into a univariate logistic regression analysis followed by multivariate logistic regression analysis . all descriptive and statistical analyses were conducted using sas / stat 9.2 ( cary , nc ) and stata 12.0 ( college station , tx ) . a retrospective review of local trauma registry for the period january 2004 through december 2013 was performed . analysis included data associated with trauma patients transported to the study center ed ( level 1 trauma center ) . this study focused on the indication of time - sensitive blood product transfusions either at the scene , en route to ed , or during early ed ( defined as the initial 4 h of ed stay ) resuscitations . patients who received any blood products before final ed dispositions were included in the experimental group while those who did not receive any blood products before final ed dispositions were included in the control group . to minimize patient selection bias , all trauma patients were included in this study regardless of mode of transportation ( e.g. because some patients received blood products emergently ( within 4 h upon arrival ) in the ed although they were not transported by ems . since this study enrolled all intend - to - treat trauma patients in the local registry , no patients were excluded from the study . since this study intended to identify trauma patients who may require earlier blood transfusion , patients who received any blood products during prehospital transportation or within the first 4 h of ed arrival were screened as positive for early transfusion . therefore , two groups were studied ( early blood transfusion versus non - early blood transfusion groups ) . those who did not receive any blood products , or received blood products later in their hospital stay ( after 4 h of arrival ) were included in the non - early transfusion group . early transfusions were defined as those occurring within 4 h of arrival because the majority of our severe trauma patients remained less than 4 h at ed and the primary end point of this study was final ed disposition . blood products included fresh frozen plasma , platelets , whole blood , or packed red blood cells ( prbc ) . variables not applicable during the prehospital phase were excluded from this study ( e.g. basic patient demographics ( age , sex , and race / ethnicity ) and clinical variables ( initial ems / ed vital signs , ems / ed glasgow coma scale ( gcs ) , and mechanisms of injury ) were analyzed and compared between these two groups . given that a number of patients receiving blood transfusions were not transferred by ems , excluding these patients would cause selection bias . the ems and ed vital signs were compared and an analysis determined whether initial ed vital signs could be used instead of ems vital signs . this allowed for expansion of our patient population to all severe trauma patients regardless of mode of transportation . to determine the independent risks predictive of early blood product transfusions in the study population , a multivariate logistic regression model was used . finally , a scoring system was derived and validated internally for identifying severe trauma patients in the field who qualified for early blood product transfusions . to identify trauma patients who qualified for early blood transfusion , retrospective derivation and validation of a blood transfusion scoring system was implemented . variables chosen for model building were reviewed by clinicians experienced in acute trauma care to ensure consistent clinical significance while protecting against over - fitting . to avoid redundant variables in the final model , spearman correlation and a regression with variance inflation ( vif ) option including all predictive variables was used . the model s discrimination was summarized using the area under the receiver operational characteristic curve ( auc ) and the model goodness of fit was measured using the hosmer and lemeshow test . accuracy of the prediction was reported with sensitivity , specificity , positive and negative likelihood ratios . finally , the sensitivity , specificity , and auc were compared between derivation versus validation data . repeated analyses were done using different randomized data to determine the stability of the scoring system . student s t - test was used to compare continuous variables , while analysis of variance with bonferroni correction was used to analyze differences among groups . independent clinical variables were initially entered into a univariate logistic regression analysis followed by multivariate logistic regression analysis . a total of 24,303 patients were listed in the trauma registry during the study period . since this study focused on the prediction of blood transfusion among trauma patients , 25 patients with unknown information on blood transfusion were excluded from the final analysis . patients who received blood transfusion were predominately male , arrived via ambulance / helicopter , sustained more penetrating injuries , had more severe injuries , and were noted to have less stable vital signs . as compared to patients who did not receive blood products , the majority of these variables demonstrated significant differences except for age and race / ethnicity ( table 1 ) . n : number ; ed : emergency department ; sd : standard deviation ; iqr : interquartile range . pulse , systolic blood pressure ( sbp ) , and gcs ) could be replaced by ed vital signs , cronbach s alpha ( ) coefficient was measured to determine the reliability and consistency of these exchanges . reliability coefficients ( ) measured were gcs = 0.99 , pulse = 0.70 , and sbp = 0.69 , indicating strong reliability between prehospital and ed vital signs . to derive and validate a blood transfusion needs scoring system , registry data were randomly split into two equal parts ( supplementary table 2 , www.jocmr.org ) . our findings demonstrated no significant differences between derivation and validation data , indicating no significant patient selection bias between the two datasets . when derivation data were used for identifying independent risk factors , patient age , gcs , penetrating injury , initial ed pulse , and sbp odds ratios of each risk factor ( table 2 ) were determined using either univariate or multivariate logistic regression analysis with the final model of c - statistic ( auc ) equal to 0.90 ( 95% confidence interval : 0.89 - 0.92 ) . results of the hosmer - lemeshow goodness of fit test for logistic regression confirmed that the distribution fit the data well and the risk prediction was well calibrated ( ( 8) = 11.57 , p = 0.172 ) . these five risk factors ( patient age , gcs , penetrating injury , initial ed pulse , and sbp ) were then incorporated into the blood transfusion score with an associated integer point value ( table 3 ) resulting in a total score ranging from -4 to 17 . in order to confirm the accuracy of using the study transfusion score , this score was applied back to the derivation data yielding a similar auc ( 0.87 , 95% confidence interval : 0.85 - 0.89 ) when compared to the auc determined by the logistic regression model . ci : confidence interval ; ed : emergency department ; sbp : systolic blood pressure ; gcs : glasgow coma scale . a validation dataset was used for internal validation yielding an auc of 0.86 ( 95% confidence interval : 0.84 - 0.88 ) . 1 , p > 0.05 ) . using a score > 5 as the predictive cutoff value for early blood transfusion needs , the sensitivity and specificity of the derivation data were 83% and 80% , respectively . similar results were obtained when analyzing the validation data ( supplementary table 3 , www.jocmr.org ) . moreover , if a score > 8 was considered as the predictive cutoff value for early blood transfusion needs , derivation data set analysis specificity was about 90% , whereas sensitivity dropped to near 70% . our studies suggest in general that a score of > 5 be considered for patients with potential need for early blood transfusion and a score of > 8 be considered for high risk patients . repeat analyses were performed using different randomized data in the derivation and validation sets yielding similar results indicating adequate stability of the study scoring system ( data not shown ) . receiver operating characteristic curve comparisons between deviation and validation data using blood transfusion scoring system . trauma patients with higher scores had higher correlation of receiving blood transfusions earlier in their care timelines . our study recommends strong consideration of ems initiated prehospital blood transfusion for patients with high blood transfusion needs scores . variables used in this scoring system are easy to harvest thereby facilitating a pragmatic protocol which may improve patient outcomes . the significance of this study is that the early blood transfusion needs score will provide appropriate guidance to ems personnel implementing dcr in the field . evidence from previous studies supported blood product transfusions being one of the most effective interventions during initial dcr and proving to be particularly beneficial in severe trauma patients [ 10 , 13 , 24 ] . this strategy can shorten length of stay in hospital , protect against acute traumatic coagulopathy , and reduce in - hospital and 30-day mortality [ 12 , 13 ] . early blood transfusions may subsequently decrease the total amount of blood transfusion requirements per patient . when considering poor outcomes observed among trauma patients with delayed blood transfusions during the initial resuscitation and among trauma patients receiving delayed excessive and unnecessary or futile blood transfusions [ 10 , 11 , 25 , 26 ] , it is apparent that a strategy that balances the timely delivery of blood products produces improved patient outcomes . given limited on scene information , the intricate challenge for ems personnel and physicians is to determine which specific patients require early blood transfusions in the prehospital setting . in general , time sensitive decisions are best supported by simple , easy - to - use tools as compared to those requiring sophisticated calculations . in this study , we show unequivocally that all variables required for blood transfusion needs score calculations can be obtained within a few minutes in the field . the variables used in the calculation of blood transfusion needs scores in this study have been reported previously in the literature [ 15 , 17 - 19 ] . among these variables , sbp and heart rate ( hr ) are two variables commonly used to determine the need for blood transfusion in the literature [ 12 , 28 ] . isolated hypotension was found to be less reliable unless combined with additional variables ( such as age , penetrating injury , hr , etc . ) . of interest a recent report on geriatric trauma patients with pre - injury cardiac medications ( e.g. hr / sbp ) was determined to be more accurate and a better predictor of early blood transfusion needs [ 33 - 35 ] . results from an analysis of the national trauma data bank also showed a combination of si and age is a better predictor of life threatening shock in older trauma patients . generally speaking , gcs is less reliable in determining the need for blood transfusion on its own , but has clinical value when considered along with other variables . taken together , it is ideal to derive a tool with a combination of all available information while also addressing the individual weight of each variable to increase the probability of predicting time - sensitive interventions . in this study however , concern regarding outcomes of patients receiving blood transfusions based on pseudo negative / positive score results can not be ruled out . a few studies reported unnecessary blood transfusions with no significant harmful outcomes [ 39 , 40 ] . if indicated , emergency physicians may decide to stop the prehospital initiated blood transfusion upon patient arrival or when further detailed information is obtained ( e.g. , imaging studies ) . this is a retrospective study with limited information , missing data and potential for patient population selection bias . however , the overall missing data rate among the major variables was less than 10% ( supplementary table 1 , www.jocmr.org ) . data imputed in this study posed less significant bias with respect to incomplete data ( supplementary table 1 , www.jocmr.org ) . the simplest variables that were easily approachable were used , therefore , vital signs , age , and mental status were chosen . ed vital signs as opposed to vital signs at the scene were used for blood transfusion needs prediction which may be a source of bias . this was done to include patients who were not transported by ems but still required early blood transfusion during ed resuscitation . our ems vital signs correlated well with the ed vital signs within the same categorical range due to limited ems transportation time in this study ( usually less than 10 - 15 min ) . an early blood transfusion needs scoring system was derived and internally validated to predict severe trauma patients requiring blood transfusion during prehospital or initial ed resuscitation . a score > 5 indicated risk of receiving early blood transfusion with about 80% sensitivity and specificity in both the derivation and validation cohorts . this is a retrospective study with limited information , missing data and potential for patient population selection bias . data imputed in this study posed less significant bias with respect to incomplete data ( supplementary table 1 , www.jocmr.org ) . we were not able to include all possible variables that may predict blood transfusions needs . the simplest variables that were easily approachable were used , therefore , vital signs , age , and mental status were chosen . ed vital signs as opposed to vital signs at the scene were used for blood transfusion needs prediction which may be a source of bias . this was done to include patients who were not transported by ems but still required early blood transfusion during ed resuscitation . our ems vital signs correlated well with the ed vital signs within the same categorical range due to limited ems transportation time in this study ( usually less than 10 - 15 min ) . an early blood transfusion needs scoring system was derived and internally validated to predict severe trauma patients requiring blood transfusion during prehospital or initial ed resuscitation . a score > 5 indicated risk of receiving early blood transfusion with about 80% sensitivity and specificity in both the derivation and validation cohorts . hw , rdr , and cds conceived the study and developed the design in consultation with all of the authors . hw , rdr , jl , sb , as , and nrz drafted the articles .

they have a lifetime incidence of 70% to 80% , and approximately 25% of women report being symptomatic . widespread use of hormonal contraception may minimize symptomatology and may skew the reported incidence downward . although hormone use results in a temporary reduction in symptoms , it is not a permanent treatment for fibroids . ultrasound studies have shown that myomas begin to develop in female patients at a young age and increase in both size and number until women reach menopause . studies have shown ethnic differences in the incidence of leiomyomas , with african - american women having a disproportionately higher occurrence than other races . the fibroid growth study included data showing decreasing myoma growth rates among white women as they approached menopause ; however , among african - american women , growth rates remained unchanged . symptoms , including heavy menstrual bleeding , dysmenorrhea , pelvic - abdominal pain or pressure , urinary frequency and incontinence , dyspareunia , infertility , and an increased risk of spontaneous abortion , occur in 20% to 50% of patients with fibroids . symptomatic uterine fibroids represent the most common indication for hysterectomy , accounting for 150 000 to 200 000 of all of the hysterectomy procedures , or 30% to 40% of cases , that are performed annually in the united states . we present the following report of phase ii and phase iii studies of laparoscopic ultrasound - guided radiofrequency volumetric thermal ablation ( rfvta ) of leiomyomas in an effort to address the dearth of new clinical research in the united states on minimally invasive approaches to symptomatic fibroid therapy and to provide descriptions of what we believe to be an emerging , preferred uterine - sparing fibroid treatment modality . both percutaneous and laparoscopic radiofrequency ( rf ) no long - term medical therapy exists for symptomatic fibroids ; consequently , treatment has been and remains largely surgical . in suitable candidates , myomectomy may be performed . myomectomy , though allowing for uterine preservation , does require one or more myometrial incisions ; can require a postprocedural inpatient hospitalization similar to that of hysterectomy ; and may be associated with increased blood loss , longer procedure times , and increased risk for postoperative hemorrhage and/or pelvic adhesions . interventional radiologists have achieved symptom improvement and fibroid tumor shrinkage through embolization of the uterine arteries . laparoscopic myolysis with either bipolar needles or a neodymium : yttrium - aluminum - garnet laser was performed in europe in the late 1980s and was subsequently reported in the united states by goldfarb . the procedure initially targeted the entire myoma by using multiple punctures through the uterine serosa with resultant and significant formation of postoperative adhesions . lee performed rfvta of fibroids in the 1990s and early 2000s under an institutional review board approved off - label study using an rfvta system designed for soft - tissue ablation . this procedure involved the insertion of an rf needle with 7 deployable curved electrodes that created a volumetric ablation within the myoma . lee subsequently developed a system designed specifically to treat uterine fibroids using rfvta ( acessa system ; halt medical , brentwood , california ) . this system was tested in multiple studies enrolling a total of 206 women with symptomatic uterine fibroids . the primary objectives of the phase ii studies , which were conducted under similar but separate protocols , were to assess the safety and efficacy of the acessa system in women with symptomatic fibroids 6 cm and to evaluate the treatment 's effect on uterine volume . study endpoints were the incidence of procedure- and device - related adverse events , change in uterine fibroid symptom and quality - of - life ( ufs - qol ) questionnaire responses over time , and change in uterine volume . the primary objectives of the phase iii study , which was conducted under 1 protocol and included 9 sites in the united states and the 2 latin american sites , from the phase ii study were to confirm the safety and efficacy of the acessa system for the treatment of symptomatic uterine fibroids 7 cm in diameter in patients with confirmed clinical menorrhagia ( indicated by menstrual blood loss of 160 ml to 500 ml ) based on alkaline hematin testing . study primary endpoints at 12 months were safety , surgical reintervention rates , and change in menstrual blood loss . secondary endpoints included changes in uterine and fibroid volumes , evaluation of patients ' ufs - qol and general health status questionnaire responses over time , and patients ' satisfaction after treatment . the acessa system has been approved for commercial use in the united states , mexico , canada , and the european union for the treatment of symptomatic uterine fibroids . insufficient data exist to evaluate the safety and effectiveness of the acessa procedure in women who plan future pregnancy . the phase ii trial was a prospective , dual - center , longitudinal , open - label , single - arm feasibility study assessing the safety and efficacy of rfvta of symptomatic myomas in women seeking alternative treatment to hysterectomy . the setting was two urban university hospitals : one in monterrey , mexico , and the other in guatemala city , guatemala . participation in the trial was voluntary , the local ethics committee and ministry of health of each country approved the recruitment of the study participants , and the principal investigators or designee obtained written informed consent from each enrolled patient according to good clinical practice guidelines , local and federal regulations of mexico and guatemala , and international standard en iso 141551:2003 ( clinical investigation of medical devices for human subjects ) . the phase iii trial , which is ongoing , is a food and drug administration and institutional review board approved , prospective , multicenter , longitudinal , pivotal study in which the patients serve as their own controls . all enrolled study participants were diagnosed with symptomatic fibroids , specifically menorrhagia confirmed by alkaline hematin testing . eleven urban study centers in the united states ( n = 9 ) and in latin america ( n = 2 ) provided the clinical setting . as with the phase ii trial , participation by the enrollees was voluntary and written informed consent was obtained from each patient according to good clinical practice guidelines ; federal regulations established by the us food and drug administration , mexico , and guatemala ; and international standard en iso 141551:2003 ( clinical investigation of medical devices for human subjects ) . patients in both phases have been followed up for a minimum of 1 year after rf ablation of their symptomatic fibroids . at each of the 3- , 6- , and 12-month follow - up visits , the patients were evaluated clinically to assess symptoms and to identify any adverse events ; at baseline and at each follow - up visit , they were also asked to complete a ufs - qol questionnaire . ( the questionnaire 's mean transformed scores range from 0 to 100 . a decrease in symptom score over time from baseline indicates improvement , whereas a higher score for health - related quality of life after the procedure indicates improvement ) . before the intervention , baseline medical data were collected and recorded , including a complete health history , a pregnancy test within 24 hours of surgery , a complete blood panel , and completion of the ufs - qol questionnaire . for the phase ii trial , transvaginal ultrasound was performed for an initial determination of the location , number , and size of fibroid(s ) and to measure the uterine volume . for the phase iii trial , magnetic resonance imaging was incorporated in the preoperative assessment along with transvaginal ultrasound . the device ( acessa system ) is composed of an rf generator and a disposable rf handpiece with deployable needle electrodes , two dispersive electrode pads , extension cables , and a foot pedal ( figure 1 ) . the two dispersive return pads are designed specifically to minimize the risk of surface skin burns at these points of current return from the patient to the generator . during surgery , laparoscopic ultrasound and video observation locate each target fibroid . the rf handpiece is inserted percutaneously and is introduced into the fibroid through the uterine serosal surface . the surgeon then may choose to deploy the 7-needle electrode array to the desired ablation size within the fibroid capsule ( figures 2 and 3 ) . rf ablation is carried out with a continuous , low - voltage , high - frequency ( 45500 khz ) alternating current with a maximum output of 200 w. once the tissue temperature reaches 100c , the wattage automatically adjusts to maintain treatment temperature and deliver rf energy to tissue through the handheld disposable rf handpiece . energy propagation is concentrated in the pelvic region only ; thus there is no safety risk to the patient in terms of high levels of current being conducted across the heart , which would otherwise be a concern . the maximum level of current conduction is approximately 2.5 amps , which only occurs under conditions of highest power ( 200 w ) and lowest resistance / impedance ( 3040 in the case of rfvta procedures ) . the typical current level at 60 w and 60 is 1 amp and is representative of most acessa rfvta cases . schema of acessa system : rf generator with foot pedal , extension cables , rf handpiece , and dispersive pads . ultrasound image of rf handpiece tip advancing from left side and located inside fibroid capsule , before deployment of array . the surgeon makes selections from a menu on the generator that is controlled through a set of scroll and select buttons on the rf handpiece . the surgeon initiates treatment by depressing the foot pedal , and the rf generator increases the tissue temperature to 100c and maintains that temperature for the duration of the ablation . the total time of treatment for each fibroid is determined by a treatment algorithm , which is based on the volume of tissue to be ablated . the generator is specifically designed for use with this rf device and has real - time temperature or power displays ( depending on the mode being used ) that facilitate monitoring and control of the ablation throughout the procedure . the generator also monitors the return temperature from each dispersive electrode pad and automatically stops rf treatment if any of the dispersive pad thermocouples register a skin temperature > 40c . multiple uterine fibroids can be treated during the procedure and often by use of only one insertion through the uterine serosa . the technology and the laparoscopic ultrasound mapping allow the surgeon to target only the fibroids and to spare the surrounding myometrium , with the exception of a 3.4-mm puncture followed by coagulation of the tract during probe withdrawal . because there is no requirement for uterine incisions , after fibroid ablation , the surgeon withdraws the electrode array into the 3.4-mm primary needle and removes the handpiece while simultaneously performing standard needle - track rf coagulation by applying a lower wattage ( 815 w ) of intermittent coagulation mode to the needle tip ( figure 4 ) . on occasion , depending on the fibroid 's shape and size , multiple ablations may be required for a single fibroid . fibroids can be treated virtually in any portion of the uterine myometrium ( anterior , posterior , or lateral ) , including those that are subserosal , intramural , transmural , or submucosal . pedunculated ( type 0 ) intracavitary fibroids usually are not treated by this method because hysteroscopic resection is the preferred therapy for these myomas . the device ( acessa system ) is composed of an rf generator and a disposable rf handpiece with deployable needle electrodes , two dispersive electrode pads , extension cables , and a foot pedal ( figure 1 ) . the two dispersive return pads are designed specifically to minimize the risk of surface skin burns at these points of current return from the patient to the generator . during surgery , laparoscopic ultrasound and video observation locate each target fibroid . the rf handpiece is inserted percutaneously and is introduced into the fibroid through the uterine serosal surface . the surgeon then may choose to deploy the 7-needle electrode array to the desired ablation size within the fibroid capsule ( figures 2 and 3 ) . rf ablation is carried out with a continuous , low - voltage , high - frequency ( 45500 khz ) alternating current with a maximum output of 200 w. once the tissue temperature reaches 100c , the wattage automatically adjusts to maintain treatment temperature and deliver rf energy to tissue through the handheld disposable rf handpiece . energy propagation is concentrated in the pelvic region only ; thus there is no safety risk to the patient in terms of high levels of current being conducted across the heart , which would otherwise be a concern . the maximum level of current conduction is approximately 2.5 amps , which only occurs under conditions of highest power ( 200 w ) and lowest resistance / impedance ( 3040 in the case of rfvta procedures ) . the typical current level at 60 w and 60 is 1 amp and is representative of most acessa rfvta cases . schema of acessa system : rf generator with foot pedal , extension cables , rf handpiece , and dispersive pads . rf handpiece tip showing full deployment of 7-needle array and effective ablation zone . ultrasound image of rf handpiece tip advancing from left side and located inside fibroid capsule , before deployment of array . the surgeon makes selections from a menu on the generator that is controlled through a set of scroll and select buttons on the rf handpiece . the surgeon initiates treatment by depressing the foot pedal , and the rf generator increases the tissue temperature to 100c and maintains that temperature for the duration of the ablation . the total time of treatment for each fibroid is determined by a treatment algorithm , which is based on the volume of tissue to be ablated . the generator is specifically designed for use with this rf device and has real - time temperature or power displays ( depending on the mode being used ) that facilitate monitoring and control of the ablation throughout the procedure . the generator also monitors the return temperature from each dispersive electrode pad and automatically stops rf treatment if any of the dispersive pad thermocouples register a skin temperature > 40c . multiple uterine fibroids can be treated during the procedure and often by use of only one insertion through the uterine serosa . the technology and the laparoscopic ultrasound mapping allow the surgeon to target only the fibroids and to spare the surrounding myometrium , with the exception of a 3.4-mm puncture followed by coagulation of the tract during probe withdrawal . because there is no requirement for uterine incisions , after fibroid ablation , the surgeon withdraws the electrode array into the 3.4-mm primary needle and removes the handpiece while simultaneously performing standard needle - track rf coagulation by applying a lower wattage ( 815 w ) of intermittent coagulation mode to the needle tip ( figure 4 ) . on occasion , depending on the fibroid 's shape and size , multiple ablations may be required for a single fibroid . fibroids can be treated virtually in any portion of the uterine myometrium ( anterior , posterior , or lateral ) , including those that are subserosal , intramural , transmural , or submucosal . pedunculated ( type 0 ) intracavitary fibroids usually are not treated by this method because hysteroscopic resection is the preferred therapy for these myomas . patient demographic data and myoma symptom characteristics at baseline for both the phase ii and phase iii trials are presented in table 1 . phase ii participants were all of hispanic or latino origin ( 100% ) , whereas 55.1% of phase iii participants reported not being hispanic or latino . after intervention in the phase ii study , 69 patients ( 100% ) were seen at the 3-month follow - up visit , 65 ( 94.2% ) at the 6-month follow - up visit , and 58 ( 84.1% ) at the 12-month visit . the flow of patients through the phase iii study has been similar through 12 months : 137 patients ( 100% ) were treated at baseline , 134 ( 97.8% ) have been followed up through 3 months , 134 ( 97.8% ) have been followed up through 6 months , and 132 ( 96.4% ) have been followed up through 12 months . the phase iii study is ongoing . phase ii and phase iii patient demographics and myoma symptom characteristics at baseline the baseline population for phase iii does not include two patients who , after rfvta , did not meet all inclusion criteria . one patient in the phase ii trial conceived after ablation , delivered a healthy , full - term infant by elective repeat cesarean section , and was excluded from further participation in the study . three patients in the phase iii trial conceived after ablation and were excluded from further participation in the study . one patient delivered a full - term , healthy infant by cesarean section ; one woman delivered a full - term , healthy infant vaginally ; and one patient miscarried at 10 weeks . a total of 128 myomas were visualized in the 69 patients by transvaginal ultrasound at baseline , and 285 myomas were mapped and treated by rf ablation in phase ii ( median , 3 ; range , 120 ) . during intraoperative laparoscopic ultrasound mapping during phase iii , 818 myomas were visualized and 640 were treated ( median , 4 ; range , 129 ) . the distribution of imaged leiomyomas by type and study phase is presented in table 2 . in phase ii surgeons treated a median of 3 fibroids per patient ( range , 120 ) in 2.33 hours ( range , 0.704.83 hours ) ( timed from first incision to close of last incision ) . phase iii surgeons treated a median of 4 fibroids per patient ( range , 129 ) in 1.88 hours ( range , 0.355.55 hours ) ( timed from initiation of pneumoperitoneum to close of last incision ) . distribution of myomas by type and by study for phase ii and phase iii as found on laparoscopic ultrasound a myoma could be of more than one type . a comparison of phase ii and phase iii uterine volume measurements ( by transvaginal ultrasound ) from baseline to 12 months after the procedure is provided in figure 5 . the mean uterine volume decreased over time from baseline through 12 months for the phase ii patients : the mean volume reduction at 3 months ( n = 68 ) , 6 months ( n = 64 ) , and 12 months ( n = 58 ) was 20.3% , 25.2% , and 28.7% , respectively . similarly , the mean uterine volume for the phase iii patients decreased from baseline ( n = 134 ) by 14.6% at the 3-month follow - up visit ( n = 129 ) , by 20.6% at the 6-month follow - up visit ( n = 128 ) , and by 25.7% at the 12-month follow - up visit ( n = 127 ) . uterine volume measurements by transvaginal ultrasound over time from baseline to 12 months after procedure for phases ii and iii . improvements in phase ii and phase iii ufs - qol mean transformed symptom severity and health - related quality - of - life scores and subscale scores over time are presented in table 3 . mean ufs - qol transformed scores and health - related quality - of - life subscale scores for phase ii and phase iii studies scores range from 0 to 100 . a lower symptom severity score indicates a decrease in symptoms , and a higher health - related quality of life score indicates an improvement in health - related quality of life . ten patients were removed from the ufs - qol analysis because of pregnancy , amenorrhea / menopause , loss to follow - up , or baseline parameters outside the inclusion criteria . symptom severity decreased from baseline to 12 months in phase ii by 83.7% ( 53.9 to 8.8 , n = 58 ) ; health - related quality of life improved from baseline to 12 months by 89.8% ( 48.5 to 92.0 , n = 58 ) . in the phase iii study , symptom severity decreased by 56.5% from the mean transformed score of 61.1 at baseline ( n = 127 ) to 26.6 at 12 months ( n = 124 ) and health - related quality of life improved by 110.4% over the mean baseline value of 37.3 to 79.5 at 12 months after the procedure ( n = 124 ) . in terms of safety , there was one serious adverse event in phase ii ( 1.4% , 1 of 69 ) : early postoperative bleeding at the abdominal wall resulting in a hematoma , which was related to the procedure and possibly related to the device . there were 6 adverse events ( 10.0% , 6 of 69 ) : 4 cases of abdominal pain and 2 urinary tract infections , which may have possibly been related to the procedure . device - related adverse events were reported in 5 patients ( 3.7% , 5 of 137 ) in phase iii ( table 4 ) . one study patient ( 1 of 137 , 0.7% ) , who withdrew early from the phase iii trial , later had a surgical reintervention ( uterine artery embolization ) for her heavy menstrual bleeding . device - related adverse events reported in phase iii trial through 12 months the study patients in phase iii who were employed ( n = 88 ) missed a median of 5 days of work ( range , 029 days ) . thirty - nine patients in phase ii described themselves as working outside of the home , and they reported missing a median of 4 days of work ( range , 210 days ) . laparoscopic , ultrasound - guided rfvta of fibroids was developed by lee after using a system designed primarily for liver ablation . the current device used in this study was the result of years of testing the principles of volumetric fibroid tissue ablation by which the treatment effect is limited to the fibroid and spares the myometrial tissue . rfvta contrasts with current methods for laparoscopic or abdominal myomectomy procedures , with which only a portion of the existent fibroids can be identified and/or removed . with rfvta , gynecologic surgeons have been able to treat all fibroid types in all locations , with the exception of type 0 ( pedunculated ) intracavitary fibroids , which are best removed with operative hysteroscopic resection . performance of laparoscopic rfvta requires 3 basic gynecologic skills : laparoscopy , ultrasound , and probe placement under ultrasound guidance . the method described in this report targets only the fibroids , leaving the endometrium intact without incision of the myometrium . because no myometrial incisions are created , there is little blood loss and there is no need for laparoscopic uterine suturing . patients are normally discharged the same day of treatment and require minimal pain management , similar to diagnostic laparoscopy . in addition , it is normal to have and we expected to see small amounts of vaginal discharge after placement of a cervical tenaculum ; we saw such discharge , and in all cases the amount was minimal . because neither the phase ii nor the phase iii studies were designed as randomized controlled trials or trials comparing rfvta with other methods of myoma treatment , we are restricted by the regulatory authorities from making comparative statements . we can report that there are two different comparative / randomized controlled trials under way in canada ( clinicaltrials.gov identifier nct01563783 ) and in germany ( clinicaltrials.gov identifier nct01750008 ) . these studies , which will be reported in upcoming articles , describe the following myoma treatments : rfvta , laparoscopic and abdominal myomectomy , and uterine artery embolization . the 12-month phase ii and interim phase iii results of rfvta of symptomatic fibroids show significant clinical reduction in symptoms and improvement in health - related quality of life within an acceptable safety profile . despite the fact that the phase iii patients displayed greater mean baseline uterine volumes , lower baseline health - related quality - of - life scores , and higher baseline symptom scores , the improvements in all 3 measures were similar across all 3 studies . phase ii and iii reductions in uterine volume over time ( 20.3% and 28.7% , respectively , at 3 months and 14.6% and 25.7% , respectively , at 12 months ) are meaningful and likely contribute significantly to the decline in symptoms and improvement in health - related quality of life as reflected in the follow - up ufs - qol questionnaire scores . twelve - month phase iii results provided a rigorous setting to substantiate the phase ii safety and efficacy results of rfvta of leiomyomas : the decreases in uterine volume and severity of symptoms over time accompanied by a significant improvement in health - related quality of life for up to 12 months after treatment indicate the potential for long - term clinically significant outcomes over the 3-year life of the phase iii trial and beyond .

background and objectives : to review phase ii and phase iii treatments of symptomatic uterine fibroids ( myomas ) using laparoscopic radiofrequency volumetric thermal ablation ( rfvta).methods : we performed a retrospective , multicenter clinical analysis of 206 consecutive cases of ultrasound - guided laparoscopic rfvta of symptomatic myomas conducted on an outpatient basis under two phase ii studies at 2 sites ( n = 69 ) and one phase iii study at 11 sites ( n = 137 ) . descriptive and exploratory , general trend , and matched - pair analyses were applied.results:from baseline to 12 months in the phase ii study , the mean transformed symptom severity scores improved from 53.9 to 8.8 ( p < .001 ) ( n = 57 ) , health - related quality - of - life scores improved from 48.5 to 92.0 ( p < .001 ) ( n = 57 ) , and mean uterine volume decreased from 204.4 cm3 to 151.4 cm3 ( p = .008 ) ( n = 58 ) . patients missed a median of 4 days of work ( range , 210 days ) . the rate of possible device - related adverse events was 1.4% ( 1 of 69 ) . in the phase iii study , approximately 98% of patients were assessed at 12 months , and their transformed symptom severity scores , health - related quality - of - life scores , mean decrease in uterine volume , and mean menstrual bleeding reduction were also significant . patients in phase iii missed a median of 5 days of work ( range , 129 days ) . the rate of periprocedural device - related adverse events was 3.5% ( 5 of 137 ) . despite the enrollment requirement for patients in both phases to have completed childbearing , 4 pregnancies occurred within the first year after treatment.conclusions:rfvta does not require any uterine incisions and provides a uterine - sparing procedure with rapid recovery , significant reduction in uterine size , significant reduction or elimination of myoma symptoms , and significant improvement in quality of life .

INTRODUCTION METHODS Device Description and Procedure RESULTS DISCUSSION CONCLUSIONS

they have a lifetime incidence of 70% to 80% , and approximately 25% of women report being symptomatic . although hormone use results in a temporary reduction in symptoms , it is not a permanent treatment for fibroids . ultrasound studies have shown that myomas begin to develop in female patients at a young age and increase in both size and number until women reach menopause . studies have shown ethnic differences in the incidence of leiomyomas , with african - american women having a disproportionately higher occurrence than other races . symptoms , including heavy menstrual bleeding , dysmenorrhea , pelvic - abdominal pain or pressure , urinary frequency and incontinence , dyspareunia , infertility , and an increased risk of spontaneous abortion , occur in 20% to 50% of patients with fibroids . symptomatic uterine fibroids represent the most common indication for hysterectomy , accounting for 150 000 to 200 000 of all of the hysterectomy procedures , or 30% to 40% of cases , that are performed annually in the united states . we present the following report of phase ii and phase iii studies of laparoscopic ultrasound - guided radiofrequency volumetric thermal ablation ( rfvta ) of leiomyomas in an effort to address the dearth of new clinical research in the united states on minimally invasive approaches to symptomatic fibroid therapy and to provide descriptions of what we believe to be an emerging , preferred uterine - sparing fibroid treatment modality . both percutaneous and laparoscopic radiofrequency ( rf ) no long - term medical therapy exists for symptomatic fibroids ; consequently , treatment has been and remains largely surgical . myomectomy , though allowing for uterine preservation , does require one or more myometrial incisions ; can require a postprocedural inpatient hospitalization similar to that of hysterectomy ; and may be associated with increased blood loss , longer procedure times , and increased risk for postoperative hemorrhage and/or pelvic adhesions . laparoscopic myolysis with either bipolar needles or a neodymium : yttrium - aluminum - garnet laser was performed in europe in the late 1980s and was subsequently reported in the united states by goldfarb . the procedure initially targeted the entire myoma by using multiple punctures through the uterine serosa with resultant and significant formation of postoperative adhesions . lee performed rfvta of fibroids in the 1990s and early 2000s under an institutional review board approved off - label study using an rfvta system designed for soft - tissue ablation . this procedure involved the insertion of an rf needle with 7 deployable curved electrodes that created a volumetric ablation within the myoma . lee subsequently developed a system designed specifically to treat uterine fibroids using rfvta ( acessa system ; halt medical , brentwood , california ) . this system was tested in multiple studies enrolling a total of 206 women with symptomatic uterine fibroids . the primary objectives of the phase ii studies , which were conducted under similar but separate protocols , were to assess the safety and efficacy of the acessa system in women with symptomatic fibroids 6 cm and to evaluate the treatment 's effect on uterine volume . study endpoints were the incidence of procedure- and device - related adverse events , change in uterine fibroid symptom and quality - of - life ( ufs - qol ) questionnaire responses over time , and change in uterine volume . the primary objectives of the phase iii study , which was conducted under 1 protocol and included 9 sites in the united states and the 2 latin american sites , from the phase ii study were to confirm the safety and efficacy of the acessa system for the treatment of symptomatic uterine fibroids 7 cm in diameter in patients with confirmed clinical menorrhagia ( indicated by menstrual blood loss of 160 ml to 500 ml ) based on alkaline hematin testing . study primary endpoints at 12 months were safety , surgical reintervention rates , and change in menstrual blood loss . secondary endpoints included changes in uterine and fibroid volumes , evaluation of patients ' ufs - qol and general health status questionnaire responses over time , and patients ' satisfaction after treatment . the acessa system has been approved for commercial use in the united states , mexico , canada , and the european union for the treatment of symptomatic uterine fibroids . the phase ii trial was a prospective , dual - center , longitudinal , open - label , single - arm feasibility study assessing the safety and efficacy of rfvta of symptomatic myomas in women seeking alternative treatment to hysterectomy . participation in the trial was voluntary , the local ethics committee and ministry of health of each country approved the recruitment of the study participants , and the principal investigators or designee obtained written informed consent from each enrolled patient according to good clinical practice guidelines , local and federal regulations of mexico and guatemala , and international standard en iso 141551:2003 ( clinical investigation of medical devices for human subjects ) . the phase iii trial , which is ongoing , is a food and drug administration and institutional review board approved , prospective , multicenter , longitudinal , pivotal study in which the patients serve as their own controls . eleven urban study centers in the united states ( n = 9 ) and in latin america ( n = 2 ) provided the clinical setting . as with the phase ii trial , participation by the enrollees was voluntary and written informed consent was obtained from each patient according to good clinical practice guidelines ; federal regulations established by the us food and drug administration , mexico , and guatemala ; and international standard en iso 141551:2003 ( clinical investigation of medical devices for human subjects ) . patients in both phases have been followed up for a minimum of 1 year after rf ablation of their symptomatic fibroids . at each of the 3- , 6- , and 12-month follow - up visits , the patients were evaluated clinically to assess symptoms and to identify any adverse events ; at baseline and at each follow - up visit , they were also asked to complete a ufs - qol questionnaire . ( the questionnaire 's mean transformed scores range from 0 to 100 . a decrease in symptom score over time from baseline indicates improvement , whereas a higher score for health - related quality of life after the procedure indicates improvement ) . before the intervention , baseline medical data were collected and recorded , including a complete health history , a pregnancy test within 24 hours of surgery , a complete blood panel , and completion of the ufs - qol questionnaire . for the phase ii trial , transvaginal ultrasound was performed for an initial determination of the location , number , and size of fibroid(s ) and to measure the uterine volume . for the phase iii trial , magnetic resonance imaging was incorporated in the preoperative assessment along with transvaginal ultrasound . the device ( acessa system ) is composed of an rf generator and a disposable rf handpiece with deployable needle electrodes , two dispersive electrode pads , extension cables , and a foot pedal ( figure 1 ) . the surgeon then may choose to deploy the 7-needle electrode array to the desired ablation size within the fibroid capsule ( figures 2 and 3 ) . rf ablation is carried out with a continuous , low - voltage , high - frequency ( 45500 khz ) alternating current with a maximum output of 200 w. once the tissue temperature reaches 100c , the wattage automatically adjusts to maintain treatment temperature and deliver rf energy to tissue through the handheld disposable rf handpiece . energy propagation is concentrated in the pelvic region only ; thus there is no safety risk to the patient in terms of high levels of current being conducted across the heart , which would otherwise be a concern . the maximum level of current conduction is approximately 2.5 amps , which only occurs under conditions of highest power ( 200 w ) and lowest resistance / impedance ( 3040 in the case of rfvta procedures ) . schema of acessa system : rf generator with foot pedal , extension cables , rf handpiece , and dispersive pads . the surgeon initiates treatment by depressing the foot pedal , and the rf generator increases the tissue temperature to 100c and maintains that temperature for the duration of the ablation . multiple uterine fibroids can be treated during the procedure and often by use of only one insertion through the uterine serosa . because there is no requirement for uterine incisions , after fibroid ablation , the surgeon withdraws the electrode array into the 3.4-mm primary needle and removes the handpiece while simultaneously performing standard needle - track rf coagulation by applying a lower wattage ( 815 w ) of intermittent coagulation mode to the needle tip ( figure 4 ) . on occasion , depending on the fibroid 's shape and size , multiple ablations may be required for a single fibroid . the surgeon then may choose to deploy the 7-needle electrode array to the desired ablation size within the fibroid capsule ( figures 2 and 3 ) . rf ablation is carried out with a continuous , low - voltage , high - frequency ( 45500 khz ) alternating current with a maximum output of 200 w. once the tissue temperature reaches 100c , the wattage automatically adjusts to maintain treatment temperature and deliver rf energy to tissue through the handheld disposable rf handpiece . energy propagation is concentrated in the pelvic region only ; thus there is no safety risk to the patient in terms of high levels of current being conducted across the heart , which would otherwise be a concern . the maximum level of current conduction is approximately 2.5 amps , which only occurs under conditions of highest power ( 200 w ) and lowest resistance / impedance ( 3040 in the case of rfvta procedures ) . schema of acessa system : rf generator with foot pedal , extension cables , rf handpiece , and dispersive pads . the surgeon initiates treatment by depressing the foot pedal , and the rf generator increases the tissue temperature to 100c and maintains that temperature for the duration of the ablation . because there is no requirement for uterine incisions , after fibroid ablation , the surgeon withdraws the electrode array into the 3.4-mm primary needle and removes the handpiece while simultaneously performing standard needle - track rf coagulation by applying a lower wattage ( 815 w ) of intermittent coagulation mode to the needle tip ( figure 4 ) . on occasion , depending on the fibroid 's shape and size , multiple ablations may be required for a single fibroid . fibroids can be treated virtually in any portion of the uterine myometrium ( anterior , posterior , or lateral ) , including those that are subserosal , intramural , transmural , or submucosal . patient demographic data and myoma symptom characteristics at baseline for both the phase ii and phase iii trials are presented in table 1 . phase ii participants were all of hispanic or latino origin ( 100% ) , whereas 55.1% of phase iii participants reported not being hispanic or latino . after intervention in the phase ii study , 69 patients ( 100% ) were seen at the 3-month follow - up visit , 65 ( 94.2% ) at the 6-month follow - up visit , and 58 ( 84.1% ) at the 12-month visit . the flow of patients through the phase iii study has been similar through 12 months : 137 patients ( 100% ) were treated at baseline , 134 ( 97.8% ) have been followed up through 3 months , 134 ( 97.8% ) have been followed up through 6 months , and 132 ( 96.4% ) have been followed up through 12 months . the phase iii study is ongoing . phase ii and phase iii patient demographics and myoma symptom characteristics at baseline the baseline population for phase iii does not include two patients who , after rfvta , did not meet all inclusion criteria . one patient in the phase ii trial conceived after ablation , delivered a healthy , full - term infant by elective repeat cesarean section , and was excluded from further participation in the study . three patients in the phase iii trial conceived after ablation and were excluded from further participation in the study . one patient delivered a full - term , healthy infant by cesarean section ; one woman delivered a full - term , healthy infant vaginally ; and one patient miscarried at 10 weeks . a total of 128 myomas were visualized in the 69 patients by transvaginal ultrasound at baseline , and 285 myomas were mapped and treated by rf ablation in phase ii ( median , 3 ; range , 120 ) . during intraoperative laparoscopic ultrasound mapping during phase iii , 818 myomas were visualized and 640 were treated ( median , 4 ; range , 129 ) . in phase ii surgeons treated a median of 3 fibroids per patient ( range , 120 ) in 2.33 hours ( range , 0.704.83 hours ) ( timed from first incision to close of last incision ) . phase iii surgeons treated a median of 4 fibroids per patient ( range , 129 ) in 1.88 hours ( range , 0.355.55 hours ) ( timed from initiation of pneumoperitoneum to close of last incision ) . distribution of myomas by type and by study for phase ii and phase iii as found on laparoscopic ultrasound a myoma could be of more than one type . a comparison of phase ii and phase iii uterine volume measurements ( by transvaginal ultrasound ) from baseline to 12 months after the procedure is provided in figure 5 . the mean uterine volume decreased over time from baseline through 12 months for the phase ii patients : the mean volume reduction at 3 months ( n = 68 ) , 6 months ( n = 64 ) , and 12 months ( n = 58 ) was 20.3% , 25.2% , and 28.7% , respectively . similarly , the mean uterine volume for the phase iii patients decreased from baseline ( n = 134 ) by 14.6% at the 3-month follow - up visit ( n = 129 ) , by 20.6% at the 6-month follow - up visit ( n = 128 ) , and by 25.7% at the 12-month follow - up visit ( n = 127 ) . uterine volume measurements by transvaginal ultrasound over time from baseline to 12 months after procedure for phases ii and iii . improvements in phase ii and phase iii ufs - qol mean transformed symptom severity and health - related quality - of - life scores and subscale scores over time are presented in table 3 . mean ufs - qol transformed scores and health - related quality - of - life subscale scores for phase ii and phase iii studies scores range from 0 to 100 . a lower symptom severity score indicates a decrease in symptoms , and a higher health - related quality of life score indicates an improvement in health - related quality of life . ten patients were removed from the ufs - qol analysis because of pregnancy , amenorrhea / menopause , loss to follow - up , or baseline parameters outside the inclusion criteria . symptom severity decreased from baseline to 12 months in phase ii by 83.7% ( 53.9 to 8.8 , n = 58 ) ; health - related quality of life improved from baseline to 12 months by 89.8% ( 48.5 to 92.0 , n = 58 ) . in the phase iii study , symptom severity decreased by 56.5% from the mean transformed score of 61.1 at baseline ( n = 127 ) to 26.6 at 12 months ( n = 124 ) and health - related quality of life improved by 110.4% over the mean baseline value of 37.3 to 79.5 at 12 months after the procedure ( n = 124 ) . in terms of safety , there was one serious adverse event in phase ii ( 1.4% , 1 of 69 ) : early postoperative bleeding at the abdominal wall resulting in a hematoma , which was related to the procedure and possibly related to the device . there were 6 adverse events ( 10.0% , 6 of 69 ) : 4 cases of abdominal pain and 2 urinary tract infections , which may have possibly been related to the procedure . device - related adverse events were reported in 5 patients ( 3.7% , 5 of 137 ) in phase iii ( table 4 ) . one study patient ( 1 of 137 , 0.7% ) , who withdrew early from the phase iii trial , later had a surgical reintervention ( uterine artery embolization ) for her heavy menstrual bleeding . device - related adverse events reported in phase iii trial through 12 months the study patients in phase iii who were employed ( n = 88 ) missed a median of 5 days of work ( range , 029 days ) . thirty - nine patients in phase ii described themselves as working outside of the home , and they reported missing a median of 4 days of work ( range , 210 days ) . laparoscopic , ultrasound - guided rfvta of fibroids was developed by lee after using a system designed primarily for liver ablation . with rfvta , gynecologic surgeons have been able to treat all fibroid types in all locations , with the exception of type 0 ( pedunculated ) intracavitary fibroids , which are best removed with operative hysteroscopic resection . performance of laparoscopic rfvta requires 3 basic gynecologic skills : laparoscopy , ultrasound , and probe placement under ultrasound guidance . in addition , it is normal to have and we expected to see small amounts of vaginal discharge after placement of a cervical tenaculum ; we saw such discharge , and in all cases the amount was minimal . because neither the phase ii nor the phase iii studies were designed as randomized controlled trials or trials comparing rfvta with other methods of myoma treatment , we are restricted by the regulatory authorities from making comparative statements . we can report that there are two different comparative / randomized controlled trials under way in canada ( clinicaltrials.gov identifier nct01563783 ) and in germany ( clinicaltrials.gov identifier nct01750008 ) . these studies , which will be reported in upcoming articles , describe the following myoma treatments : rfvta , laparoscopic and abdominal myomectomy , and uterine artery embolization . the 12-month phase ii and interim phase iii results of rfvta of symptomatic fibroids show significant clinical reduction in symptoms and improvement in health - related quality of life within an acceptable safety profile . despite the fact that the phase iii patients displayed greater mean baseline uterine volumes , lower baseline health - related quality - of - life scores , and higher baseline symptom scores , the improvements in all 3 measures were similar across all 3 studies . phase ii and iii reductions in uterine volume over time ( 20.3% and 28.7% , respectively , at 3 months and 14.6% and 25.7% , respectively , at 12 months ) are meaningful and likely contribute significantly to the decline in symptoms and improvement in health - related quality of life as reflected in the follow - up ufs - qol questionnaire scores . twelve - month phase iii results provided a rigorous setting to substantiate the phase ii safety and efficacy results of rfvta of leiomyomas : the decreases in uterine volume and severity of symptoms over time accompanied by a significant improvement in health - related quality of life for up to 12 months after treatment indicate the potential for long - term clinically significant outcomes over the 3-year life of the phase iii trial and beyond .

the hypothalamus is often times a target for newer potential obesity treatments due to its crucial role in food intake and metabolism . it is also established that food intake modulation is confounded by numerous players within the hypothalamus , allowing for the research and development of a diverse array of obesity management drug leads that have completely different underlying mechanisms . the melanocortin system is established amongst these systems ; however , the cannabinoid system , among others , also has an established role . speculation of a connection between the cannabinoid system and melanocortin system regarding food intake has existed due to their presence in nearby regions of the hypothalamus . moreover , suboptimal doses of sr 141716 ( rimonabant ) together with suboptimal doses of -melanocyte - stimulating hormone ( -msh ) are known to behave synergistically in order to reduce food intake . attempts to develop a safe drug to treat obesity via blocking the cb1 receptor have proven to be elusive and controversial , as drugs such as rimonabant have had to struggle for approval due to numerous reported and suspected side effects , particularly depression . the role of the melanocortin system in food intake is well - established and prevention of the rapid inactivation of -msh may prove to be a better alternative pathway for potential obesity treatments . recent studies suggest that prolylcarboxypeptidase ( prcp ) involved in regulating blood pressure and inflammation is an appetite stimulant and , by consequence , prcp inhibitors may prove to be a viable lead to treat obesity . there have been numerous excellent reviews on melanocortin receptors.16 however , this article only reports the most current information about how the two tectonic physiological processes , namely the proteolytic enzymes of renin - angiotensin system ( ras ) and proopiomelanocortin ( pomc)-derived neuropeptide regulatory processes in the central nervous system might be shifting toward each other . recent findings suggest that prcp ( a ras enzyme ) regulates -msh ( a pomc - derived neuropeptide ) levels , a theme addressed by the present review . while this article briefly introduces both prcp- and -msh - mediated processes , it also outlines how brain prcp may play a key role in controlling food intake and weight gain . the prcp - catalyzed reaction was initially found to be part of the pathway for angiotensin ii ( ang ii ) metabolism in renal tissues , where prcp appeared to control the total amount of ang ii . odya and others demonstrated that prcp metabolizes ang ii to angiotensin 17 ( ang 17 ) ( figure 1).7 the activation of ang 17 receptor mas ( a g - protein - coupled protein ) by ang 17 results in the generation of nitric oxide ( no ) and prostaglandins.8 thus , ang 17 counteracts ang ii function , providing evidence that prcp regulates the negative effects of ang ii such as high blood pressure and heart failure.9 in addition , the activation of the ang 17 receptor mas may also lead to diminished cell proliferation through down - regulation of the phosphorylation and activation of erk1 and erk2 in the erk1/erk2 map kinase signaling pathway.10,11 in theory , the prcp inhibitors to target the production of pro - inflammatory prostaglandins and promote proliferation through the ang 17 receptor mas - dependent pathway represent a novel approach to suppress unwanted inflammation - causing prostaglandins . later , it was shown that prcp is one of several enzymes that convert ang ii to a unique bioactive molecule . in vitro studies showed that angiotensin - converting enzyme 2 ( ace2 ) is an exopeptidase that converts ang ii to ang 17 at a much faster rate than prcp.12 these data suggest that ang ii is a poor substrate for prcp . clinical studies have provided reliable evidence that ace2 is an essential regulator of angiotensin i ( ang i ) , ang ii , and angiotensin - induced cardiac hypertrophy.13 recent studies clearly show increased myocardial levels of ang ii and a significant decrease in ang 17 in ace2-deficient hearts , suggesting that the role of prcp in metabolizing ang ii may be insignificant.9 taken together , these observations suggest that prcp is a redundant catalyst contributing to alternate pathways for ang ii metabolism . while the well - established cardiovascular and renal actions of ang ii are attributed to the angiotensin type 1 receptor ( at1r ) , much less is known about angiotensin iii and its cardiovascular effects . for more than 30 years , it was known that prcp metabolizes ang iii to ang 27 ( figure 1).7 soon after , studies demonstrated that ang iii is a pressor agent whose response , like that of ang ii , is mediated by at1 receptors.14,15 apparently , ang iii has multiple effects on renal function in the diseased kidney and can enhance renal disease through the overproduction of aldosterone , leading to arterial hypertension and/or atrial fibrillation.16 aldosterone maintains blood volume , pressure , and electrolyte balance . its production is known to be regulated by renin , an enzyme produced in the kidneys . renin increases in response to low blood pressure , decreased blood flow to the kidneys , or sodium deficiency . studies indicate that ang iii also activates the secretion of aldosterone.15 recently , we have demonstrated that recombinant prcp ( rpcrp ) metabolizes ang iii to ang 27 , removing phenylalanine ( phe).8 it is tempting to speculate that prcp might funnel the generation of angiotensin 34 ( ang 34 ) through ang 27 ( figure 1 ) . if the over - secretion of aldosterone by ang iii is viewed as a trigger of arterial hypertension , then inactivation of ang iii by prcp might lead to a decrease in blood pressure . further studies are required to determine whether prcp is critically important for regulating ang iii - induced hypertension and preserving renal structure and function . this is an important area of research to pursue given the increasing prevalence of cardiovascular disease and stroke in the older population . the possible actions of another substrate of prcp , plasma prekallikrein ( pk , fletcher factor ) , have recently begun to receive much attention . when the complex of high molecular weight kininogen ( hk ) and pk binds to endothelial membrane , pk is rapidly converted to kallikrein by prcp.17 the formed kallikrein then cleaves hk to liberate bradykinin ( bk ) , which leads to no and prostaglandin - i2 formation , as well as subsequent vasodilation , by activating constitutive bradykinin b2 and inducible bradykinin b1 receptors.18,19 the prcp - dependent pk activation pathway might be considered an additional mechanism to preserve the availability of no and prostacyclin as vasodilatory agents in vascular smooth muscle . we proposed that chronic prcp inhibition might elevate blood pressure . in accordance , we have found that prcp mice have mild hypertension , suggesting a causative relationship between prcp levels and signs of hypertension.20 local skeletal muscle ischemia and acidosis are shown to increase the generation of bk and prostaglandins , the two circulating products of the prcp - induced cell activation ( figure 1).21 the increased acidotic response during exercise and inflammatory mediators such as bk and prostacyclin have been shown to cause abnormal exercise - related symptoms and autonomic responses in congestive heart failure syndrome.22 nonetheless , the long - term elevated concentrations of no and prostacyclin through prcp - dependent pathways may be detrimental and eventually responsible for cardiovascular diseases such as congestive heart disease . since bk and prostaglandins exacerbate the genesis of the symptoms of exercise intolerance in heart failure,23 the inhibitors of prcp might be effective in ameliorating the exercise - limiting symptoms . clinical studies demonstrate that prcp is involved in the pathogenesis of inflammatory conditions such as rheumatoid arthritis and infection.24 melanocortin peptides have numerous effects on the host such as the modulation of fever , inflammation and appetite.25 recently , we showed that prcp metabolizes alpha - melanocyte - stimulating hormone 113 ( -msh113 ) to alpha - melanocyte - stimulating hormone 112 ( -msh112),26 ( figure 1 ) . -msh113 is a potent anti - inflammatory agent.27 in addition to the specificity of cleavage , the cellular release of pro - inflammatory mediators seemed to be critical to prcp actions . in theory , agents that increase production and effects of -msh113 could be used to counteract the effects of pro - inflammatory mediators such as bradykinin and cytokines ( figure 1 ) . obesity is known to cause inflammation and insulin resistance in the vasculature and non - vascular tissues involved in glucose metabolism.28 evidence suggests that hyperglycemia may contribute to defective no - dependent vasodilation in diabetes.29 the inducible no synthase ( inos ) expression is elevated in adipose tissue of obese people compared to those of lean people30 and is a mediator of inflammation and a key enzyme in insulin resistance.31 the colocalization of -msh113 receptors ( mc4r ) with inos has been reported , suggesting a role for -msh113 in obese people.32 the inactivation of -msh113 by prcp provides a positive feedback loop for postprandial enhancement of food intake and inflammation by inhibiting -msh113 function , as shown in figure 2.26 since prcp regulates the anorectic action of -msh113 , this study highlights the presence of a newly recognized interaction between inflammation , obesity , and the expression and activity of prcp ( figure 2).26 in view of the above studies , we consider that prcp may be a key player in the obesity - associated metabolic complications , inflammatory response , and the host defense mechanism . the following sections emphasize pertinent findings , which best describe the theoretical perspective on the components of the central melanocortin system and stress the importance of prcp influence in the melanogenic signaling pathway . pro - opiomelanocortin ( pomc ) , a prohormone with molecular weight of 31 kda , is ubiquitously expressed in various tissues of mammals.33,34 pomc expression in the central nervous system , however , is limited to the arcuate nucleus of the hypothalamus ( arc ) , nucleus tractus solitarius of the caudal medulla ( nts ) , and corticotrophs and melanotrophs of the anterior pituitary ( figure 2).35 the 1200 base pair pomc transcript encodes for the 267 amino acid prohormone with an n - terminal signal peptide of 26 residues.36 as this precursor peptide passes through the golgi stacks , it is targeted , via a specific signal peptide , into regulated secretory granules.37 pomc undergoes extensive posttranslational modification within these secretory granules mediated by a family of serine proteases , the prohormone convertases ( pcs ) , as illustrated in figure 3 . pomc is cleaved by prohormone convertase 1 ( pc1 ) to produce 22 kda pro - acth and -lipoprotein hormone ( -lph ) ( figure 3).38 pro - acth is further cleaved by pc1 to produce the n - terminus of pomc - joining peptide and 4.5 kda acth . prohormone convertase 2 ( pc2 ) cleaves acth to acth 117 and corticotropin - like intermediate lobe peptide ( clip ) and -lph to -lipoprotein hormone ( -lph ) and the synthesis of -msh from pomc involves several specific enzymes in addition to pc1 and pc2 . first , carboxypeptidase e cleaves the c - terminal basic amino acid residues of acth 117 . the peptide is then amidated by peptidyl -amidating monooxygenase ( pam ) to produce desacetyl -msh ( des--msh ) . finally , des--msh is acetylated by n - acetyltransferase ( nat ) to produce acetylated -msh ( act--msh ) , as illustrated in figure 2 . act--msh is more potent than des--msh in activating melanocortin receptor signaling and in reducing food intake , effects that are likely due to rapid degradation of des--msh . guo et al have shown that total hypothalamic -msh levels are decreased in leptin - deficient ob / ob mice and increased in leptin - treated ob / ob and c57bl/6j mice . leptin specifically enhances hypothalamic levels of act--msh without significantly affecting the amounts of des--msh , possibly by activating nat in the pomc neurons.39 the melanocortin receptors are g - protein - coupled receptors with characteristic seven transmembrane domains . mc5r.4043 the melanocortin receptors are gs - coupled and signal via the adenylate cyclase - camp - protein kinase a second messenger pathway . however , depending on the cell type and the melanocortin receptor expression , signal transduction pathways other than camp may be activated which include inositol triphosphate - diacyl glycerol - protein kinase c ( ip3-dag - pkc ) pathway , extracellular ca influx , map kinase pathway , and the jak / stat pathway.4448 mc1r , mc3r , mc4r and mc5r show 40%60% amino acid homology . the natural msh peptides have a conserved sequence , his - phe - arg - trp , which plays an important role in the binding of these peptides to specific melanocortin receptors.49 mc1r was the first melanocortin receptor to be cloned and was isolated from human melanoma cell line.40 - , - and -msh and acth are the known agonists whereas agouti is the known antagonist of the mc1r . mc1r is expressed in human and mouse melanoma cells , human melanocytes , skin glands , and hair follicles.40,5053 -msh , agouti , and mc1r , therefore , play an important role in regulating skin pigmentation and hair color . the presence of mc1r in the testes and the pituitary has been demonstrated by chhajlani et al.54 mc1r expression in the central nervous system is limited to neurons of the periaqueductal grey in both rat and human brain.55 however , mc1r is widely expressed in cells involved in inflammation such as endothelial cells , neutrophils , monocytes , macrophages , fibroblasts and astrocytes.25,5659 -msh has been shown to inhibit inflammation via mc1r - mediated decrease in the production of inflammatory cytokines such as il-1 , il-6 , and tumor necrosis factor - alpha ( tnf ) , as well as suppression of nf-b.6062 mc2r is encoded by a single gene localized to chromosome 18p11.2.53 acth is the only known agonist of the mc2r.63 using in situ hybridization , xia et al showed that mc2r is highly expressed in the adrenal cortex with the highest expression in the zona fasciculata and zona glomerulosa and relatively low expression in the zona reticularis.64 these findings are consistent with the role of mc2r in mediating the effect of acth on the synthesis and release of corticosteroids . besides the adrenal cortex , mc2r is expressed in murine adipocytes , which explains the lipolytic effect of acth.59 however , human adipocytes do not express mc2r and there is no evidence to suggest that human adipose tissue is responsive to the lipolytic effect of acth.54 these species differences in the expression of mc2r may be important when studying the role of melanocortins in obesity . mc2r expression has also been demonstrated in the skin along with three cytochrome enzymes involved in steroid hormone synthesis.65 kapas et al have shown that acth produced in the skin by keratinocytes stimulates dna synthesis and induces cell proliferation via mc2r.66 mc2r , therefore , appears to play an important role in cutaneous pathophysiology . mc3r is encoded by a single gene localized to the q13.2q13.3 region of chromosome 20.67 since this locus is associated with type 2 noninsulin - dependent diabetes mellitus ( niddm ) , mc3r could represent a candidate gene for niddm.68 mc3r is unique in that it binds to - , - and -msh with similar affinities . using northern blot hybridization and polymerase chain reaction , gantz et al first showed that mc3r is expressed in brain , placental , and gut tissues but not in melanoma cells or in adrenal glands.69 mc3r expression in the brain is highest in the hypothalamus especially in the arcuate nucleus , ventromedial nucleus , preoptic nucleus , lateral hypothalamic area , and posterior hypothalamic area.43 agouti - related protein ( agrp ) , which is normally expressed in the hypothalamus , is a potent antagonist of both mc3r and mc4r.70 however , pomc and agrp neurons in the arcuate nucleus of hypothalamus selectively express mc3r , and not mc4r , suggesting that mc3r might function as a presynaptic autoreceptor regulating the release of melanocortins.71,72 physiological support for this hypothesis was provided by cowley et al,73 who demonstrated that the selective mc3r agonist , d - trp--msh inhibited firing of gfp - labeled pomc neurons in the pvn . also , marks et al showed that peripheral administration of the mc3r agonist stimulates feeding via mc3r - mediated inhibition of the arc pomc neurons.74 an association between mc3r and human obesity has been identified by linkage studies . several sequence variants have been found in the mc3r coding region and in 5 flanking sequences.75 mc3r variants are associated with subtle changes in weight , leptin levels , and insulin - glucose ratios , but none of these explain human morbid obesity.76 a novel heterozygous mutation i183n in mc3r was identified in two obese patients of the same family.77 functional characterization of i183n showed that this mutation completely abolished the activity of the mutated receptor to stimulate intracellular camp production , suggesting that i183n might play an important role in obesity.78 similarly , tao et al showed that a novel mutation i335s in mc3r results in complete loss of ligand binding and signaling suggesting that this mutation might contribute to obesity.79 however , a recent study evaluating the functional consequences of all mutations found in mc3r and mc4r in severely obese north american adults concluded that mc4r , but not mc3r mutations are associated with severe obesity in this population.80 thus , the significance of mc3r mutations in human obesity is still not conclusively established to date . mc4r was first cloned by gantz et al in 1993.42 mc4r is a 332 amino acid protein encoded by a single gene , localized to chromosome 18q21.3 . using northern blot analysis and in situ hybridization , mc4r mc4r expression was notably absent in the adrenal cortex , melanocytes , and placenta.42 mc4r is widely distributed in the central nervous system , especially in the cortex , hippocampus , amygdala , septum , corpus striatum , nucleus accumbens , hypothalamus , nucleus tractus solitarius , visual and motor nuclei of the brainstem , and the dorsal horn of the spinal cord.81 -msh , -msh , and acth are the known agonists and agrp is the known antagonist of the mc4r ( figure 2).70 since mc4r is highly expressed in the hypothalamus and has a strong affinity for -msh it is believed to be a strong candidate for energy balance , appetite control , and body weight regulation . mc4r knockout mice have been shown to develop a maturity onset obesity syndrome characterized by hyperphagia , hyperglycemia and hyperinsulinemia.82 since this syndrome is similar to the agouti obesity syndrome seen in avy/ mice and agrp - transgenic mice that overexpress agouti and agrp respectively , it is speculated that the primary mechanism by which agouti and agrp produce obesity is chronic antagonism of mc4r.83 cachexia , a chronic wasting syndrome characterized by loss of body weight and muscle mass , is commonly associated with diseases such as cancer and aids . mc4r mice and mice treated with agrp are resistant to lipopolysaccharide- or tumor - induced cachexia , further supporting the role of mc4r in energy balance and body weight regulation.84 mc5r is a 325 amino acid protein encoded by a single gene located on chromosome 18p11.2.85 mc5r has been shown to bind to all melanocortins except -msh.86 mc5r is the most widely expressed melanocortin receptor . mc5r mrna is expressed in the adrenal gland , adipose tissue , kidney , leukocytes , lung , lymph node , mammary gland , ovary , pituitary , testis , and uterus.54 mc5r is highly expressed in exocrine glands such as lacrimal , preputial , harderian and sebaceous glands.87 mc5r - deficient mice have a severe defect in water repulsion and thermoregulation due to decreased production of sebaceous lipids . studies in humans have shown that mc5r immunoreactivity is detectable in the epithelium and appendages , including the sebaceous , eccrine , and apocrine glands . however , analysis of mc5r variations in patients with acne , hidradenitis suppurativa , and sebaceous gland dysfunction have failed to suggest a causative role of mc5r in these conditions.88 low levels of mc5r mrna have also been reported in the central nervous system,89 however , the physiological function of mc5r in the brain remains unclear . linkage analysis in the quebec family study90 revealed a significant association of mc5r polymorphisms with body mass index , fat mass , and resting metabolic rate , thus providing some evidence for the possible role of mc5r in energy balance and body weight regulation . the pomc neurons , which produce -msh , also express another anorectic peptide cocaine - amphetamine - related transcript ( cart ) . cell bodies of the pomc / cart neurons are found throughout the rostrocaudal extent of the arcuate nucleus , as well as the periarcuate area of the hypothalamus . within the hypothalamus , these neurons project to the periventricular nucleus , paraventricular nucleus ( pvh ) , and the perifornical region.91,92 the pomc / cart neurons also project to the brainstem to innervate the rostral nts , lateral reticular nucleus , ventrolateral medulla , nucleus ambiguous , and the spinal cord , as reviewed elsewhere.93 another critical component of the central melanocortin system within the arc is the neurons expressing neuropeptide y ( npy ) and the potent mc3r / mc4r antagonist agrp ( figure 2 ) . the npy / agrp neurons have the same distribution as the pomc / cart neurons within the hypothalamus , with the densest fibers innervating the pvh , dorsomedial hypothalamus ( dmh ) , posterior hypothalamus , and septal regions around the anterior commissure.94 the npy / agrp neurons form synapses with the pomc / cart neurons within the arc , thus producing a neuronal network that is responsive to the modulatory effects of several appetite and body weight regulating hormones such as leptin , ghrelin , insulin , and peptide yy ( pyy).73,9598 leptin acts via hypothalamic receptors ( ob - r ) to decrease feeding and increase thermogenesis , resulting in a decrease in body weight . pomc / cart and npy / agrp neurons in the arc are the principal sites of leptin receptor expression and the source of potent neuropeptide hormones , -msh and npy , which exert opposing effects on feeding and metabolism as shown in figure 2 . subpopulations of npy / agrp neurons that also express gamma - aminobutyric acid ( gaba ) send inhibitory collaterals to the pomc / cart neurons . gaba inhibits the pomc / cart neurons and blocks the anorexic effect of -msh ( figure 2).99 using electrophysiological techniques , cowley et al showed that leptin stimulates the pomc / cart neurons via two mechanisms : 1 ) depolarization through a nonspecific cation channel and 2 ) hyperpolarization of npy / agrp neurons , leading to a reduction in the release of gaba that , in turn , causes disinhibition of the pomc / cart neurons ( figure 2).73 ghrelin , the endogenous ligand for growth hormone secretagogue receptor ( ghs - r ) , is a potent stimulant of growth hormone release and plays an important role in appetite control and body weight regulation . circulating ghrelin levels are markedly increased with fasting and before meals and decrease following meals.100,101 plasma ghrelin levels are also influenced by long - term energy balance and are increased in anorexia and decreased in obesity.102,103 within the arc , gsh - r is expressed on the npy / agrp neurons which are thought to mediate the orexigenic effects of ghrelin . central and peripheral administration of ghrelin induces c - fos in these neurons and increases hypothalamic npy and agrp mrna expression , thus antagonizing the anorexic effects of leptin.95,98,104 also , electrophysiological studies have shown that ghrelin directly activates the orexigenic npy / agrp neurons while coordinately inhibiting the anorexogenic pomc / cart neurons via increased gaba release on them ( figure 2).105 stimulation of food intake by ghrelin is blocked by administration of npy antagonist and is reduced in npy mice.95 lastly , mc3r and mc4r knockout mice show reduced sensitivity to ghrelin as evidenced by decreased ghrelin - induced food intake and growth hormone secretion , thus suggesting an important role of mc3r and mc4r in mediating the orexigenic effects of ghrelin.106 npy is a potent hypothalamic orexigenic peptide , probably the most powerful stimulant of appetite known . nyp mrna expression in the hypothalamus is significantly increased during lactation and fasting.107 central administration of npy causes robust increase in food intake and body weight in rats.108,109 chronic intracerebroventricular administration of npy to normal rats produces hyperphagia , hyperinsulinemia , and liver and adipose tissue lipogenesis , thus mimicking the hormonal and metabolic changes of obesity.110 recent evidence suggests that ectopic overexpression of npy in other areas of the hypothalamus such as pvh , lateral hypothalamus , and dmh also increases food intake and body weight and that npy knockdown in the dmh ameliorates the hyperphagia , obesity , and diabetes of otsuka long - evans tokushima fatty ( oletf ) rats.111,112 thus , npy in the hypothalamus plays an important role in modulating food intake and body weight ( figure 2 ) . npy exerts its orexigenic effects probably by inhibiting the arc pomc / cart neurons via its y2 receptor.113 npy is metabolized by several peptidases in the plasma . recent evidence suggests that npy(136 ) is metabolized into three major fragments : npy(336 ) , npy(335 ) , and npy(235 ) , upon incubation with human serum.114 specific inhibitors of dipeptidyl peptidase 4 , plasma kallikrein , and aminopeptidase p prevent the production of npy(336 ) , npy(335 ) , and npy(236 ) , respectively . plasma kallikrein metabolizes npy(336 ) to npy(335 ) . since npy(335 ) is unable to bind to npy y1 , y2 , and y5 receptors , npy(335 ) may represent the major metabolic end product of the y2/y5 agonist , npy(336 ) . a recent study identified two splice variants of prcp ; the second isoform was named prcp2 ( ncbi : nm_199418 ) . unlike prcp , prcp2 has a longer transcript and a unique amino - terminal region . although its full - length sequence is known , there is no evidence suggesting whether prcp2 mrna encodes a functional protein.24 the prcp gene is speculated to be a candidate gene for essential hypertension.115 mutational analysis of the human prcp has led to a better understanding of prcp - catalyzed reactions . certain putative mutant forms of human prcp apparently predispose the polymorphic carriers to cardiovascular diseases including hypertension and the risk of preeclampsia.116 the e112d polymorphism in the prcp gene leads to increased antihypertensive effect of benazepril treatment in hypertensive patients.117 we have also demonstrated that prcp mice have mild hypertension.20 our recent studies demonstrated that the prcp - null ( prcp ) mice ate less and had even less fat than the mice with partial loss of the enzyme.26 these observations suggest that prcp is a genetic marker for weight regulation and putative prcp single nucleotide polymorphism ( snp ) variants are associated with mild hypertension . continued identification of prcp mutations , full - characterization of prcp knockout mice , and studies with knock - in mice with prcp / prcp mutations will provide evidence that prcp is a disease - causing gene for both obesity and hypertension . meanwhile , we propose that the use of prcp inhibitors should be strongly indicated by a diagnosis of obesity in patients with no systolic or diastolic deterioration . the prcp - catalyzed reaction was initially found to be part of the pathway for angiotensin ii ( ang ii ) metabolism in renal tissues , where prcp appeared to control the total amount of ang ii . odya and others demonstrated that prcp metabolizes ang ii to angiotensin 17 ( ang 17 ) ( figure 1).7 the activation of ang 17 receptor mas ( a g - protein - coupled protein ) by ang 17 results in the generation of nitric oxide ( no ) and prostaglandins.8 thus , ang 17 counteracts ang ii function , providing evidence that prcp regulates the negative effects of ang ii such as high blood pressure and heart failure.9 in addition , the activation of the ang 17 receptor mas may also lead to diminished cell proliferation through down - regulation of the phosphorylation and activation of erk1 and erk2 in the erk1/erk2 map kinase signaling pathway.10,11 in theory , the prcp inhibitors to target the production of pro - inflammatory prostaglandins and promote proliferation through the ang 17 receptor mas - dependent pathway represent a novel approach to suppress unwanted inflammation - causing prostaglandins . later , it was shown that prcp is one of several enzymes that convert ang ii to a unique bioactive molecule . in vitro studies showed that angiotensin - converting enzyme 2 ( ace2 ) is an exopeptidase that converts ang ii to ang 17 at a much faster rate than prcp.12 these data suggest that ang ii is a poor substrate for prcp . clinical studies have provided reliable evidence that ace2 is an essential regulator of angiotensin i ( ang i ) , ang ii , and angiotensin - induced cardiac hypertrophy.13 recent studies clearly show increased myocardial levels of ang ii and a significant decrease in ang 17 in ace2-deficient hearts , suggesting that the role of prcp in metabolizing ang ii may be insignificant.9 taken together , these observations suggest that prcp is a redundant catalyst contributing to alternate pathways for ang ii metabolism . while the well - established cardiovascular and renal actions of ang ii are attributed to the angiotensin type 1 receptor ( at1r ) , much less is known about angiotensin iii and its cardiovascular effects . for more than 30 years , it was known that prcp metabolizes ang iii to ang 27 ( figure 1).7 soon after , studies demonstrated that ang iii is a pressor agent whose response , like that of ang ii , is mediated by at1 receptors.14,15 apparently , ang iii has multiple effects on renal function in the diseased kidney and can enhance renal disease through the overproduction of aldosterone , leading to arterial hypertension and/or atrial fibrillation.16 aldosterone maintains blood volume , pressure , and electrolyte balance . its production is known to be regulated by renin , an enzyme produced in the kidneys . renin increases in response to low blood pressure , decreased blood flow to the kidneys , or sodium deficiency . the elevation of renin results in an increase in synthesis and secretion of aldosterone . studies indicate that ang iii also activates the secretion of aldosterone.15 recently , we have demonstrated that recombinant prcp ( rpcrp ) metabolizes ang iii to ang 27 , removing phenylalanine ( phe).8 it is tempting to speculate that prcp might funnel the generation of angiotensin 34 ( ang 34 ) through ang 27 ( figure 1 ) . if the over - secretion of aldosterone by ang iii is viewed as a trigger of arterial hypertension , then inactivation of ang iii by prcp might lead to a decrease in blood pressure . further studies are required to determine whether prcp is critically important for regulating ang iii - induced hypertension and preserving renal structure and function . this is an important area of research to pursue given the increasing prevalence of cardiovascular disease and stroke in the older population . the possible actions of another substrate of prcp , plasma prekallikrein ( pk , fletcher factor ) , have recently begun to receive much attention . when the complex of high molecular weight kininogen ( hk ) and pk binds to endothelial membrane , pk is rapidly converted to kallikrein by prcp.17 the formed kallikrein then cleaves hk to liberate bradykinin ( bk ) , which leads to no and prostaglandin - i2 formation , as well as subsequent vasodilation , by activating constitutive bradykinin b2 and inducible bradykinin b1 receptors.18,19 the prcp - dependent pk activation pathway might be considered an additional mechanism to preserve the availability of no and prostacyclin as vasodilatory agents in vascular smooth muscle . , we have found that prcp mice have mild hypertension , suggesting a causative relationship between prcp levels and signs of hypertension.20 local skeletal muscle ischemia and acidosis are shown to increase the generation of bk and prostaglandins , the two circulating products of the prcp - induced cell activation ( figure 1).21 the increased acidotic response during exercise and inflammatory mediators such as bk and prostacyclin have been shown to cause abnormal exercise - related symptoms and autonomic responses in congestive heart failure syndrome.22 nonetheless , the long - term elevated concentrations of no and prostacyclin through prcp - dependent pathways may be detrimental and eventually responsible for cardiovascular diseases such as congestive heart disease . since bk and prostaglandins exacerbate the genesis of the symptoms of exercise intolerance in heart failure,23 the inhibitors of prcp might be effective in ameliorating the exercise - limiting symptoms . clinical studies demonstrate that prcp is involved in the pathogenesis of inflammatory conditions such as rheumatoid arthritis and infection.24 melanocortin peptides have numerous effects on the host such as the modulation of fever , inflammation and appetite.25 recently , we showed that prcp metabolizes alpha - melanocyte - stimulating hormone 113 ( -msh113 ) to alpha - melanocyte - stimulating hormone 112 ( -msh112),26 ( figure 1 ) . -msh113 is a potent anti - inflammatory agent.27 in addition to the specificity of cleavage , the cellular release of pro - inflammatory mediators seemed to be critical to prcp actions . in theory , agents that increase production and effects of -msh113 could be used to counteract the effects of pro - inflammatory mediators such as bradykinin and cytokines ( figure 1 ) . obesity is known to cause inflammation and insulin resistance in the vasculature and non - vascular tissues involved in glucose metabolism.28 evidence suggests that hyperglycemia may contribute to defective no - dependent vasodilation in diabetes.29 the inducible no synthase ( inos ) expression is elevated in adipose tissue of obese people compared to those of lean people30 and is a mediator of inflammation and a key enzyme in insulin resistance.31 the colocalization of -msh113 receptors ( mc4r ) with inos has been reported , suggesting a role for -msh113 in obese people.32 the inactivation of -msh113 by prcp provides a positive feedback loop for postprandial enhancement of food intake and inflammation by inhibiting -msh113 function , as shown in figure 2.26 since prcp regulates the anorectic action of -msh113 , this study highlights the presence of a newly recognized interaction between inflammation , obesity , and the expression and activity of prcp ( figure 2).26 in view of the above studies , we consider that prcp may be a key player in the obesity - associated metabolic complications , inflammatory response , and the host defense mechanism . the following sections emphasize pertinent findings , which best describe the theoretical perspective on the components of the central melanocortin system and stress the importance of prcp influence in the melanogenic signaling pathway . pro - opiomelanocortin ( pomc ) , a prohormone with molecular weight of 31 kda , is ubiquitously expressed in various tissues of mammals.33,34 pomc expression in the central nervous system , however , is limited to the arcuate nucleus of the hypothalamus ( arc ) , nucleus tractus solitarius of the caudal medulla ( nts ) , and corticotrophs and melanotrophs of the anterior pituitary ( figure 2).35 the 1200 base pair pomc transcript encodes for the 267 amino acid prohormone with an n - terminal signal peptide of 26 residues.36 as this precursor peptide passes through the golgi stacks , it is targeted , via a specific signal peptide , into regulated secretory granules.37 pomc undergoes extensive posttranslational modification within these secretory granules mediated by a family of serine proteases , the prohormone convertases ( pcs ) , as illustrated in figure 3 . pomc is cleaved by prohormone convertase 1 ( pc1 ) to produce 22 kda pro - acth and -lipoprotein hormone ( -lph ) ( figure 3).38 pro - acth is further cleaved by pc1 to produce the n - terminus of pomc - joining peptide and 4.5 kda acth . prohormone convertase 2 ( pc2 ) cleaves acth to acth 117 and corticotropin - like intermediate lobe peptide ( clip ) and -lph to -lipoprotein hormone ( -lph ) and the synthesis of -msh from pomc involves several specific enzymes in addition to pc1 and pc2 . first , carboxypeptidase e cleaves the c - terminal basic amino acid residues of acth 117 . the peptide is then amidated by peptidyl -amidating monooxygenase ( pam ) to produce desacetyl -msh ( des--msh ) . finally , des--msh is acetylated by n - acetyltransferase ( nat ) to produce acetylated -msh ( act--msh ) , as illustrated in figure 2 . act--msh is more potent than des--msh in activating melanocortin receptor signaling and in reducing food intake , effects that are likely due to rapid degradation of des--msh . guo et al have shown that total hypothalamic -msh levels are decreased in leptin - deficient ob / ob mice and increased in leptin - treated ob / ob and c57bl/6j mice . leptin specifically enhances hypothalamic levels of act--msh without significantly affecting the amounts of des--msh , possibly by activating nat in the pomc neurons.39 the melanocortin receptors are g - protein - coupled receptors with characteristic seven transmembrane domains . mc5r.4043 the melanocortin receptors are gs - coupled and signal via the adenylate cyclase - camp - protein kinase a second messenger pathway . however , depending on the cell type and the melanocortin receptor expression , signal transduction pathways other than camp may be activated which include inositol triphosphate - diacyl glycerol - protein kinase c ( ip3-dag - pkc ) pathway , extracellular ca influx , map kinase pathway , and the jak / stat pathway.4448 mc1r , mc3r , mc4r and mc5r show 40%60% amino acid homology . the natural msh peptides have a conserved sequence , his - phe - arg - trp , which plays an important role in the binding of these peptides to specific melanocortin receptors.49 mc1r was the first melanocortin receptor to be cloned and was isolated from human melanoma cell line.40 - , - and -msh and acth are the known agonists whereas agouti is the known antagonist of the mc1r . mc1r is expressed in human and mouse melanoma cells , human melanocytes , skin glands , and hair follicles.40,5053 -msh , agouti , and mc1r , therefore , play an important role in regulating skin pigmentation and hair color . the presence of mc1r in the testes and the pituitary has been demonstrated by chhajlani et al.54 mc1r expression in the central nervous system is limited to neurons of the periaqueductal grey in both rat and human brain.55 however , mc1r is widely expressed in cells involved in inflammation such as endothelial cells , neutrophils , monocytes , macrophages , fibroblasts and astrocytes.25,5659 -msh has been shown to inhibit inflammation via mc1r - mediated decrease in the production of inflammatory cytokines such as il-1 , il-6 , and tumor necrosis factor - alpha ( tnf ) , as well as suppression of nf-b.6062 mc2r is encoded by a single gene localized to chromosome 18p11.2.53 acth is the only known agonist of the mc2r.63 using in situ hybridization , xia et al showed that mc2r is highly expressed in the adrenal cortex with the highest expression in the zona fasciculata and zona glomerulosa and relatively low expression in the zona reticularis.64 these findings are consistent with the role of mc2r in mediating the effect of acth on the synthesis and release of corticosteroids . besides the adrenal cortex , mc2r is expressed in murine adipocytes , which explains the lipolytic effect of acth.59 however , human adipocytes do not express mc2r and there is no evidence to suggest that human adipose tissue is responsive to the lipolytic effect of acth.54 these species differences in the expression of mc2r may be important when studying the role of melanocortins in obesity . mc2r expression has also been demonstrated in the skin along with three cytochrome enzymes involved in steroid hormone synthesis.65 kapas et al have shown that acth produced in the skin by keratinocytes stimulates dna synthesis and induces cell proliferation via mc2r.66 mc2r , therefore , appears to play an important role in cutaneous pathophysiology . mc3r is encoded by a single gene localized to the q13.2q13.3 region of chromosome 20.67 since this locus is associated with type 2 noninsulin - dependent diabetes mellitus ( niddm ) , mc3r could represent a candidate gene for niddm.68 mc3r is unique in that it binds to - , - and -msh with similar affinities . using northern blot hybridization and polymerase chain reaction , gantz et al first showed that mc3r is expressed in brain , placental , and gut tissues but not in melanoma cells or in adrenal glands.69 mc3r expression in the brain is highest in the hypothalamus especially in the arcuate nucleus , ventromedial nucleus , preoptic nucleus , lateral hypothalamic area , and posterior hypothalamic area.43 agouti - related protein ( agrp ) , which is normally expressed in the hypothalamus , is a potent antagonist of both mc3r and mc4r.70 however , pomc and agrp neurons in the arcuate nucleus of hypothalamus selectively express mc3r , and not mc4r , suggesting that mc3r might function as a presynaptic autoreceptor regulating the release of melanocortins.71,72 physiological support for this hypothesis was provided by cowley et al,73 who demonstrated that the selective mc3r agonist , d - trp--msh inhibited firing of gfp - labeled pomc neurons in the pvn . also , marks et al showed that peripheral administration of the mc3r agonist stimulates feeding via mc3r - mediated inhibition of the arc pomc neurons.74 an association between mc3r and human obesity has been identified by linkage studies . several sequence variants have been found in the mc3r coding region and in 5 flanking sequences.75 mc3r variants are associated with subtle changes in weight , leptin levels , and insulin - glucose ratios , but none of these explain human morbid obesity.76 a novel heterozygous mutation i183n in mc3r was identified in two obese patients of the same family.77 functional characterization of i183n showed that this mutation completely abolished the activity of the mutated receptor to stimulate intracellular camp production , suggesting that i183n might play an important role in obesity.78 similarly , tao et al showed that a novel mutation i335s in mc3r results in complete loss of ligand binding and signaling suggesting that this mutation might contribute to obesity.79 however , a recent study evaluating the functional consequences of all mutations found in mc3r and mc4r in severely obese north american adults concluded that mc4r , but not mc3r mutations are associated with severe obesity in this population.80 thus , the significance of mc3r mutations in human obesity is still not conclusively established to date . mc4r was first cloned by gantz et al in 1993.42 mc4r is a 332 amino acid protein encoded by a single gene , localized to chromosome 18q21.3 . using northern blot analysis and in situ hybridization , mc4r expression was notably absent in the adrenal cortex , melanocytes , and placenta.42 mc4r is widely distributed in the central nervous system , especially in the cortex , hippocampus , amygdala , septum , corpus striatum , nucleus accumbens , hypothalamus , nucleus tractus solitarius , visual and motor nuclei of the brainstem , and the dorsal horn of the spinal cord.81 -msh , -msh , and acth are the known agonists and agrp is the known antagonist of the mc4r ( figure 2).70 since mc4r is highly expressed in the hypothalamus and has a strong affinity for -msh it is believed to be a strong candidate for energy balance , appetite control , and body weight regulation . mc4r knockout mice have been shown to develop a maturity onset obesity syndrome characterized by hyperphagia , hyperglycemia and hyperinsulinemia.82 since this syndrome is similar to the agouti obesity syndrome seen in avy/ mice and agrp - transgenic mice that overexpress agouti and agrp respectively , it is speculated that the primary mechanism by which agouti and agrp produce obesity is chronic antagonism of mc4r.83 cachexia , a chronic wasting syndrome characterized by loss of body weight and muscle mass , is commonly associated with diseases such as cancer and aids . mc4r mice and mice treated with agrp are resistant to lipopolysaccharide- or tumor - induced cachexia , further supporting the role of mc4r in energy balance and body weight regulation.84 mc5r is a 325 amino acid protein encoded by a single gene located on chromosome 18p11.2.85 mc5r has been shown to bind to all melanocortins except -msh.86 mc5r is the most widely expressed melanocortin receptor . mc5r mrna is expressed in the adrenal gland , adipose tissue , kidney , leukocytes , lung , lymph node , mammary gland , ovary , pituitary , testis , and uterus.54 mc5r is highly expressed in exocrine glands such as lacrimal , preputial , harderian and sebaceous glands.87 mc5r - deficient mice have a severe defect in water repulsion and thermoregulation due to decreased production of sebaceous lipids . studies in humans have shown that mc5r immunoreactivity is detectable in the epithelium and appendages , including the sebaceous , eccrine , and apocrine glands . however , analysis of mc5r variations in patients with acne , hidradenitis suppurativa , and sebaceous gland dysfunction have failed to suggest a causative role of mc5r in these conditions.88 low levels of mc5r mrna have also been reported in the central nervous system,89 however , the physiological function of mc5r in the brain remains unclear . linkage analysis in the quebec family study90 revealed a significant association of mc5r polymorphisms with body mass index , fat mass , and resting metabolic rate , thus providing some evidence for the possible role of mc5r in energy balance and body weight regulation . the pomc neurons , which produce -msh , also express another anorectic peptide cocaine - amphetamine - related transcript ( cart ) . cell bodies of the pomc / cart neurons are found throughout the rostrocaudal extent of the arcuate nucleus , as well as the periarcuate area of the hypothalamus . within the hypothalamus , these neurons project to the periventricular nucleus , paraventricular nucleus ( pvh ) , and the perifornical region.91,92 the pomc / cart neurons also project to the brainstem to innervate the rostral nts , lateral reticular nucleus , ventrolateral medulla , nucleus ambiguous , and the spinal cord , as reviewed elsewhere.93 another critical component of the central melanocortin system within the arc is the neurons expressing neuropeptide y ( npy ) and the potent mc3r / mc4r antagonist agrp ( figure 2 ) . the npy / agrp neurons have the same distribution as the pomc / cart neurons within the hypothalamus , with the densest fibers innervating the pvh , dorsomedial hypothalamus ( dmh ) , posterior hypothalamus , and septal regions around the anterior commissure.94 the npy / agrp neurons form synapses with the pomc / cart neurons within the arc , thus producing a neuronal network that is responsive to the modulatory effects of several appetite and body weight regulating hormones such as leptin , ghrelin , insulin , and peptide yy ( pyy).73,9598 leptin acts via hypothalamic receptors ( ob - r ) to decrease feeding and increase thermogenesis , resulting in a decrease in body weight . pomc / cart and npy / agrp neurons in the arc are the principal sites of leptin receptor expression and the source of potent neuropeptide hormones , -msh and npy , which exert opposing effects on feeding and metabolism as shown in figure 2 . subpopulations of npy / agrp neurons that also express gamma - aminobutyric acid ( gaba ) send inhibitory collaterals to the pomc / cart neurons . gaba inhibits the pomc / cart neurons and blocks the anorexic effect of -msh ( figure 2).99 using electrophysiological techniques , cowley et al showed that leptin stimulates the pomc / cart neurons via two mechanisms : 1 ) depolarization through a nonspecific cation channel and 2 ) hyperpolarization of npy / agrp neurons , leading to a reduction in the release of gaba that , in turn , causes disinhibition of the pomc / cart neurons ( figure 2).73 ghrelin , the endogenous ligand for growth hormone secretagogue receptor ( ghs - r ) , is a potent stimulant of growth hormone release and plays an important role in appetite control and body weight regulation . circulating ghrelin levels are markedly increased with fasting and before meals and decrease following meals.100,101 plasma ghrelin levels are also influenced by long - term energy balance and are increased in anorexia and decreased in obesity.102,103 within the arc , gsh - r is expressed on the npy / agrp neurons which are thought to mediate the orexigenic effects of ghrelin . central and peripheral administration of ghrelin induces c - fos in these neurons and increases hypothalamic npy and agrp mrna expression , thus antagonizing the anorexic effects of leptin.95,98,104 also , electrophysiological studies have shown that ghrelin directly activates the orexigenic npy / agrp neurons while coordinately inhibiting the anorexogenic pomc / cart neurons via increased gaba release on them ( figure 2).105 stimulation of food intake by ghrelin is blocked by administration of npy antagonist and is reduced in npy mice.95 lastly , mc3r and mc4r knockout mice show reduced sensitivity to ghrelin as evidenced by decreased ghrelin - induced food intake and growth hormone secretion , thus suggesting an important role of mc3r and mc4r in mediating the orexigenic effects of ghrelin.106 npy is a potent hypothalamic orexigenic peptide , probably the most powerful stimulant of appetite known . nyp mrna expression in the hypothalamus is significantly increased during lactation and fasting.107 central administration of npy causes robust increase in food intake and body weight in rats.108,109 chronic intracerebroventricular administration of npy to normal rats produces hyperphagia , hyperinsulinemia , and liver and adipose tissue lipogenesis , thus mimicking the hormonal and metabolic changes of obesity.110 recent evidence suggests that ectopic overexpression of npy in other areas of the hypothalamus such as pvh , lateral hypothalamus , and dmh also increases food intake and body weight and that npy knockdown in the dmh ameliorates the hyperphagia , obesity , and diabetes of otsuka long - evans tokushima fatty ( oletf ) rats.111,112 thus , npy in the hypothalamus plays an important role in modulating food intake and body weight ( figure 2 ) . npy exerts its orexigenic effects probably by inhibiting the arc pomc / cart neurons via its y2 receptor.113 npy is metabolized by several peptidases in the plasma . recent evidence suggests that npy(136 ) is metabolized into three major fragments : npy(336 ) , npy(335 ) , and npy(235 ) , upon incubation with human serum.114 specific inhibitors of dipeptidyl peptidase 4 , plasma kallikrein , and aminopeptidase p prevent the production of npy(336 ) , npy(335 ) , and npy(236 ) , respectively . plasma kallikrein metabolizes npy(336 ) to npy(335 ) . since npy(335 ) is unable to bind to npy y1 , y2 , and y5 receptors , npy(335 ) may represent the major metabolic end product of the y2/y5 agonist , npy(336 ) . a recent study identified two splice variants of prcp ; the second isoform was named prcp2 ( ncbi : nm_199418 ) . unlike prcp , prcp2 has a longer transcript and a unique amino - terminal region . although its full - length sequence is known , there is no evidence suggesting whether prcp2 mrna encodes a functional protein.24 the prcp gene is speculated to be a candidate gene for essential hypertension.115 mutational analysis of the human prcp has led to a better understanding of prcp - catalyzed reactions . certain putative mutant forms of human prcp apparently predispose the polymorphic carriers to cardiovascular diseases including hypertension and the risk of preeclampsia.116 the e112d polymorphism in the prcp gene leads to increased antihypertensive effect of benazepril treatment in hypertensive patients.117 we have also demonstrated that prcp mice have mild hypertension.20 our recent studies demonstrated that the prcp - null ( prcp ) mice ate less and had even less fat than the mice with partial loss of the enzyme.26 these observations suggest that prcp is a genetic marker for weight regulation and putative prcp single nucleotide polymorphism ( snp ) variants are associated with mild hypertension . continued identification of prcp mutations , full - characterization of prcp knockout mice , and studies with knock - in mice with prcp / prcp mutations will provide evidence that prcp is a disease - causing gene for both obesity and hypertension . meanwhile , we propose that the use of prcp inhibitors should be strongly indicated by a diagnosis of obesity in patients with no systolic or diastolic deterioration . pro - opiomelanocortin ( pomc ) , a prohormone with molecular weight of 31 kda , is ubiquitously expressed in various tissues of mammals.33,34 pomc expression in the central nervous system , however , is limited to the arcuate nucleus of the hypothalamus ( arc ) , nucleus tractus solitarius of the caudal medulla ( nts ) , and corticotrophs and melanotrophs of the anterior pituitary ( figure 2).35 the 1200 base pair pomc transcript encodes for the 267 amino acid prohormone with an n - terminal signal peptide of 26 residues.36 as this precursor peptide passes through the golgi stacks , it is targeted , via a specific signal peptide , into regulated secretory granules.37 pomc undergoes extensive posttranslational modification within these secretory granules mediated by a family of serine proteases , the prohormone convertases ( pcs ) , as illustrated in figure 3 . pomc is cleaved by prohormone convertase 1 ( pc1 ) to produce 22 kda pro - acth and -lipoprotein hormone ( -lph ) ( figure 3).38 pro - acth is further cleaved by pc1 to produce the n - terminus of pomc - joining peptide and 4.5 kda acth . prohormone convertase 2 ( pc2 ) cleaves acth to acth 117 and corticotropin - like intermediate lobe peptide ( clip ) and -lph to -lipoprotein hormone ( -lph ) and the synthesis of -msh from pomc involves several specific enzymes in addition to pc1 and pc2 . first , carboxypeptidase e cleaves the c - terminal basic amino acid residues of acth 117 . the peptide is then amidated by peptidyl -amidating monooxygenase ( pam ) to produce desacetyl -msh ( des--msh ) . finally , des--msh is acetylated by n - acetyltransferase ( nat ) to produce acetylated -msh ( act--msh ) , as illustrated in figure 2 . act--msh is more potent than des--msh in activating melanocortin receptor signaling and in reducing food intake , effects that are likely due to rapid degradation of des--msh . guo et al have shown that total hypothalamic -msh levels are decreased in leptin - deficient ob / ob mice and increased in leptin - treated ob / ob and c57bl/6j mice . leptin specifically enhances hypothalamic levels of act--msh without significantly affecting the amounts of des--msh , possibly by activating nat in the pomc neurons.39 the melanocortin receptors are g - protein - coupled receptors with characteristic seven transmembrane domains . mc5r.4043 the melanocortin receptors are gs - coupled and signal via the adenylate cyclase - camp - protein kinase a second messenger pathway . however , depending on the cell type and the melanocortin receptor expression , signal transduction pathways other than camp may be activated which include inositol triphosphate - diacyl glycerol - protein kinase c ( ip3-dag - pkc ) pathway , extracellular ca influx , map kinase pathway , and the jak / stat pathway.4448 mc1r , mc3r , mc4r and mc5r show 40%60% amino acid homology . the natural msh peptides have a conserved sequence , his - phe - arg - trp , which plays an important role in the binding of these peptides to specific melanocortin receptors.49 mc1r was the first melanocortin receptor to be cloned and was isolated from human melanoma cell line.40 - , - and -msh and acth are the known agonists whereas agouti is the known antagonist of the mc1r . mc1r is expressed in human and mouse melanoma cells , human melanocytes , skin glands , and hair follicles.40,5053 -msh , agouti , and mc1r , therefore , play an important role in regulating skin pigmentation and hair color . the presence of mc1r in the testes and the pituitary has been demonstrated by chhajlani et al.54 mc1r expression in the central nervous system is limited to neurons of the periaqueductal grey in both rat and human brain.55 however , mc1r is widely expressed in cells involved in inflammation such as endothelial cells , neutrophils , monocytes , macrophages , fibroblasts and astrocytes.25,5659 -msh has been shown to inhibit inflammation via mc1r - mediated decrease in the production of inflammatory cytokines such as il-1 , il-6 , and tumor necrosis factor - alpha ( tnf ) , as well as suppression of nf-b.6062 mc2r is encoded by a single gene localized to chromosome 18p11.2.53 acth is the only known agonist of the mc2r.63 using in situ hybridization , xia et al showed that mc2r is highly expressed in the adrenal cortex with the highest expression in the zona fasciculata and zona glomerulosa and relatively low expression in the zona reticularis.64 these findings are consistent with the role of mc2r in mediating the effect of acth on the synthesis and release of corticosteroids . besides the adrenal cortex , mc2r is expressed in murine adipocytes , which explains the lipolytic effect of acth.59 however , human adipocytes do not express mc2r and there is no evidence to suggest that human adipose tissue is responsive to the lipolytic effect of acth.54 these species differences in the expression of mc2r may be important when studying the role of melanocortins in obesity . mc2r expression has also been demonstrated in the skin along with three cytochrome enzymes involved in steroid hormone synthesis.65 kapas et al have shown that acth produced in the skin by keratinocytes stimulates dna synthesis and induces cell proliferation via mc2r.66 mc2r , therefore , appears to play an important role in cutaneous pathophysiology . mc3r is encoded by a single gene localized to the q13.2q13.3 region of chromosome 20.67 since this locus is associated with type 2 noninsulin - dependent diabetes mellitus ( niddm ) , mc3r could represent a candidate gene for niddm.68 mc3r is unique in that it binds to - , - and -msh with similar affinities . using northern blot hybridization and polymerase chain reaction , gantz et al first showed that mc3r is expressed in brain , placental , and gut tissues but not in melanoma cells or in adrenal glands.69 mc3r expression in the brain is highest in the hypothalamus especially in the arcuate nucleus , ventromedial nucleus , preoptic nucleus , lateral hypothalamic area , and posterior hypothalamic area.43 agouti - related protein ( agrp ) , which is normally expressed in the hypothalamus , is a potent antagonist of both mc3r and mc4r.70 however , pomc and agrp neurons in the arcuate nucleus of hypothalamus selectively express mc3r , and not mc4r , suggesting that mc3r might function as a presynaptic autoreceptor regulating the release of melanocortins.71,72 physiological support for this hypothesis was provided by cowley et al,73 who demonstrated that the selective mc3r agonist , d - trp--msh inhibited firing of gfp - labeled pomc neurons in the pvn . also , marks et al showed that peripheral administration of the mc3r agonist stimulates feeding via mc3r - mediated inhibition of the arc pomc neurons.74 an association between mc3r and human obesity has been identified by linkage studies . several sequence variants have been found in the mc3r coding region and in 5 flanking sequences.75 mc3r variants are associated with subtle changes in weight , leptin levels , and insulin - glucose ratios , but none of these explain human morbid obesity.76 a novel heterozygous mutation i183n in mc3r was identified in two obese patients of the same family.77 functional characterization of i183n showed that this mutation completely abolished the activity of the mutated receptor to stimulate intracellular camp production , suggesting that i183n might play an important role in obesity.78 similarly , tao et al showed that a novel mutation i335s in mc3r results in complete loss of ligand binding and signaling suggesting that this mutation might contribute to obesity.79 however , a recent study evaluating the functional consequences of all mutations found in mc3r and mc4r in severely obese north american adults concluded that mc4r , but not mc3r mutations are associated with severe obesity in this population.80 thus , the significance of mc3r mutations in human obesity is still not conclusively established to date . mc4r was first cloned by gantz et al in 1993.42 mc4r is a 332 amino acid protein encoded by a single gene , localized to chromosome 18q21.3 . using northern blot analysis and in situ hybridization , mc4r was originally found to be expressed primarily in the brain . mc4r expression was notably absent in the adrenal cortex , melanocytes , and placenta.42 mc4r is widely distributed in the central nervous system , especially in the cortex , hippocampus , amygdala , septum , corpus striatum , nucleus accumbens , hypothalamus , nucleus tractus solitarius , visual and motor nuclei of the brainstem , and the dorsal horn of the spinal cord.81 -msh , -msh , and acth are the known agonists and agrp is the known antagonist of the mc4r ( figure 2).70 since mc4r is highly expressed in the hypothalamus and has a strong affinity for -msh it is believed to be a strong candidate for energy balance , appetite control , and body weight regulation . mc4r knockout mice have been shown to develop a maturity onset obesity syndrome characterized by hyperphagia , hyperglycemia and hyperinsulinemia.82 since this syndrome is similar to the agouti obesity syndrome seen in avy/ mice and agrp - transgenic mice that overexpress agouti and agrp respectively , it is speculated that the primary mechanism by which agouti and agrp produce obesity is chronic antagonism of mc4r.83 cachexia , a chronic wasting syndrome characterized by loss of body weight and muscle mass , is commonly associated with diseases such as cancer and aids . mc4r mice and mice treated with agrp are resistant to lipopolysaccharide- or tumor - induced cachexia , further supporting the role of mc4r in energy balance and body weight regulation.84 mc5r is a 325 amino acid protein encoded by a single gene located on chromosome 18p11.2.85 mc5r has been shown to bind to all melanocortins except -msh.86 mc5r is the most widely expressed melanocortin receptor . mc5r mrna is expressed in the adrenal gland , adipose tissue , kidney , leukocytes , lung , lymph node , mammary gland , ovary , pituitary , testis , and uterus.54 mc5r is highly expressed in exocrine glands such as lacrimal , preputial , harderian and sebaceous glands.87 mc5r - deficient mice have a severe defect in water repulsion and thermoregulation due to decreased production of sebaceous lipids . studies in humans have shown that mc5r immunoreactivity is detectable in the epithelium and appendages , including the sebaceous , eccrine , and apocrine glands . however , analysis of mc5r variations in patients with acne , hidradenitis suppurativa , and sebaceous gland dysfunction have failed to suggest a causative role of mc5r in these conditions.88 low levels of mc5r mrna have also been reported in the central nervous system,89 however , the physiological function of mc5r in the brain remains unclear . linkage analysis in the quebec family study90 revealed a significant association of mc5r polymorphisms with body mass index , fat mass , and resting metabolic rate , thus providing some evidence for the possible role of mc5r in energy balance and body weight regulation . the pomc neurons , which produce -msh , also express another anorectic peptide cocaine - amphetamine - related transcript ( cart ) . cell bodies of the pomc / cart neurons are found throughout the rostrocaudal extent of the arcuate nucleus , as well as the periarcuate area of the hypothalamus . within the hypothalamus , these neurons project to the periventricular nucleus , paraventricular nucleus ( pvh ) , and the perifornical region.91,92 the pomc / cart neurons also project to the brainstem to innervate the rostral nts , lateral reticular nucleus , ventrolateral medulla , nucleus ambiguous , and the spinal cord , as reviewed elsewhere.93 another critical component of the central melanocortin system within the arc is the neurons expressing neuropeptide y ( npy ) and the potent mc3r / mc4r antagonist agrp ( figure 2 ) . the npy / agrp neurons have the same distribution as the pomc / cart neurons within the hypothalamus , with the densest fibers innervating the pvh , dorsomedial hypothalamus ( dmh ) , posterior hypothalamus , and septal regions around the anterior commissure.94 the npy / agrp neurons form synapses with the pomc / cart neurons within the arc , thus producing a neuronal network that is responsive to the modulatory effects of several appetite and body weight regulating hormones such as leptin , ghrelin , insulin , and peptide yy ( pyy).73,9598 leptin acts via hypothalamic receptors ( ob - r ) to decrease feeding and increase thermogenesis , resulting in a decrease in body weight . pomc / cart and npy / agrp neurons in the arc are the principal sites of leptin receptor expression and the source of potent neuropeptide hormones , -msh and npy , which exert opposing effects on feeding and metabolism as shown in figure 2 . subpopulations of npy / agrp neurons that also express gamma - aminobutyric acid ( gaba ) send inhibitory collaterals to the pomc / cart neurons . gaba inhibits the pomc / cart neurons and blocks the anorexic effect of -msh ( figure 2).99 using electrophysiological techniques , cowley et al showed that leptin stimulates the pomc / cart neurons via two mechanisms : 1 ) depolarization through a nonspecific cation channel and 2 ) hyperpolarization of npy / agrp neurons , leading to a reduction in the release of gaba that , in turn , causes disinhibition of the pomc / cart neurons ( figure 2).73 ghrelin , the endogenous ligand for growth hormone secretagogue receptor ( ghs - r ) , is a potent stimulant of growth hormone release and plays an important role in appetite control and body weight regulation . circulating ghrelin levels are markedly increased with fasting and before meals and decrease following meals.100,101 plasma ghrelin levels are also influenced by long - term energy balance and are increased in anorexia and decreased in obesity.102,103 within the arc , gsh - r is expressed on the npy / agrp neurons which are thought to mediate the orexigenic effects of ghrelin . central and peripheral administration of ghrelin induces c - fos in these neurons and increases hypothalamic npy and agrp mrna expression , thus antagonizing the anorexic effects of leptin.95,98,104 also , electrophysiological studies have shown that ghrelin directly activates the orexigenic npy / agrp neurons while coordinately inhibiting the anorexogenic pomc / cart neurons via increased gaba release on them ( figure 2).105 stimulation of food intake by ghrelin is blocked by administration of npy antagonist and is reduced in npy mice.95 lastly , mc3r and mc4r knockout mice show reduced sensitivity to ghrelin as evidenced by decreased ghrelin - induced food intake and growth hormone secretion , thus suggesting an important role of mc3r and mc4r in mediating the orexigenic effects of ghrelin.106 npy is a potent hypothalamic orexigenic peptide , probably the most powerful stimulant of appetite known . nyp mrna expression in the hypothalamus is significantly increased during lactation and fasting.107 central administration of npy causes robust increase in food intake and body weight in rats.108,109 chronic intracerebroventricular administration of npy to normal rats produces hyperphagia , hyperinsulinemia , and liver and adipose tissue lipogenesis , thus mimicking the hormonal and metabolic changes of obesity.110 recent evidence suggests that ectopic overexpression of npy in other areas of the hypothalamus such as pvh , lateral hypothalamus , and dmh also increases food intake and body weight and that npy knockdown in the dmh ameliorates the hyperphagia , obesity , and diabetes of otsuka long - evans tokushima fatty ( oletf ) rats.111,112 thus , npy in the hypothalamus plays an important role in modulating food intake and body weight ( figure 2 ) . npy exerts its orexigenic effects probably by inhibiting the arc pomc / cart neurons via its y2 receptor.113 npy is metabolized by several peptidases in the plasma . recent evidence suggests that npy(136 ) is metabolized into three major fragments : npy(336 ) , npy(335 ) , and npy(235 ) , upon incubation with human serum.114 specific inhibitors of dipeptidyl peptidase 4 , plasma kallikrein , and aminopeptidase p prevent the production of npy(336 ) , npy(335 ) , and npy(236 ) , respectively . plasma kallikrein metabolizes npy(336 ) to npy(335 ) . since npy(335 ) is unable to bind to npy y1 , y2 , and y5 receptors , npy(335 ) may represent the major metabolic end product of the y2/y5 agonist , npy(336 ) . a recent study identified two splice variants of prcp ; the second isoform was named prcp2 ( ncbi : nm_199418 ) . unlike prcp , prcp2 has a longer transcript and a unique amino - terminal region . although its full - length sequence is known , there is no evidence suggesting whether prcp2 mrna encodes a functional protein.24 the prcp gene is speculated to be a candidate gene for essential hypertension.115 mutational analysis of the human prcp has led to a better understanding of prcp - catalyzed reactions . certain putative mutant forms of human prcp apparently predispose the polymorphic carriers to cardiovascular diseases including hypertension and the risk of preeclampsia.116 the e112d polymorphism in the prcp gene leads to increased antihypertensive effect of benazepril treatment in hypertensive patients.117 we have also demonstrated that prcp mice have mild hypertension.20 our recent studies demonstrated that the prcp - null ( prcp ) mice ate less and had even less fat than the mice with partial loss of the enzyme.26 these observations suggest that prcp is a genetic marker for weight regulation and putative prcp single nucleotide polymorphism ( snp ) variants are associated with mild hypertension . continued identification of prcp mutations , full - characterization of prcp knockout mice , and studies with knock - in mice with prcp / prcp mutations will provide evidence that prcp is a disease - causing gene for both obesity and hypertension . meanwhile , we propose that the use of prcp inhibitors should be strongly indicated by a diagnosis of obesity in patients with no systolic or diastolic deterioration . obesity is an emerging worldwide public health hazard and is associated with significant morbidity and mortality . although the physiological determinants of normal / abnormal eating behavior have been investigated , the underlying causes and mechanisms of dysregulation of food intake in obesity , type 2 diabetes , and metabolic syndrome are not well understood . the long - lasting challenge for clinicians and scientists in basic research to unfold the major cause of the dysregulation of the food intake is becoming close to the last battle . clinical studies indicate that the molecular and cellular mechanisms by which leptin and alpha - melanocyte stimulation hormone ( -msh ) modulate each other s activity result in the regulation of food intake and energy expenditure.118 these studies suggest that -msh is intimately involved in the regulatory mechanism of obesity , energy expenditure , and body weight . recently , prolylcarboxypeptidase ( prcp ) was found to be responsible for the control of food intake and energy expenditure at a central level . the molecular mechanisms underlying the suppression of food intake in prcp - deficient mice or by the inhibitor of prcp clearly provide physiological evidence that prcp is an inactivator of -msh.26 thus , prcp is emerging as a new identity involved in the control of food intake and energy metabolism . since -msh can activate both melanocortin 4 receptors ( mc4r ) and melanocortin 1 receptors ( mc1r ) in a decreasing order , the catalysis of -msh113 to -msh112 by prcp would lead to the suppression of both mc4r and mc1r activation as shown in figure 4 . although there are various contributing factors for obesity , the recent research findings indicate that prcp is involved in the development of weight gain and obesity . an increase in prcp expression or activity may result in obesity due to an imbalance between energy intake and energy expenditure . although regulation of -msh113-mediated mc4r activation described above is demonstrably important , the importance of the role of prcp on the signaling mechanisms of mc1r in anti - inflammatory response remains an enigma ( figure 4).119 additional studies are needed to determine whether or not prcp regulates -msh113-mediated mc1r activation . the cellular role of prcp is beginning to be unraveled both at the molecular and physiological levels . the upregulation of human prcp expression during inflammation has been described.24 on other hand , the prcp knockout mice study demonstrates that prcp is an appetite stimulant . therefore , the pleiotropic effects of prcp include increased no and prostaglandin bioavailability , decreased vasoconstriction , and increased appetite . in summary , knowledge of the role of prcp in mouse appetite regulation thus , research on prcp--msh interactions may be important to the further understanding of human obesity . in addition , the identification of prcp as an inactivator of -msh should provide an attractive therapeutic target in the fight against obesity . however , due to its pleiotropic effects , the prcp inhibitors must be scrutinized carefully to optimize their use in the treatment and prevention of obesity and obesity - related diseases .

recently , we serendipitously discovered that mice with the deficiency of the enzyme prolylcarboxypeptidase ( prcp ) have elevated -melanocyte - stimulating hormone ( -msh ) levels which lead to decreased food intake and weight loss . this suggests that prcp is an endogenous inactivator of -msh and an appetite stimulant . since a modest weight loss can have the most profound influence on reducing cardiovascular risk factors , the inhibitors of prcp would be emerging as a possible alternative for pharmacotherapy in high - risk patients with obesity and obesity - related disorders . the discovery of a new biological activity of prcp in the prcp - deficient mice and studies of -msh function indicate the importance and complexity of the hypothalamic pro - opiomelanocortin ( pomc ) system in altering food intake . identifying a role for prcp in regulating -msh in the brain may be a critical step in enhancing our understanding of how the brain controls food intake and body weight . in light of recent findings , the potential role of prcp in regulating fuel homeostasis is critically evaluated . further studies of the role of prcp in obesity are much needed .

Introduction The current scope of prolylcarboxypeptidase Prolylcarboxypeptidases physiological function and relationship with hypothalamic appetite-regulating pathways Synthesis of melanocyte stimulating hormone Five distinct central melanocortin receptors with different physiological functions Appetite is tightly controlled by the relative hypothalamic levels of -MSH Insight into the physiological functions of PRCP through genetic studies in mice Future perspectives

moreover , suboptimal doses of sr 141716 ( rimonabant ) together with suboptimal doses of -melanocyte - stimulating hormone ( -msh ) are known to behave synergistically in order to reduce food intake . the role of the melanocortin system in food intake is well - established and prevention of the rapid inactivation of -msh may prove to be a better alternative pathway for potential obesity treatments . recent studies suggest that prolylcarboxypeptidase ( prcp ) involved in regulating blood pressure and inflammation is an appetite stimulant and , by consequence , prcp inhibitors may prove to be a viable lead to treat obesity . odya and others demonstrated that prcp metabolizes ang ii to angiotensin 17 ( ang 17 ) ( figure 1).7 the activation of ang 17 receptor mas ( a g - protein - coupled protein ) by ang 17 results in the generation of nitric oxide ( no ) and prostaglandins.8 thus , ang 17 counteracts ang ii function , providing evidence that prcp regulates the negative effects of ang ii such as high blood pressure and heart failure.9 in addition , the activation of the ang 17 receptor mas may also lead to diminished cell proliferation through down - regulation of the phosphorylation and activation of erk1 and erk2 in the erk1/erk2 map kinase signaling pathway.10,11 in theory , the prcp inhibitors to target the production of pro - inflammatory prostaglandins and promote proliferation through the ang 17 receptor mas - dependent pathway represent a novel approach to suppress unwanted inflammation - causing prostaglandins . clinical studies have provided reliable evidence that ace2 is an essential regulator of angiotensin i ( ang i ) , ang ii , and angiotensin - induced cardiac hypertrophy.13 recent studies clearly show increased myocardial levels of ang ii and a significant decrease in ang 17 in ace2-deficient hearts , suggesting that the role of prcp in metabolizing ang ii may be insignificant.9 taken together , these observations suggest that prcp is a redundant catalyst contributing to alternate pathways for ang ii metabolism . in accordance , we have found that prcp mice have mild hypertension , suggesting a causative relationship between prcp levels and signs of hypertension.20 local skeletal muscle ischemia and acidosis are shown to increase the generation of bk and prostaglandins , the two circulating products of the prcp - induced cell activation ( figure 1).21 the increased acidotic response during exercise and inflammatory mediators such as bk and prostacyclin have been shown to cause abnormal exercise - related symptoms and autonomic responses in congestive heart failure syndrome.22 nonetheless , the long - term elevated concentrations of no and prostacyclin through prcp - dependent pathways may be detrimental and eventually responsible for cardiovascular diseases such as congestive heart disease . clinical studies demonstrate that prcp is involved in the pathogenesis of inflammatory conditions such as rheumatoid arthritis and infection.24 melanocortin peptides have numerous effects on the host such as the modulation of fever , inflammation and appetite.25 recently , we showed that prcp metabolizes alpha - melanocyte - stimulating hormone 113 ( -msh113 ) to alpha - melanocyte - stimulating hormone 112 ( -msh112),26 ( figure 1 ) . obesity is known to cause inflammation and insulin resistance in the vasculature and non - vascular tissues involved in glucose metabolism.28 evidence suggests that hyperglycemia may contribute to defective no - dependent vasodilation in diabetes.29 the inducible no synthase ( inos ) expression is elevated in adipose tissue of obese people compared to those of lean people30 and is a mediator of inflammation and a key enzyme in insulin resistance.31 the colocalization of -msh113 receptors ( mc4r ) with inos has been reported , suggesting a role for -msh113 in obese people.32 the inactivation of -msh113 by prcp provides a positive feedback loop for postprandial enhancement of food intake and inflammation by inhibiting -msh113 function , as shown in figure 2.26 since prcp regulates the anorectic action of -msh113 , this study highlights the presence of a newly recognized interaction between inflammation , obesity , and the expression and activity of prcp ( figure 2).26 in view of the above studies , we consider that prcp may be a key player in the obesity - associated metabolic complications , inflammatory response , and the host defense mechanism . pro - opiomelanocortin ( pomc ) , a prohormone with molecular weight of 31 kda , is ubiquitously expressed in various tissues of mammals.33,34 pomc expression in the central nervous system , however , is limited to the arcuate nucleus of the hypothalamus ( arc ) , nucleus tractus solitarius of the caudal medulla ( nts ) , and corticotrophs and melanotrophs of the anterior pituitary ( figure 2).35 the 1200 base pair pomc transcript encodes for the 267 amino acid prohormone with an n - terminal signal peptide of 26 residues.36 as this precursor peptide passes through the golgi stacks , it is targeted , via a specific signal peptide , into regulated secretory granules.37 pomc undergoes extensive posttranslational modification within these secretory granules mediated by a family of serine proteases , the prohormone convertases ( pcs ) , as illustrated in figure 3 . gaba inhibits the pomc / cart neurons and blocks the anorexic effect of -msh ( figure 2).99 using electrophysiological techniques , cowley et al showed that leptin stimulates the pomc / cart neurons via two mechanisms : 1 ) depolarization through a nonspecific cation channel and 2 ) hyperpolarization of npy / agrp neurons , leading to a reduction in the release of gaba that , in turn , causes disinhibition of the pomc / cart neurons ( figure 2).73 ghrelin , the endogenous ligand for growth hormone secretagogue receptor ( ghs - r ) , is a potent stimulant of growth hormone release and plays an important role in appetite control and body weight regulation . nyp mrna expression in the hypothalamus is significantly increased during lactation and fasting.107 central administration of npy causes robust increase in food intake and body weight in rats.108,109 chronic intracerebroventricular administration of npy to normal rats produces hyperphagia , hyperinsulinemia , and liver and adipose tissue lipogenesis , thus mimicking the hormonal and metabolic changes of obesity.110 recent evidence suggests that ectopic overexpression of npy in other areas of the hypothalamus such as pvh , lateral hypothalamus , and dmh also increases food intake and body weight and that npy knockdown in the dmh ameliorates the hyperphagia , obesity , and diabetes of otsuka long - evans tokushima fatty ( oletf ) rats.111,112 thus , npy in the hypothalamus plays an important role in modulating food intake and body weight ( figure 2 ) . certain putative mutant forms of human prcp apparently predispose the polymorphic carriers to cardiovascular diseases including hypertension and the risk of preeclampsia.116 the e112d polymorphism in the prcp gene leads to increased antihypertensive effect of benazepril treatment in hypertensive patients.117 we have also demonstrated that prcp mice have mild hypertension.20 our recent studies demonstrated that the prcp - null ( prcp ) mice ate less and had even less fat than the mice with partial loss of the enzyme.26 these observations suggest that prcp is a genetic marker for weight regulation and putative prcp single nucleotide polymorphism ( snp ) variants are associated with mild hypertension . odya and others demonstrated that prcp metabolizes ang ii to angiotensin 17 ( ang 17 ) ( figure 1).7 the activation of ang 17 receptor mas ( a g - protein - coupled protein ) by ang 17 results in the generation of nitric oxide ( no ) and prostaglandins.8 thus , ang 17 counteracts ang ii function , providing evidence that prcp regulates the negative effects of ang ii such as high blood pressure and heart failure.9 in addition , the activation of the ang 17 receptor mas may also lead to diminished cell proliferation through down - regulation of the phosphorylation and activation of erk1 and erk2 in the erk1/erk2 map kinase signaling pathway.10,11 in theory , the prcp inhibitors to target the production of pro - inflammatory prostaglandins and promote proliferation through the ang 17 receptor mas - dependent pathway represent a novel approach to suppress unwanted inflammation - causing prostaglandins . clinical studies have provided reliable evidence that ace2 is an essential regulator of angiotensin i ( ang i ) , ang ii , and angiotensin - induced cardiac hypertrophy.13 recent studies clearly show increased myocardial levels of ang ii and a significant decrease in ang 17 in ace2-deficient hearts , suggesting that the role of prcp in metabolizing ang ii may be insignificant.9 taken together , these observations suggest that prcp is a redundant catalyst contributing to alternate pathways for ang ii metabolism . , we have found that prcp mice have mild hypertension , suggesting a causative relationship between prcp levels and signs of hypertension.20 local skeletal muscle ischemia and acidosis are shown to increase the generation of bk and prostaglandins , the two circulating products of the prcp - induced cell activation ( figure 1).21 the increased acidotic response during exercise and inflammatory mediators such as bk and prostacyclin have been shown to cause abnormal exercise - related symptoms and autonomic responses in congestive heart failure syndrome.22 nonetheless , the long - term elevated concentrations of no and prostacyclin through prcp - dependent pathways may be detrimental and eventually responsible for cardiovascular diseases such as congestive heart disease . clinical studies demonstrate that prcp is involved in the pathogenesis of inflammatory conditions such as rheumatoid arthritis and infection.24 melanocortin peptides have numerous effects on the host such as the modulation of fever , inflammation and appetite.25 recently , we showed that prcp metabolizes alpha - melanocyte - stimulating hormone 113 ( -msh113 ) to alpha - melanocyte - stimulating hormone 112 ( -msh112),26 ( figure 1 ) . obesity is known to cause inflammation and insulin resistance in the vasculature and non - vascular tissues involved in glucose metabolism.28 evidence suggests that hyperglycemia may contribute to defective no - dependent vasodilation in diabetes.29 the inducible no synthase ( inos ) expression is elevated in adipose tissue of obese people compared to those of lean people30 and is a mediator of inflammation and a key enzyme in insulin resistance.31 the colocalization of -msh113 receptors ( mc4r ) with inos has been reported , suggesting a role for -msh113 in obese people.32 the inactivation of -msh113 by prcp provides a positive feedback loop for postprandial enhancement of food intake and inflammation by inhibiting -msh113 function , as shown in figure 2.26 since prcp regulates the anorectic action of -msh113 , this study highlights the presence of a newly recognized interaction between inflammation , obesity , and the expression and activity of prcp ( figure 2).26 in view of the above studies , we consider that prcp may be a key player in the obesity - associated metabolic complications , inflammatory response , and the host defense mechanism . pro - opiomelanocortin ( pomc ) , a prohormone with molecular weight of 31 kda , is ubiquitously expressed in various tissues of mammals.33,34 pomc expression in the central nervous system , however , is limited to the arcuate nucleus of the hypothalamus ( arc ) , nucleus tractus solitarius of the caudal medulla ( nts ) , and corticotrophs and melanotrophs of the anterior pituitary ( figure 2).35 the 1200 base pair pomc transcript encodes for the 267 amino acid prohormone with an n - terminal signal peptide of 26 residues.36 as this precursor peptide passes through the golgi stacks , it is targeted , via a specific signal peptide , into regulated secretory granules.37 pomc undergoes extensive posttranslational modification within these secretory granules mediated by a family of serine proteases , the prohormone convertases ( pcs ) , as illustrated in figure 3 . gaba inhibits the pomc / cart neurons and blocks the anorexic effect of -msh ( figure 2).99 using electrophysiological techniques , cowley et al showed that leptin stimulates the pomc / cart neurons via two mechanisms : 1 ) depolarization through a nonspecific cation channel and 2 ) hyperpolarization of npy / agrp neurons , leading to a reduction in the release of gaba that , in turn , causes disinhibition of the pomc / cart neurons ( figure 2).73 ghrelin , the endogenous ligand for growth hormone secretagogue receptor ( ghs - r ) , is a potent stimulant of growth hormone release and plays an important role in appetite control and body weight regulation . nyp mrna expression in the hypothalamus is significantly increased during lactation and fasting.107 central administration of npy causes robust increase in food intake and body weight in rats.108,109 chronic intracerebroventricular administration of npy to normal rats produces hyperphagia , hyperinsulinemia , and liver and adipose tissue lipogenesis , thus mimicking the hormonal and metabolic changes of obesity.110 recent evidence suggests that ectopic overexpression of npy in other areas of the hypothalamus such as pvh , lateral hypothalamus , and dmh also increases food intake and body weight and that npy knockdown in the dmh ameliorates the hyperphagia , obesity , and diabetes of otsuka long - evans tokushima fatty ( oletf ) rats.111,112 thus , npy in the hypothalamus plays an important role in modulating food intake and body weight ( figure 2 ) . certain putative mutant forms of human prcp apparently predispose the polymorphic carriers to cardiovascular diseases including hypertension and the risk of preeclampsia.116 the e112d polymorphism in the prcp gene leads to increased antihypertensive effect of benazepril treatment in hypertensive patients.117 we have also demonstrated that prcp mice have mild hypertension.20 our recent studies demonstrated that the prcp - null ( prcp ) mice ate less and had even less fat than the mice with partial loss of the enzyme.26 these observations suggest that prcp is a genetic marker for weight regulation and putative prcp single nucleotide polymorphism ( snp ) variants are associated with mild hypertension . pro - opiomelanocortin ( pomc ) , a prohormone with molecular weight of 31 kda , is ubiquitously expressed in various tissues of mammals.33,34 pomc expression in the central nervous system , however , is limited to the arcuate nucleus of the hypothalamus ( arc ) , nucleus tractus solitarius of the caudal medulla ( nts ) , and corticotrophs and melanotrophs of the anterior pituitary ( figure 2).35 the 1200 base pair pomc transcript encodes for the 267 amino acid prohormone with an n - terminal signal peptide of 26 residues.36 as this precursor peptide passes through the golgi stacks , it is targeted , via a specific signal peptide , into regulated secretory granules.37 pomc undergoes extensive posttranslational modification within these secretory granules mediated by a family of serine proteases , the prohormone convertases ( pcs ) , as illustrated in figure 3 . gaba inhibits the pomc / cart neurons and blocks the anorexic effect of -msh ( figure 2).99 using electrophysiological techniques , cowley et al showed that leptin stimulates the pomc / cart neurons via two mechanisms : 1 ) depolarization through a nonspecific cation channel and 2 ) hyperpolarization of npy / agrp neurons , leading to a reduction in the release of gaba that , in turn , causes disinhibition of the pomc / cart neurons ( figure 2).73 ghrelin , the endogenous ligand for growth hormone secretagogue receptor ( ghs - r ) , is a potent stimulant of growth hormone release and plays an important role in appetite control and body weight regulation . nyp mrna expression in the hypothalamus is significantly increased during lactation and fasting.107 central administration of npy causes robust increase in food intake and body weight in rats.108,109 chronic intracerebroventricular administration of npy to normal rats produces hyperphagia , hyperinsulinemia , and liver and adipose tissue lipogenesis , thus mimicking the hormonal and metabolic changes of obesity.110 recent evidence suggests that ectopic overexpression of npy in other areas of the hypothalamus such as pvh , lateral hypothalamus , and dmh also increases food intake and body weight and that npy knockdown in the dmh ameliorates the hyperphagia , obesity , and diabetes of otsuka long - evans tokushima fatty ( oletf ) rats.111,112 thus , npy in the hypothalamus plays an important role in modulating food intake and body weight ( figure 2 ) . certain putative mutant forms of human prcp apparently predispose the polymorphic carriers to cardiovascular diseases including hypertension and the risk of preeclampsia.116 the e112d polymorphism in the prcp gene leads to increased antihypertensive effect of benazepril treatment in hypertensive patients.117 we have also demonstrated that prcp mice have mild hypertension.20 our recent studies demonstrated that the prcp - null ( prcp ) mice ate less and had even less fat than the mice with partial loss of the enzyme.26 these observations suggest that prcp is a genetic marker for weight regulation and putative prcp single nucleotide polymorphism ( snp ) variants are associated with mild hypertension . clinical studies indicate that the molecular and cellular mechanisms by which leptin and alpha - melanocyte stimulation hormone ( -msh ) modulate each other s activity result in the regulation of food intake and energy expenditure.118 these studies suggest that -msh is intimately involved in the regulatory mechanism of obesity , energy expenditure , and body weight . the molecular mechanisms underlying the suppression of food intake in prcp - deficient mice or by the inhibitor of prcp clearly provide physiological evidence that prcp is an inactivator of -msh.26 thus , prcp is emerging as a new identity involved in the control of food intake and energy metabolism . the upregulation of human prcp expression during inflammation has been described.24 on other hand , the prcp knockout mice study demonstrates that prcp is an appetite stimulant . in summary , knowledge of the role of prcp in mouse appetite regulation thus , research on prcp--msh interactions may be important to the further understanding of human obesity . however , due to its pleiotropic effects , the prcp inhibitors must be scrutinized carefully to optimize their use in the treatment and prevention of obesity and obesity - related diseases .

folic acid ( fa ) targeting has been extensively studied for improving the therapeutic index of drugs . although the molecular - level structure of this interaction has only recently been fully elucidated , substantial progress has still been made over the past 20 years in fa targeting , with seven drug conjugates advancing to clinical trials . targeting of a drug or drug conjugate exploits the interaction of this vitamin with a high affinity ( kd 0.1 nm ) folic acid receptor ( far ) , which is overexpressed in many cancer cells . this receptor is also found in healthy epithelial cells ; however , these are generally inaccessible to fa bearing conjugates in the blood , enabling this system to exploit cytotoxic effects of drugs while minimizing collateral damage in healthy tissues . in addition to cell surface targeting , fa conjugation provides a selective uptake pathway for the conjugated drug via folate receptor mediated endocytosis and release of the fa / conjugate from the receptor and endosome . many targeted small molecule delivery designs take advantage of this highly specific interaction including examples such as doxorubicin , methotrexate , protein toxins , imaging agents , and immunotherapeutics both in vitro and in vivo by exploiting carrier mechanisms including liposomes , inorganic nanoparticles , and organic polymers . multivalent conjugates of ligands to nanomaterials are often employed purposefully to increase the avidity and/or specificity of an interaction or accidentally as a result of stochastic synthetic approaches . the enthalpic and entropic mechanisms through which multivalency increases the interaction of a ligand and its target have been extensively studied from a theoretical viewpoint . briefly , there are two main multivalent effects that may contribute to the system studied here ; those dependent on the increased effective ( or local ) concentration , and those due to multiple binding events occurring for a single conjugate . higher local concentrations can result in higher affinities , and an increased chance of rebinding upon dissociation of the initial interaction ( statistical rebinding ) or secondary binding events . multivalent classifications have been discussed and reviewed elsewhere by kiessling , whitesides , and cloninger . although multivalent conjugates of many dyes , drugs , and targeting ligands ( including fa ) have been developed , the actual impact of the specific number of ligands on improvements in avidity and/or biological activity has been difficult to analyze due to the heterogeneous mixtures generated by stochastic conjugation chemistries employed in their synthesis . for example , a stochastic conjugation of 3 equiv of fa to a scaffold with multiple functionalizable sites ( 30 ) results in a sample with a mean of 3 fas per scaffold , but also a distribution of unique conjugates with fa - to - scaffold ratios ranging from 0 to 11 fa molecules per scaffold ( figure 1 ) . distribution of conjugates resulting from a stochastic conjugation of 3 equiv of fa to 1 equiv of scaffold . previous efforts to quantify multivalent binding constants have employed surface plasmon resonance ( spr ) to measure increases in binding between materials containing different average numbers of ligands ( folic acid and methotrexate ) and folate binding protein ( fbp ) modified surfaces . the binding constant of folic acid to fbp , kd 510 m , is roughly 1000-fold weaker than observed for folate receptor . although these studies have reported a general trend of greater avidity with increased valency , the utilization of materials containing a distribution of ligand - to - scaffold ratios complicated understanding the mechanisms involved in multivalent binding , or elucidation of the relative activity of the various components in the sample . for example , does the entire population illustrated in figure 1 with two or more conjugated fas ( 80% of the population ) enable equivalent receptor clustering in a cell ? or does a higher valency , and consequently higher effective concentration , such as 511 fas per scaffold ( 18% of the population ) produce all of the observed activity ? poly(amidoamine ) ( pamam ) dendrimer is an extensively studied vector for the multivalent , targeted delivery of drugs , genes , and imaging agents . the dendritic architecture has many advantages for biomedical applications , including low polydispersity , internal core space available for the entrapment of drugs , and multiple branches providing terminal groups for functionalization . pamam dendrimer is particularly suited for such applications due to its protein - like architecture , low immunogenicity , ability to solubilize hydrophobic small molecules , and easily functionalized primary amine terminal surface groups . the size of generation 5 ( g5 ) pamam ( 5.4 nm diameter ) is also ideal for vascular delivery and excretion due to kidney filtration . recent advancements have enabled the isolation of monomeric g5 pamam dendrimers from oligomeric ( dimer , trimer , etc . ) and trailing generation defects ( g1g4 ) , narrowing the experimentally realized size distribution of this vector from 1115 kda ( commercial material ) to 2529 kda . possible convolution of results by large mass differences and vector - accessible surface area is eliminated by removing both trailing generations and oligomers from the g5 pamam monomer material . in 2007 , employed spr to measure the increased avidity to fbp and cellular uptake of g5 pamam fa conjugates as a function of average number of attached fas ( figure 2 ) . the dissociation constant ( kd ) was observed to exponentially decrease as the average valency of fa increased ; however , this calculation assumed that given a long enough experiment all bound materials would dissociate from the surface and that the experimental sensorgram would return to the level of signal present prior to g5fan(avg ) exposure . the nonlinear ( exponential ) behavior in kd was attributed to a saturation of fa fbp binding events limited by the immobilized protein density on the spr flow cell surface and not to the valency of fa ( figure 2a ) . interestingly , the same trend in signal saturation as a function of fa valency was observed for mean fluorescence , as measured by flow cytometry when equivalent conjugates labeled with a dye were evaluated for binding to far upregulated kb cells . this observation was interpreted as an indication that the dendrimer conjugates do not trigger receptor clustering on the cell surface , which would allow for higher affinities as more proteins became available . ( b ) any multivalent binding ( 2 or more interactions ) is irreversible , and monovalent binding is reversible . ( c ) fa keys the initial interaction between conjugate and fbp , which is followed by strong nonspecific interaction between the dendrimer and protein . c represents g5fan conjugate , p is fbp , cp a complex between a conjugate and n 1 fbp . cp * is a tight complex formed by a conformation change in the polymer and the resulting polymer protein interaction . subsequent analyses of this data , employing different assumptions , resulted in two alternate mechanisms for explaining the changes in binding as a function of average valency . in 2010 , waddell , sander et al . reanalyzed the original data set and proposed that the binding of the conjugates occurs via two distinct interactions . this mechanism acknowledges the broad distribution of ligand - to - dendrimer ratios present in stochastically synthesized materials , including dendrimers that have zero fa , one fa , or two or more fas . it was proposed that ( 1 ) monovalent interaction between g5fa1 and one fbp is attributable to binding that is reversible on the time scale of the experiment and ( 2 ) multivalent binding between g5fa2 to two or more fbps is irreversible on the spr experimental time scale ( figure 2b ) . waddell , sander , et al . hypothesized that the increased avidity attributed to valency increase by banaszak holl et al . actually arises from decreased amounts of zero - functional and monofunctional conjugates in the stochastic average material . the original flow cytometry data can be similarly interpreted ; receptor clustering is achieved by bivalent conjugates , and further increasing of valency has no measurable effect on the cell . a very different mechanism based on kinetic limitations of cooperativity was proposed by licata and tkachenko in 2008 . this study concludes that the increased avidity proposed for the g5fan(avg ) conjugates is higher than can be attributed to cumulative effects of multivalent binding and that kinetic limitations actually prevent the type of multivalent interactions proposed in figures 2a and 2b . they propose that the enhanced interaction observed by spr is a result of van der waals interactions between the polymer vector and protein / chip surface that are enabled by a single key the broad distribution of folic acid - to - dendrimer ratios present in each sample , including both monovalent and multivalent conjugates in the low average materials , prevented a clear experimental elucidation between the three models depicted in figure 2 . in particular , a conjugate with a precise ratio of 1 fa per dendrimer ( g5fa1 ) was lacking to determine if the observed increase in avidity was a product of multivalent binding between the conjugate and spr surface ( banaszak holl and sander mechanisms ) or a single fa fbp lock - and - key combined with van der waals polymer / surface interaction ( licata and tkachenko mechanism ) . in order to address these materials - based challenges to understanding multivalency , we have developed click chemistry and reverse - phase high performance liquid chromatography ( rp - hplc ) methods to isolate dendrimers conjugated to precise numbers of ligands ( i.e. , g5-lx , x = 04 , where x is not a mean value ) . these methodologies , which have been previously demonstrated to be successful for azide and fluorinated , ring - strain - promoted click ligands , are now extended to a second ring strain promoted ligand ( cyclooct-1-yne-3-glycolic acid ( cog ) ) , which has been used in previous g5fan(avg ) spr studies . in principle , isolating the precise ratio samples g5fax , x = 1 , 2 , 3 , etc . , would allow spr experiments where the multivalent binding effect is decoupled from the heterogeneity of stochastic samples ( g5fan(avg ) ) . the isolated g5-lx were clicked with a -azide - lys - asp - fa derivative ( -azide - fa ) . the resulting samples include a g5-pamam dendrimer with a fa - to - dendrimer ratio of 0.96 that contains no detectable multivalent g5fa2 species : the sample needed to differentiate the three mechanistic hypotheses proposed to date . the remainder of the click reactions did not proceed with 100% efficiency , but still yielded samples that contained a well - defined high - n cutoff and had a narrower - than - stochastic distribution of fa - to - dendrimer ratios . the binding of these conjugates was analyzed by spr on both high and low fbp density surfaces . the results indicate that , at either surface fbp density , total folic acid concentration is the dominant factor leading to increased amounts of bound material with increased valency . only a small multivalent effect is observed for g5fa2 material because of increased statistical rebinding as compared to g5fa1 . most importantly , the g5fa1 sample exhibited the same irreversible binding to the fbp surface , on the spr time scale , as the g5fa2 samples . this experimental result conclusively rules out the earlier mechanistic hypotheses by banaszak holl et al . and by sander et al . and provides strong experimental support for the key this mechanism falls into the general class of slow - onset , tight binding interactions between ligand and protein albeit with the novel feature of polymer adsorption onto the protein surface to yield the final tight - binding interaction . upon the initial binding event of a single conjugated fa to the fbp , the fbp undergoes a conformational change which exposes a more hydrophilic surface , enabling the irreversible van der waals interaction with the polymer . all chemicals and materials were purchased from sigma - aldrich or fischer scientific and used as received unless otherwise specified . g5 pamam dendrimer was purchased from dendritech and purified as previously reported to remove trailing generation and g5 oligomer impurities . cyclooct-1-yne-3-glycolic acid ( cog ) was synthesized from a modified literature preparation ( see supporting information ) . synthesis and characterization of -azide - lys - asp - folic acid ( -azide - fa ) conjugates were prepared from g5 dendrimer and cog via amide coupling . in brief , amine - terminated g5 ( 202.6 mg ) was dissolved to 0.16 m in di water ( 45 ml ) . cog ( 4.9 mg ) was activated by dissolving to 10.5 m in acetonitrile ( 1.25 ml ) with 2.65 equiv of 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide ( edc ) ( 14.0 mg ) and 2.78 equiv of n - hydroxysuccinimide ( nhs ) ( 9.1 mg ) and stirring for 2 h. the activated cog was added dropwise via syringe pump to the dendrimer solution and stirred overnight . the product was purified using amicon ultra centrifugal units , 10 kda cutoff membranes , with 2 phosphate buffered saline ( pbs ) washes and 4 deionized water ( di ) washes . the material ( 126.4 mg ) was then fully acetylated ( converting 100% of the remaining primary amines to acetyl groups , henceforth designated ac ) by redissolving in anhydrous methanol ( 0.19 m , 24 ml ) and adding 450 equiv of triethylamine ( 305 l ) and 360 equiv of acetic anhydride ( 166 l ) , stirring for 4 h , purified by centrifugation , and isolated by lyophilization . g5-ac - cog4.0(avg ) ( 96.4 mg ) was characterized by rp - uplc . dendrimers with precise ratios of cog ligands per dendrimer were isolated via rp - hplc according to literature procedures . briefly , three 910 l injections at a 32 mg / ml concentration of the averaged material were performed with a c18 column on a water / acetonitrile gradient with 0.1% tfa . fractions were collected as the material eluted and combined to obtain samples with precisely x = 04 cog ligands per dendrimer . products were purified using pd-10 desalting protocols , with di as the equilibration buffer and samples dissolved in 10 pbs , then lyophilized to dry . curve fitting of chromatograms by igor pro was performed to assess purity of precise ratio materials and to determine the average number of cog ligands of stochastic materials ( table s1 in the supporting information ) . dendrimers with well - characterized numbers of covalently bound folic acids were synthesized via click reaction of g5-ac - cogx conjugates and -azide - fa . briefly , 10 equiv of a stock -azide - fa solution ( 77 mm in dmso ) was added to dendrimer conjugates . the resulting mixtures were then brought to a final dendrimer concentration of approximately 310 m to fully dissolve the dendrimer conjugates ( see table s2 in the supporting information for exact amounts used in each reaction ) . solutions were agitated for 48 h , then diluted to 2.5 ml with di , and purified using pd-10 desalting columns , gravity protocols , followed by 16 rounds of dialysis against di . the samples were then further purified by repeating the pd-10 desalting column using 10 pbs to dilute the sample , followed by 2 rounds of dialysis against 1 pbs and 4 rounds against di . curve fitting of chromatograms provided yield , purity , and fa average and distribution species for g5fan materials . h nmr spectra were obtained used 10 s preacquisition delays and a total of 64 scans . all sample solutions were set to a dendrimer concentration of 15 mg / ml in deuterium oxide . cm5 sensor chips were purchased for use in spr experiments from ge healthcare life sciences . spr experiments were conducted in a biacore x instrument ( pharmacia biosensor ab ) . two immobilized folate binding protein ( fbp ) chips were prepared following the suggested protocols : a solution of 0.2 m edc and 0.05 m nhs was used as an activating solution , an immobilization solution of fbp at 1 mg / ml for the low density chip and 1.5 mg / ml for the high density chip , with ethanolamine as the deactivation solution . the surface density of fbp was approximately 10 and 20 ng / mm for the low and high density chips , respectively . flow cell two was employed as a control cell by activating and deactivating the surface without the addition of protein . the chips were characterized using free fa solutions and checked for nonspecific binding with a control of g5-ac containing no cog or fa . the high density chip contains roughly double the amount of immobilized fbp according to total change in response units . conjugate samples were dissolved in fresh hbs - ep buffer at 100 m and serially diluted to 20 , 10 , 5 , 2.5 , and 1.25 m in hbs - ep buffer from fischer scientific . the system was allowed to equilibrate at the beginning of each run for no less than 300 s , followed by a 2 min , 30 l ( 50555 bubble method ) injection . the system was monitored for no less than 500 s postinjection . between each run , the chip was washed with a 5 l injection of ph 1.5 buffer to remove bound materials followed by an instrument prime step . the sensograms represent a subtraction of fc2 ( no protein ) from fc1 ( protein immobilized ) . 96.4 mg of g5-ac - cog conjugate was prepared with an average of 4.0 cogs per dendrimer as calculated by rp - uplc peak fitting ( overall yield 41% ) . all samples were characterized by h nmr spectroscopy ( figure s1 in the supporting information ) and rp - uplc . ( b ) semiprep rp - hplc isolation of pamam with 1 , 2 , 3 , 4 , or 5 cogs . ( c ) isolated samples elute from rp - uplc as a function of ligand - to - dendrimer ratio . dendrimer samples with x = 04 were isolated in quantities ranging from 3 to 8 mg . all samples were characterized by h nmr spectroscopy ( figure s2 in the supporting information ) and rp - uplc ( table s1 in the supporting information ) . one equivalent of g5-ac - cogx and 10x ( x = 14 ) equivalents of -azide - fa were dissolved to give a dendrimer concentration of 310 m in dmso . samples were then desalted according to the manufacturer s gravity protocol with pd-10 desalting columns ( equilibration buffer as di , sample dissolved in 10 pbs ) , and then dialyzed against di using 10 000 da cutoff membranes ( 16 media changes ) . large amounts of unreacted -azide - fa remained after initial purification as detected by rp - uplc . two additional rounds of dialysis against 1 pbs buffer followed by 4 rounds against di removed unreacted -azide - fa as assessed by rp - uplc . samples were characterized by rp - uplc ( figure s3 in the supporting information ) and h nmr spectroscopy ( figure s4 in the supporting information ) . the n = 1 click reaction had an efficiency of 96% , while all other efficiencies ranged from 54 to 64% with mass recoveries over 95% . a detailed analysis of each sample s fractional composition is summarized in table s3 in the supporting information . for these materials , hplc provides the most accurate method for determining conjugate dendrimer ratios ( vide infra and supporting information ) and % fa values are calculated on the basis of hplc data . sensorgrams for g5-ac - fan ( n = 0 , 1.0 , 1.2 , 1.9 , 2.7 ) were collected for both the low ( figure 4 ) and high ( figure 5 ) density chips . the unfunctionalized , neutral conjugate ( n = 0 ) showed no specific binding at either chip density across all concentrations tested . all g5fa conjugates showed specific binding to the fbp immobilized flow cell 1 , which increased in a fa concentration dependent manner . the association and dissociation phases were fit with various models for evaluation of ka , kd , and kd . spr sensograms of conjugates ( n = 1.0 , red ; n = 1.2 , orange ; n = 1.9 , green ; n = 2.7 , blue ) and controls ( n = 0 , gray ; free fa , purple ) on lower density chip . the color gradient represents concentration from low ( light ) to high ( dark ) . free fa samples were run at millimolar as opposed to micromolar concentrations to obtain adequate signal . spr sensograms of conjugates ( n = 1.0 , red ; n = 1.2 , orange ; n = 1.9 , green ; n = 2.7 , blue ) and controls ( n = 0 , gray ; free fa , purple ) on higher density chip . the color gradient represents concentration from low ( light ) to high ( dark ) . rp - hplc is an effective tool for isolating dendrimers with precise numbers of clickable ligands . to date , four unique click ligands have been employed using the same gradient , with functional groups of azide , alkyne , a fluorinated ring strain promoted ligand , and the cyclooctyne ligand presented here for the first time . this robust methodology allows isolation of various species containing single ligand / dendrimer ratios from heterogeneous , averaged samples containing 10 or more species . due to the flexible nature of the pamam dendrimer and transient interaction of the ligand with the hydrophobic column , this technique has proven to be nonspecific to the relative location of the multiple ligands conjugated to the same sample , i.e. , all dendrimer conjugated to three ligands coelutes , simplifying the separation process . isolation of the g5-ac - cogx conjugates utilized in this paper reflect the success of prior studies with other click ligands . all isolated samples of g5-ac - cogx had single species purities over 95% . in the average sample , the most common species was dendrimer conjugated to 2 cog ligands , and this portion comprised only 16% of the sample . however , the isolated sample labeled g5-ac - cog2 contained only g5 conjugated to 2 ligands as measured by rp - uplc , with no detectable presence of dendrimer conjugated to 0 , 1 , 3 , or other numbers of ligands . here , we present the first application of the g5 pamam precise ligand - to - dendrimer ratio materials to a multivalent targeting system . fbp , employed as a model for the far overexpressed in various cancer cell lines , and the interaction of this target with fa has been a highly studied system for both cancer cell targeting of chemotherapeutics and for the more basic understanding of multivalent nanoparticle interactions . to understand how multivalency affects nanoparticle ligand conjugate behavior in biological systems , it is vital to compare monovalent particles to those with 2 or more targeting ligands . however , stochastically synthesized conjugates contain a distribution of ligands per particle , making it difficult to distinguish the behaviors of the individual populations . the controlled ligand / dendrimer ratio conjugates allowed for the synthesis of functional g5fan materials with well - defined subpopulations , including a conjugate with a fa - to - dendrimer ratio of 1 , with no higher valencies present . these materials , when studied by spr , allowed comparison of the binding strength and potential for multivalent interaction of conjugates containing no more than 1 , 2 , 3 , or 4 fa ligands ( table s3 in the supporting information ) . reaction of the cog conjugates with precise ligand - to - dendrimer ratios with complementary click functionalized fa allows for the generation of dendrimers with well - defined numbers of covalently conjugated fas via orthogonal click chemistry between the ring - strained cyclooctyne on the dendrimer and an azido group on the modified fa . the reaction between g5-ac - cog1 and -azide - fa yielded a product that has 96% conjugate with a fa - to - dendrimer ratio of precisely 1 and 4% of a conjugate with no fa . because the original sample had no dendrimer conjugated to 2 or more cog ligands , the resulting product has no material with the ability to undergo multivalent binding . this fact allows us to test both the licata and tkachenko key lock / van der waals interaction mechanism ( figure 2c ) , which attributes the irreversible binding to dendrimer protein van der waals interactions and not multivalent fa binding , and the sander mechanism that assumes that monovalent behavior will significantly differ from bivalent and higher behavior . this critical piece of data would also have prevented the ( incorrect ) assessment by banaszak holl et al . the remaining click reactions with the higher cog valent material went to about 60% completion despite a 10-fold excess of the -azide - fa . this result has been duplicated for g5cogx conjugates with this -azide - fa and other small molecules ( unpublished data ) within the lab , where reaction times greater than 48 h were tested . similar reaction conditions employed in the literature between a g5-ac - cog20(avg ) conjugate and a -azide - modified methotrexate yielded 100% reaction efficiency , however in this case the limiting reagent was the small molecule . this observation suggests that limiting the number of cog ligands on the dendrimer may limit accessibility for click reaction , perhaps via folding of hydrophobic ligands into the dendrimer core . the interior cavity of g5 pamam is limited , therefore with a high number ( i.e. , 20 ) of conjugated cog ligands , the dendrimer can not internalize all the ligands at once , so at any given time cog ligands are available for conjugation . however , at lower numbers of cog ligands ( i.e. , 14 as described here ) there is likely enough void volume in the dendrimer to hold all cog ligands at once , possibly preventing click reaction with solution species . additionally , utilization of click chemistry with -azide - fa eliminates the less active -fa that is bound through the -carboxylic acid . both structural isomers of the click reaction are likely present , although that alone would not be expected to have great effect on binding to the fbp . the presence of both isomers may contribute to peak broadening of the products in rp - uplc ( see figure s3 in the supporting information ) . rp - uplc also provides a useful tool for monitoring the click reaction , as the reaction of the hydrophobic ligand leads to a decrease in retention of the dendrimer conjugate on the c18 column . this technique provides a more accurate measurement of fa - to - dendrimer ratio of the product than techniques such as nmr , which only provides an average number and provides no detail about the individual ligand - to - dendrimer ratios that are present within a sample . for this measurement the nmr spectroscopy based averages suffer from low signal for the conjugated species as compared to the polymer scaffold , and from the polydispersity of the scaffold employed ( see supporting information ) . figure 6a compares the monovalent sample , g5-ac - fa1.0 , to the poisson distribution expected for a stochastically synthesized g5fa conjugate with an average ratio of 1 . by way of comparison , g5-ac - fa1.0 has only 4% unfunctionalized material compared to 37% in the stochastic material . more importantly , 26% of the stochastic material has two or more fas covalently attached , meaning this material is not truly representative of monovalent behavior . the g5-ac - fa1.0 material may only undergo a single , monovalent specific interaction with a single fbp . although the higher fa conjugates are not monodisperse , their heterogeneity has been significantly reduced as compared to an equivalent average stochastic conjugation . rp - uplc has also revealed the relative amount of each ratio present in the samples ( figure 6b d ) , allowing for a much better understanding of the contribution of each for example , the product of the g5-ac - cog3 click reaction ( g5-ac - fa1.9 ) has an average of 2 fas per dendrimer , but uplc reveals that 23% of the material has three fas attached , while 49% has two fas , 24% is monovalent , and 4% of the material has zero fa . the presence of dendrimer conjugated to more than 3 fas is not possible as the starting material contained no dendrimer conjugated to 4 or more cogs . the equivalent stochastic average of n = 1.9 has significant concentrations of 10 unique fa - to - dendrimer ratios ( ranging from 0 to 9 ) , and 15% of the sample has zero fa . the decreased sample complexity and improved characterization for the samples summarized in table s3 in the supporting information allow for more accurate interpretation of subsequent spr results . comparison of distributions in click reaction products vs theoretical stochastically conjugated products ( purple bars ) of the same average for ratios of ( a ) 1.0 ( red bars ) , ( b ) 1.2 ( orange bars ) , ( c ) 1.9 ( green bars ) , and ( d ) 2.7 ( blue bars ) . as illustrated in figures 4 and 5 , g5-ac - fa0 shows no binding to either of the fbp immobilized chips at the concentrations tested . however , g5fan=1.02.7 have binding curves that saturate at higher concentration . the total signal during binding phase ( 0200 s ) ( figure 7 ) increases as a function of polymer concentration , fa valency ( n ) , and density of protein immobilization . definition of fitting parameters . at 200 s , injection is complete and the dissociation phase begins ( figures 4 and 5 ) . first , at free fa concentrations 100-fold higher than the equivalent conjugated fa conditions , free fa returns to baseline in the low density chip and nearly to baseline in the high density chip . this observation is consistent with the expected , reversible binding of fa to fbp . g5-ac - fa0 also returns to baseline , indicating no irreversible interaction with the surface on the time scale of the experiment . most importantly , monovalent g5-ac - fa1.0 has a significantly reduced dissociation rate as compared to fa ( figures 4c and 5c ) . in addition , g5-ac - fa1.0 does not return to baseline during the time scale of the experiment ( 500 s ) at any concentration for either fbp surface density . the dissociation phase levels off substantially above the initial baseline , indicating that a portion of the material remains bound to the surface . this observation is true even though the highest relative fa concentration tested for g5-ac - fa1.0 ( 10 m ) is 25 times lower than the lowest fa concentration ( 0.25 mm ) . in other words , the dendrimer conjugate binds much more tightly than free fa ( kd 510 m ) . the irreversible binding on the time scale of the spr experiment has previously been attributed to multivalent binding between the conjugate and receptor , however that can not be the case for this purely monovalent conjugate . lock / van der waals binding mechanism proposed by licata and tkachenko in which only one fa to fbp interaction is necessary to initiate the stronger interaction between the dendrimer and fbp , which itself is a result of the summation of many weak van der waals interactions . have demonstrated using fluorescence spectroscopy that the tryptophan residues reorient upon folic acid binding to the fbp interior generating a more hydrophilic protein surface . we hypothesize that this reorientation leads to the large increase in polymer protein binding strength when fa is conjugated to the polymer . all g5-ac - fan=1.02.7 have dissociation sensorgrams similar to those previously reported results on both the high and low density chips . all samples have a portion of material that is irreversibly bound to the fbp surface on the time scale of this experiment ( figures 4 and 5 ) . the saturation value ( y value in figure 7 ) changes as a function of fbp surface density ( figure 8) . on the low density chip , the maximum signal from irreversibly bound material is 14 2 response units , which is achieved at a total fa solution concentration of 10 m . on the high density surface , the only exception is g5-ac - fa1.0 , for which 10 m is the highest concentration tested . for both low and high fbp density saturation of irreversible bound material ( y ) as a function of fa concentration . this surface density - dependent saturation of signal is indicative of a limiting number of fbp binding sites available for binding to the conjugates . figure 8 also suggests that the total amount of irreversibly bound material is determined primarily by ( i ) total fa concentration in solution and ( ii ) surface fbp density . all differences in the irreversibly bound fraction for the multivalent ( n = 1.22.7 ; orange , green , and blue ) samples can be attributed to the difference in fa concentration of these samples , which completely saturates when total fa concentration is 10 m . the monovalent material ( g5-ac - fa1.0 , red ) appears to have slightly lower binding compared to the multivalent samples based on total fa concentration . this occurrence may result from the enhanced effective concentration in the multivalent samples due to dendritic architecture forcing the multiple fas into a 5 diameter spherical area . this effect is small , and there appears to be no additional effect when valency is increased above n = 2 . qualitative observations ( i.e. , irreversible binding fraction in the g5-ac - fa1.0 sample and nonzero y ) indicate that this data will not adhere to the simple single phase langmuir isotherm . to demonstrate this relationship quantitatively , the data was fit with several models . the development of the models and resulting fits may be found in the supporting information ( tables s4s6 , figures s7 and s8 ) . first , as expected , a single phase model ( which mathematically describes figure 2a ) that assumes complete dissociation of the complex is a poor fit for the dissociation phase of all samples . the single phase association appears to have a good fit with the experimental data , however because this equation includes the single phase dissociation constant determined by the poorly fit dissociation phase , the overall mechanism is still invalid . two phase dissociation fits the data significantly better for all valencies and concentrations . second , the mathematical model equivalent to figure 2b results in a poor fit for all the g5-ac - fa1.0 data . the two phase model ( equivalent to the mechanism illustrated figure 2c ) had the best overall fit with an average residual of 2.39 response units . from this analysis , two main conclusions can be drawn : ( 1 ) there are at least two types ( or steps ) of association for g5fan to the immobilized fbp , which leads to ( 2 ) the presence of both a transiently and irreversibly bound material for all g5fan , including monovalent material . clearly , in the original analysis of spr data by banaszak holl et al . ( figure 2a ) , the assumption that all bound material would eventually dissociate ( i.e. , y = 0 ) from the surface was erroneous . the model proposed by sander et al . ( figure 2b ) correctly noted that a fraction of the material remained bound to the surface for the length of the experiment ( essentially irreversibly ) ; however , the additional assumption that g5fa1 , or g5fan ( n 1 ) bound through a single fa / fbp bridge was entirely responsible for the observed dissociation in stochastic mixtures of g5fan was incorrect . this model is clearly contradicted by the g5-ac - fa1.0 results , which are poorly fit by the equivalent mathematical model , and which clearly show enhanced binding to the fbp over free fa . when the other samples were fit with the same model , allowing for n = 0 or n = 0 and 1 to reversibly bind and n 2 to irreversibly bind , poor association phase fits were observed ( especially at lower fbp densities ) . the third theory , put forth by licata and tkachenko , proposed that an initial binding event between conjugate and fbp is keyed by fa , and then the binding strength becomes dominated by van der waals forces between the 30 kda polymer and 40 kda protein ( figure 2c ) . these summed weak interactions are responsible for the increased avidity for the conjugates , which the authors hypothesized are too great to be attributed to the comparatively weak ( kd 510 m ) fa / fbp interaction . mathematically , this model would not show a dependence of kd on degree of fa valency and is best represented schematically in figure 2c ( and quantitatively by eq 6 in the supporting information ) , which allows all conjugates with at least one fa to undergo both transient ( fa / fbp bridge formation ) and irreversible ( formation of a strong complex between the pamam and fbp ) binding events . the increased avidity for the g5-ac - fa1.0 conjugate as compared to free fa on both the low and high surface density chips , which is not further improved even with the g5-ac - fa2.7 conjugate , best agrees with this model qualitatively and quantitatively . therefore , we propose that the binding between g5-ac - fan conjugates and immobilized fbp can be explained by a 2-fold mechanism . this interaction has an association constant ( ka2 ) of 14 nm s. because the initial binding is dependent on the concentration of fa , there is an enhancement of avidity due to an increased total concentration of fa when multiple copies of the ligand are attached to the same dendrimer . this effective concentration may also lead to an increased chance of rebinding , as the fa / fbp dissociation constant ( kd2 ) of 9 s allows for dissociation of the conjugate from the surface on the spr experimental time scale . therefore , although strong binding is observed for all samples , the g5-ac - fa1.0 binds slightly less total material at the same relative fa concentration as compared to higher valency samples . after the fa binds to the fbp , the protein undergoes a conformational change , exposing a more hydrophilic surface . in the second step , the acetylated dendrimer arms , which are in close proximity to the protein because of the initial fa fbp key lock interaction , interact via van der waals forces with the fbp . we hypothesize that the interaction is further energetically driven by the rearrangement of fbp to yield a more hydrophilic surface after fa binding . although individual van der waals interactions are weak , the sum of many interactions available between the two 5 nm entities and the associated desolvation create a force that is irreversible over the time scale of these spr experiments . similar hydrophobic interactions are known to significantly contribute to the interactions between two proteins . experimental and theoretical measurements indicate that van der waals interactions are effective only over a very short range ( 12 ) , which supports the need for the fa / fbp interaction to key the hydrophobic interaction . additionally , it has been observed that flexibility in at least one interacting protein strongly enhances the ability for van der waals interactions to occur between proteins . as pamam dendrimers are known to be highly flexible , these observations also support the hypothesis of nonspecific interaction between the polymer and protein surface . the model proposed here is related to the well - known case of slow , tight binding previously described in detail for enzyme inhibitors . indeed , this behavior has been observed for folate analogues interacting with dihydrofolate reductase . for the case of the fa fbp equilibrium is followed by the irreversible pamam fbp binding to form a tight , stable complex . as illustrated in figure 2c , the pamam dendrimer is believed to rearrange to allow the van der waals interactions with the protein . additionally , it is likely that a rearrangement of the fbp upon binding to the conjugated fa exposes a more hydrophilic surface , enabling this interaction only when at least one fa is conjugated to the pamam . in summary , we have synthesized a monovalent g5-ac - fa1.0 conjugate that allows for the distinction between three previously proposed mechanisms for the high avidity interaction with fbp . we have also synthesized multivalent g5fa conjugates with narrow , nonstochastic fa - to - dendrimer ratio distributions to examine the kinetics of interaction between dendrimer - conjugated fa and fbp . the removal of trailing generations and oligomers in the pamam dendrimer starting material enabled the decoupling of mass and polymer surface area effects from fa valency . rp - hplc enabled the isolation of dendrimers containing precise ratios ( 1 , 2 , 3 , and 4 ) of copper - free , ring strain promoted click ligands to a dendrimer scaffold . a -azide - fa was clicked to these precise ratio conjugates to synthesize fa functionalized dendrimers with narrow , well - defined distributions of fa with average ratios of up to 2.7 fas per dendrimer . importantly , the monovalent conjugate g5-ac - fa1.0 was synthesized with no portion of the sample having more than 1 conjugated fa , allowing for the distinction of polymer contributions ( i.e. , solubility and van der waals interactions with the surface ) from multivalent contributions ( i.e. , effective concentration and chelate binding ) to the increased binding of dendrimer conjugates to fbp surfaces . spr studies revealed that g5-ac - fa1 experiences the enhanced avidity over free fa that has previously been attributed to multivalent fa binding . through examination of four quantitative models , it was concluded that the mechanism of interaction between g5-ac - fan and surface immobilized fbp is 2-fold : an initial , reversible , fa concentration dependent key lock or slow - onset , tight - binding interaction between the conjugate and protein , followed by irreversible interaction between the dendrimer and protein surfaces . the confirmation that these samples , even for a monovalent sample , exhibit irreversible binding on the time scale of the fa experiment disproves the original interpretation of banaszak holl et al . these findings also provide evidence against the model proposed by sander et al . , which attributed the increase in avidity to dendrimer species with 2 or more conjugated fas and assigned all dissociated material as singly bound . however , the model proposed by licata et al . explains the original data and agrees well with these new findings . this van der waals interaction model is in agreement with similar observations between two proteins in the literature and consistent the reported rearrangement of fbp structure following fa binding . the mechanism proposed here is based on spr experiments with an immobilized fbp on a three - dimensional surface . by way of contrast , cellular uptake mechanisms for fa targeted entities involve binding to far on a fluctuating cell membrane . the data and conclusions are directly comparable to previous studies that employed this model system ; however , the results only serve to provide a possible hypothesis for the interaction mechanism of folate polymer conjugates with cell - membrane bound far . the interaction of g5-ac - fitc - fan with folic acid receptor upregulated kb cells , reported along with the original spr experiments , exhibited the same saturation behavior as a function of ligand number ( n ) . based on the data and mechanistic interpretation presented here and in the work of licata and tkachenko , the observed enhancement of residence on the kb cell surface as a function of n could result from a combination of overall increased fa concentration and increased rebinding with increasing n. alternatively , it is possible that conjugate - initiated receptor clustering occurs on the cell membrane which is impossible for the fbp immobilized to a dextran surface . experiments to synthesize fluorescent materials containing precise ratios of fa targeting ligand for cell culture and in vivo experiments are in progress .

multivalent conjugation of folic acid has been employed to target cells overexpressing folate receptors . such polymer conjugates have been previously demonstrated to have high avidity to folate binding protein . however , the lack of a monovalent folic acid polymer material has prevented a full binding analysis of these conjugates , as multivalent binding mechanisms and polymer - mass mechanisms are convoluted in samples with broad distributions of folic acid - to - dendrimer ratios . in this work , the synthesis of a monovalent folic acid dendrimer conjugate allowed the elucidation of the mechanism for increased binding between the folic acid polymer conjugate and a folate binding protein surface . the increased avidity is due to a folate - keyed interaction between the dendrimer and protein surfaces that fits into the general framework of slow - onset , tight - binding mechanisms of ligand / protein interactions .

Introduction Experimental Section Results Discussion Conclusions

folic acid ( fa ) targeting has been extensively studied for improving the therapeutic index of drugs . targeting of a drug or drug conjugate exploits the interaction of this vitamin with a high affinity ( kd 0.1 nm ) folic acid receptor ( far ) , which is overexpressed in many cancer cells . the enthalpic and entropic mechanisms through which multivalency increases the interaction of a ligand and its target have been extensively studied from a theoretical viewpoint . briefly , there are two main multivalent effects that may contribute to the system studied here ; those dependent on the increased effective ( or local ) concentration , and those due to multiple binding events occurring for a single conjugate . although multivalent conjugates of many dyes , drugs , and targeting ligands ( including fa ) have been developed , the actual impact of the specific number of ligands on improvements in avidity and/or biological activity has been difficult to analyze due to the heterogeneous mixtures generated by stochastic conjugation chemistries employed in their synthesis . for example , a stochastic conjugation of 3 equiv of fa to a scaffold with multiple functionalizable sites ( 30 ) results in a sample with a mean of 3 fas per scaffold , but also a distribution of unique conjugates with fa - to - scaffold ratios ranging from 0 to 11 fa molecules per scaffold ( figure 1 ) . previous efforts to quantify multivalent binding constants have employed surface plasmon resonance ( spr ) to measure increases in binding between materials containing different average numbers of ligands ( folic acid and methotrexate ) and folate binding protein ( fbp ) modified surfaces . the binding constant of folic acid to fbp , kd 510 m , is roughly 1000-fold weaker than observed for folate receptor . although these studies have reported a general trend of greater avidity with increased valency , the utilization of materials containing a distribution of ligand - to - scaffold ratios complicated understanding the mechanisms involved in multivalent binding , or elucidation of the relative activity of the various components in the sample . in 2007 , employed spr to measure the increased avidity to fbp and cellular uptake of g5 pamam fa conjugates as a function of average number of attached fas ( figure 2 ) . interestingly , the same trend in signal saturation as a function of fa valency was observed for mean fluorescence , as measured by flow cytometry when equivalent conjugates labeled with a dye were evaluated for binding to far upregulated kb cells . ( c ) fa keys the initial interaction between conjugate and fbp , which is followed by strong nonspecific interaction between the dendrimer and protein . this mechanism acknowledges the broad distribution of ligand - to - dendrimer ratios present in stochastically synthesized materials , including dendrimers that have zero fa , one fa , or two or more fas . it was proposed that ( 1 ) monovalent interaction between g5fa1 and one fbp is attributable to binding that is reversible on the time scale of the experiment and ( 2 ) multivalent binding between g5fa2 to two or more fbps is irreversible on the spr experimental time scale ( figure 2b ) . hypothesized that the increased avidity attributed to valency increase by banaszak holl et al . this study concludes that the increased avidity proposed for the g5fan(avg ) conjugates is higher than can be attributed to cumulative effects of multivalent binding and that kinetic limitations actually prevent the type of multivalent interactions proposed in figures 2a and 2b . they propose that the enhanced interaction observed by spr is a result of van der waals interactions between the polymer vector and protein / chip surface that are enabled by a single key the broad distribution of folic acid - to - dendrimer ratios present in each sample , including both monovalent and multivalent conjugates in the low average materials , prevented a clear experimental elucidation between the three models depicted in figure 2 . in particular , a conjugate with a precise ratio of 1 fa per dendrimer ( g5fa1 ) was lacking to determine if the observed increase in avidity was a product of multivalent binding between the conjugate and spr surface ( banaszak holl and sander mechanisms ) or a single fa fbp lock - and - key combined with van der waals polymer / surface interaction ( licata and tkachenko mechanism ) . these methodologies , which have been previously demonstrated to be successful for azide and fluorinated , ring - strain - promoted click ligands , are now extended to a second ring strain promoted ligand ( cyclooct-1-yne-3-glycolic acid ( cog ) ) , which has been used in previous g5fan(avg ) spr studies . the resulting samples include a g5-pamam dendrimer with a fa - to - dendrimer ratio of 0.96 that contains no detectable multivalent g5fa2 species : the sample needed to differentiate the three mechanistic hypotheses proposed to date . the remainder of the click reactions did not proceed with 100% efficiency , but still yielded samples that contained a well - defined high - n cutoff and had a narrower - than - stochastic distribution of fa - to - dendrimer ratios . the binding of these conjugates was analyzed by spr on both high and low fbp density surfaces . most importantly , the g5fa1 sample exhibited the same irreversible binding to the fbp surface , on the spr time scale , as the g5fa2 samples . and provides strong experimental support for the key this mechanism falls into the general class of slow - onset , tight binding interactions between ligand and protein albeit with the novel feature of polymer adsorption onto the protein surface to yield the final tight - binding interaction . upon the initial binding event of a single conjugated fa to the fbp , the fbp undergoes a conformational change which exposes a more hydrophilic surface , enabling the irreversible van der waals interaction with the polymer . synthesis and characterization of -azide - lys - asp - folic acid ( -azide - fa ) conjugates were prepared from g5 dendrimer and cog via amide coupling . the resulting mixtures were then brought to a final dendrimer concentration of approximately 310 m to fully dissolve the dendrimer conjugates ( see table s2 in the supporting information for exact amounts used in each reaction ) . two immobilized folate binding protein ( fbp ) chips were prepared following the suggested protocols : a solution of 0.2 m edc and 0.05 m nhs was used as an activating solution , an immobilization solution of fbp at 1 mg / ml for the low density chip and 1.5 mg / ml for the high density chip , with ethanolamine as the deactivation solution . ( c ) isolated samples elute from rp - uplc as a function of ligand - to - dendrimer ratio . this robust methodology allows isolation of various species containing single ligand / dendrimer ratios from heterogeneous , averaged samples containing 10 or more species . due to the flexible nature of the pamam dendrimer and transient interaction of the ligand with the hydrophobic column , this technique has proven to be nonspecific to the relative location of the multiple ligands conjugated to the same sample , i.e. isolation of the g5-ac - cogx conjugates utilized in this paper reflect the success of prior studies with other click ligands . in the average sample , the most common species was dendrimer conjugated to 2 cog ligands , and this portion comprised only 16% of the sample . however , the isolated sample labeled g5-ac - cog2 contained only g5 conjugated to 2 ligands as measured by rp - uplc , with no detectable presence of dendrimer conjugated to 0 , 1 , 3 , or other numbers of ligands . here , we present the first application of the g5 pamam precise ligand - to - dendrimer ratio materials to a multivalent targeting system . however , stochastically synthesized conjugates contain a distribution of ligands per particle , making it difficult to distinguish the behaviors of the individual populations . the controlled ligand / dendrimer ratio conjugates allowed for the synthesis of functional g5fan materials with well - defined subpopulations , including a conjugate with a fa - to - dendrimer ratio of 1 , with no higher valencies present . reaction of the cog conjugates with precise ligand - to - dendrimer ratios with complementary click functionalized fa allows for the generation of dendrimers with well - defined numbers of covalently conjugated fas via orthogonal click chemistry between the ring - strained cyclooctyne on the dendrimer and an azido group on the modified fa . the reaction between g5-ac - cog1 and -azide - fa yielded a product that has 96% conjugate with a fa - to - dendrimer ratio of precisely 1 and 4% of a conjugate with no fa . because the original sample had no dendrimer conjugated to 2 or more cog ligands , the resulting product has no material with the ability to undergo multivalent binding . similar reaction conditions employed in the literature between a g5-ac - cog20(avg ) conjugate and a -azide - modified methotrexate yielded 100% reaction efficiency , however in this case the limiting reagent was the small molecule . this observation suggests that limiting the number of cog ligands on the dendrimer may limit accessibility for click reaction , perhaps via folding of hydrophobic ligands into the dendrimer core . , 20 ) of conjugated cog ligands , the dendrimer can not internalize all the ligands at once , so at any given time cog ligands are available for conjugation . both structural isomers of the click reaction are likely present , although that alone would not be expected to have great effect on binding to the fbp . rp - uplc also provides a useful tool for monitoring the click reaction , as the reaction of the hydrophobic ligand leads to a decrease in retention of the dendrimer conjugate on the c18 column . this technique provides a more accurate measurement of fa - to - dendrimer ratio of the product than techniques such as nmr , which only provides an average number and provides no detail about the individual ligand - to - dendrimer ratios that are present within a sample . more importantly , 26% of the stochastic material has two or more fas covalently attached , meaning this material is not truly representative of monovalent behavior . rp - uplc has also revealed the relative amount of each ratio present in the samples ( figure 6b d ) , allowing for a much better understanding of the contribution of each for example , the product of the g5-ac - cog3 click reaction ( g5-ac - fa1.9 ) has an average of 2 fas per dendrimer , but uplc reveals that 23% of the material has three fas attached , while 49% has two fas , 24% is monovalent , and 4% of the material has zero fa . the equivalent stochastic average of n = 1.9 has significant concentrations of 10 unique fa - to - dendrimer ratios ( ranging from 0 to 9 ) , and 15% of the sample has zero fa . in other words , the dendrimer conjugate binds much more tightly than free fa ( kd 510 m ) . the irreversible binding on the time scale of the spr experiment has previously been attributed to multivalent binding between the conjugate and receptor , however that can not be the case for this purely monovalent conjugate . lock / van der waals binding mechanism proposed by licata and tkachenko in which only one fa to fbp interaction is necessary to initiate the stronger interaction between the dendrimer and fbp , which itself is a result of the summation of many weak van der waals interactions . have demonstrated using fluorescence spectroscopy that the tryptophan residues reorient upon folic acid binding to the fbp interior generating a more hydrophilic protein surface . the single phase association appears to have a good fit with the experimental data , however because this equation includes the single phase dissociation constant determined by the poorly fit dissociation phase , the overall mechanism is still invalid . ( figure 2b ) correctly noted that a fraction of the material remained bound to the surface for the length of the experiment ( essentially irreversibly ) ; however , the additional assumption that g5fa1 , or g5fan ( n 1 ) bound through a single fa / fbp bridge was entirely responsible for the observed dissociation in stochastic mixtures of g5fan was incorrect . the third theory , put forth by licata and tkachenko , proposed that an initial binding event between conjugate and fbp is keyed by fa , and then the binding strength becomes dominated by van der waals forces between the 30 kda polymer and 40 kda protein ( figure 2c ) . these summed weak interactions are responsible for the increased avidity for the conjugates , which the authors hypothesized are too great to be attributed to the comparatively weak ( kd 510 m ) fa / fbp interaction . mathematically , this model would not show a dependence of kd on degree of fa valency and is best represented schematically in figure 2c ( and quantitatively by eq 6 in the supporting information ) , which allows all conjugates with at least one fa to undergo both transient ( fa / fbp bridge formation ) and irreversible ( formation of a strong complex between the pamam and fbp ) binding events . this interaction has an association constant ( ka2 ) of 14 nm s. because the initial binding is dependent on the concentration of fa , there is an enhancement of avidity due to an increased total concentration of fa when multiple copies of the ligand are attached to the same dendrimer . this effective concentration may also lead to an increased chance of rebinding , as the fa / fbp dissociation constant ( kd2 ) of 9 s allows for dissociation of the conjugate from the surface on the spr experimental time scale . in the second step , the acetylated dendrimer arms , which are in close proximity to the protein because of the initial fa fbp key lock interaction , interact via van der waals forces with the fbp . although individual van der waals interactions are weak , the sum of many interactions available between the two 5 nm entities and the associated desolvation create a force that is irreversible over the time scale of these spr experiments . as pamam dendrimers are known to be highly flexible , these observations also support the hypothesis of nonspecific interaction between the polymer and protein surface . the model proposed here is related to the well - known case of slow , tight binding previously described in detail for enzyme inhibitors . in summary , we have synthesized a monovalent g5-ac - fa1.0 conjugate that allows for the distinction between three previously proposed mechanisms for the high avidity interaction with fbp . we have also synthesized multivalent g5fa conjugates with narrow , nonstochastic fa - to - dendrimer ratio distributions to examine the kinetics of interaction between dendrimer - conjugated fa and fbp . importantly , the monovalent conjugate g5-ac - fa1.0 was synthesized with no portion of the sample having more than 1 conjugated fa , allowing for the distinction of polymer contributions ( i.e. , effective concentration and chelate binding ) to the increased binding of dendrimer conjugates to fbp surfaces . through examination of four quantitative models , it was concluded that the mechanism of interaction between g5-ac - fan and surface immobilized fbp is 2-fold : an initial , reversible , fa concentration dependent key lock or slow - onset , tight - binding interaction between the conjugate and protein , followed by irreversible interaction between the dendrimer and protein surfaces . the confirmation that these samples , even for a monovalent sample , exhibit irreversible binding on the time scale of the fa experiment disproves the original interpretation of banaszak holl et al . however , the model proposed by licata et al . the data and conclusions are directly comparable to previous studies that employed this model system ; however , the results only serve to provide a possible hypothesis for the interaction mechanism of folate polymer conjugates with cell - membrane bound far . the interaction of g5-ac - fitc - fan with folic acid receptor upregulated kb cells , reported along with the original spr experiments , exhibited the same saturation behavior as a function of ligand number ( n ) . based on the data and mechanistic interpretation presented here and in the work of licata and tkachenko , the observed enhancement of residence on the kb cell surface as a function of n could result from a combination of overall increased fa concentration and increased rebinding with increasing n. alternatively , it is possible that conjugate - initiated receptor clustering occurs on the cell membrane which is impossible for the fbp immobilized to a dextran surface .

2,1-borazaronaphthalenes serve as versatile isosteres of all - carbon naphthalene substructures , which allows facile emplacement of a variety of substituents about the ring in an efficient and selective manner . because of electronic desymmetrization brought about by the incorporation of a b n bond in these aromatic systems , borazines have proven to be far more easily elaborated than naphthalenes themselves . previous studies have focused on decorating these ring systems by utilizing arylation reactions . 2-alkenylnaphthalenes can be synthesized through a variety of transition - metal - catalyzed transformations , including heck , mizoroki heck , hiyama , and suzuki miyaura reactions . however , installation of an alkenyl substituent on a functionalized naphthalene is more challenging and often requires four or five steps . one method to afford alkenyl - substituted naphthalenes is to perform free - radical bromination of a methyl - substituted derivative followed by a wittig olefination . aside from the generation of a stoichiometric amount of difficult - to - remove phosphine oxide waste , additional substitution on similarly , titanium - mediated carbonylation with dichloromethyl methyl ether followed by wittig olefination afforded 2-alkyl-3-alkenylnaphthalene in moderate yield over two steps . in addition to the drawbacks described above , the use of a strong lewis acid limits functional group tolerability in this approach . further , there is only one example demonstrating the installation of an alkenyl substituent on a 2-arylnapthalene through the cross - coupling of naphthyl iodide and alkenylboronic acid , which provides 2-aryl-3-alkenylnaphthalene in good yield . the naphthyl iodide in this case was synthesized through gold - catalyzed cyclization / iodination , which required preparation of the requisite starting material ( 1-arylalka-2,3-dienyl acetate ) . additionally , to the best of our knowledge , installation of an alkenyl substituent at the c3 position of a 1,2-disubstituted naphthalene has not been reported . accessing 2,1-borazaronaphthalenes , a class of azaborines that are bn - isosteres of naphthalene , with alkenyl substituents at the c3 position under mild reaction conditions would therefore serve as an example of how functionalized azaborines could be prepared more efficiently than their corresponding all - carbon analogues . recently , we reported the bromination of 2,1-borazaronaphthalenes in high yield under mild reaction conditions with complete regiochemical control . because of the inherent reactivity of azaborine , bromination occurs selectively at the c3 position of the azaborine in the presence of alkyl / aryl substituents on boron and nitrogen . miyaura cross - coupling reactions with potassium alkenyltrifluoroborates as a route to c3-alkenyl - substituted 2,1-borazaronaphthalenes , a compound class that has not been previously reported . optimization of the reaction of 3-bromo-2-methyl-2,1-borazaronaphthalene 1 with functionalized alkenyltrifluoroborate 2 was carried out by screening palladium sources , solvents , and bases . side products represent the sum of homocoupling of the azaborine , protodebromination of the azaborine , and unidentifiable side products . conditions for the cross - coupling of potassium aryltrifluoroborates with brominated 2,1-borazaronaphthalenes did not afford the desired product in appreciable yield ( table 1 , entry 1 ) . therefore , several other ligands were screened in this reaction ( entries 24 and 7 ) . of the ligands tested , triphenylphosphine ( pph3 ) and 1,1-bis(diphenylphosphino)ferrocene ( dppf ) provided the highest conversions to products . several solvents / ratios were tested for these ligands , and the best result was obtained using toluene as the cosolvent and pd(dppf)cl2 as the palladium source ( entry 7 ) . a representative set of potassium alkenyltrifluoroborates were subjected to the developed reaction conditions with 3-bromo-2-methyl-2,1-borazaronaphthalene as the electrophilic partner ( table 2 ) . cyclic alkenyltrifluoroborates were successful nucleophiles in the reaction by providing the desired product in yields up to 90% ( entries 5 , 6 , 8 , and 10 ) . alkenyltrifluoroborates with alkyl substituents afforded the desired product in high yields ( entries 2 , 3 , and 9 ) . vinyltrifluoroborate was a suitable nucleophile for the reaction because the azaborine was obtained in 70% yield ( entry 7 ) . most importantly , cis-1-propenyltrifluoroborate was successfully employed in the reaction , affording the product in high yield without isomerization of the alkene ( entry 3 ) . the scalable nature of the cross - coupling reaction was demonstrated by performing the coupling on a 4.5 mmol scale ( 1 g of azaborine ) with one - third palladium loading ( 2 mol % ) , which provided the desired product in similar yield ( entry 8) . reaction completed on a 4.5 mmol scale with 2.0 mol % pd(dppf)cl2 . reaction conditions ( unless otherwise noted ) : 1.0 equiv of 3-bromo-2-methyl-2,1-borazaronaphthalene , 1.1 equiv of potassium alkenyltrifluoroborate , 6.0 mol % pd(dppf)cl2 , 3.0 equiv of base , 1:1 toluene / h2o , and 60 c for 18 h. the developed reaction conditions were extended to cross - coupling of 3-bromo-2-phenyl-2,1-borazaronaphthalene with an array of potassium alkenyltrifluoroborates to demonstrate that azaborine can also be substituted with an aryl group on the boron . six different alkenyltrifluoroborates were employed in the coupling and provided the desired product in yields up to 90% . the mild reaction conditions of the coupling were demonstrated in the successful coupling of an alkenyltrifluoroborate with an alkyl chloride substituent , affording the desired product in 90% yield ( table 3 , entry 2 ) . cyclic alkenyltrifluoroborates were successfully engaged in the reaction as the desired products were obtained in high yields ( entries 3 , 4 , and 6 ) . reaction conditions ( unless otherwise noted ) : 1.0 equiv of 3-bromo-2-phenyl-2,1-borazaronaphthalene , 1.1 equiv of potassium alkenyltrifluoroborate , 6.0 mol % pd(dppf)cl2 , 3.0 equiv of base , 1:1 toluene / h2o , and 60 c for 18 h. to demonstrate the versatility of this method , an array of brominated 2,1-borazaronaphthalenes were synthesized and subjected to the developed reaction conditions with 1-decenyltrifluoroborate as the nucleophile in the reaction . the cross - coupled products were obtained with secondary cyclic and acyclic groups on boron in yields up to 90% ( table 4 , entries 1 and 2 ) . azaborines with various substitution patterns on the arene of boron were suitable electrophiles for coupling as the cross - coupled products were isolated in high yield ( entries 35 ) . substitution on the nitrogen of the 2,1-borazaronaphthalenes did not affect the coupling as reactions with both n - allyl and n - benzyl substituents provided the desired product in high yields ( entries 6 and 7 ) . reaction conditions ( unless otherwise noted ) : 1.0 equiv of 3-bromo-2,1-borazaronaphthalene , 1.1 equiv of potassium alkenyltrifluoroborate , 6.0 mol % pd(dppf)cl2 , 3.0 equiv of base , 1:1 toluene / h2o , and 60 c for 18 h. to demonstrate that the developed reaction conditions can be directly applied to the functionalization of other azaborines , 6-bromo-2-methyl-3-phenyl-2,1-borazaronaphthalene was subjected to the reaction ( eq 1 ) . the desired product was obtained in 83% yield to afford c6-alkenyl - substituted 2,1-borazaronaphthalenes , examples of which are absent in the literature.1 the reaction conditions were then extended to cross - coupling of 3,6-dibrominated 2,1-borazaronaphthalene . the addition of 2.2 equiv of 1-propenyltrifluoroborate provides a doubly cross - coupled product in 76% yield ( eq 2).2 in conclusion , a general method for suzuki miyaura cross - coupling of brominated 2,1-borazaronaphthalenes with potassium alkenyltrifluoroborates has been developed . azaborines with alkyl and aryl groups on boron and with or without substitution on nitrogen are suitable reagents for the coupling . through this route , b nmr spectra were obtained on a spectrometer equipped with the appropriate decoupling accessories . all b nmr chemical shifts were referenced to external bf3oet2 ( 0.0 ppm ) with a negative sign indicating an upfield shift . data are presented as follows : chemical shift ( ppm ) , multiplicity ( s = singlet , d = doublet , t = triplet , q = quadruplet , m = multiplet , br = broad ) , coupling constant j ( hz ) , and integration . analytical thin - layer chromatography ( tlc ) was performed on tlc silica gel plates ( 0.25 mm ) precoated with a fluorescent indicator . hrms data were obtained by either esi or ci using a tof mass spectrometer . 2,1-borazaronaphthalenes were synthesized and brominated according to methods previously described in the literature . the title compound was obtained as a white solid in 90% yield ( 1.0 mmol scale , 316.7 mg ) : mp 98103 c ; h nmr ( 500 mhz , cdcl3 ) 8.49 ( s , 1h ) , 8.07 ( s , 1h ) , 7.98 ( d , j = 7.8 hz , 2h ) , 7.72 ( d , j = 7.8 hz , 2h ) , 7.64 ( d , j = 7.9 hz , 1h ) , 7.517.49 ( m , 1h ) , 7.34 ( d , j = 8.1 hz , 1h ) , 7.307.27 ( m , 1h ) ; c nmr ( 125.8 mhz , cdcl3 ) 147.3 , 139.0 , 133.6 , 131.2 ( d , j = 32.5 hz ) , 129.7 , 129.2 , 126.5 ( q , j = 275.8 hz ) , 125.3 , 124.6 ( q , j = 3.6 hz ) , 122.5 , 118.4 ; b nmr ( 128.38 mhz , cdcl3 ) 32.9 ; ir ( neat ) 3367 , 2923 , 1556 , 1427 , 1120 , 1070 cm ; hrms ( ci ) m / z [ m + h ] calcd for c15h11bnf3br 352.9825 , found 352.9802 . to a biotage 10 ml microwave vial equipped with a magnetic stir bar were added 3-bromo-2,1-alkenyltrifluoroborate ( 1.1 mmol , 1.1 equiv ) , cs2co3 ( 3.0 mmol , 977 mg ) , pd(dppf)cl2 ( 6.0 mol % , 42 mg ) , and potassium alkenyltrifluoroborate ( 1.0 mmol , 1.0 equiv ) . the vial was sealed with a cap , lined with a teflon septum , and evacuated and purged with argon gas three times . anhydrous degassed toluene ( 1.0 ml ) and deionized h2o ( 1.0 ml ) were added under argon gas . the reaction mixture was heated at 60 c for 18 h. after cooling to rt , the reaction mixture was extracted with etoac ( 3 10 ml ) and dried ( mgso4 ) . after being concentrated in vacuo , the product was isolated by flash column chromatography and eluted with a gradient of etoac in hexanes ( 0 to 10% etoac ) . the title compound was obtained as a yellow solid in 88% yield ( 235.8 mg ) : mp 8993 c ; h nmr ( 500 mhz , cdcl3 ) 7.79 ( s , 1h ) , 7.72 ( s , 1h ) , 7.58 ( d , j = 7.5 hz , 1h ) , 7.347.37 ( m , 1h ) , 7.18 ( d , j = 8.1 hz , 1h ) , 7.157.12 ( m , 1h ) , 5.245.23 ( m , 1h ) , 5.12 ( d , j = 2.5 hz , 1h ) , 3.67 ( t , j = 4.6 hz , 4h ) , 3.31 ( s , 2h ) , 2.48 ( br , 4h ) , 0.79 ( s , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 149.0 , 140.1 , 129.6 , 128.1 , 125.0 , 121.0 , 117.4 , 114.2 , 67.3 , 64.8 , 53.9 ; b nmr ( 128.38 mhz , cdcl3 ) 36.7 ; ir ( neat ) 3319 , 2928 , 1566 , 1434 , 1109 , 891 cm ; hrms ( esi+ ) m / z [ m + h ] calcd for c16h22bn2o 269.1825 , found 269.1820 . the title compound was obtained as a yellow solid in 98% yield ( 179.3 mg ) : mp 6975 c;h nmr ( 500 mhz , cdcl3 ) 7.76 ( s , 1h ) , 7.65 ( s , 1h ) , 7.56 ( d , j = 7.8 hz , 1h ) , 7.367.29 ( m , 1h ) , 7.16 ( d , j = 8.0 hz , 1h ) , 7.137.10 ( m , 1h ) , 6.59 ( d , j = 15.6 hz , 1h ) , 6.196.12 ( m , 1h ) , 1.91 ( dd , j = 6.6 , 1.5 hz , 3h ) , 0.85 ( s , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 139.5 , 138.7 , 134.8 , 129.3 , 127.6 , 126.5 , 125.6 , 121.0 , 117.4 , 19.4 ; b nmr ( 128.38 mhz , cdcl3 ) 37.2 ; ir ( neat ) 3364 , 2924 , 1614 , 1457 , 1343 cm ; hrms ( ci ) m / z [ m ] calcd for c12h14bn 183.1219 , found 183.1216 . the title compound was obtained as a brown solid in 87% yield ( 159.2 mg ) : mp 98103 c ; h nmr ( 500 mhz , cdcl3 ) 7.42 ( s , 1h ) , 7.68 ( s , 1h ) , 7.59 ( d , j = 7.8 hz , 1h ) , 7.377.34 ( m , 1h ) , 7.19 ( d , j = 8.0 hz , 1h ) , 7.187.13 ( m , 1h ) , 6.576.54 ( m , 1h ) , 5.725.79 ( m , 1h ) , 1.85 ( dd , j = 7.0 , 1.8 hz , 3h ) , 0.74 ( s , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 140.8 , 139.6 , 132.1 , 129.4 , 127.8 , 125.3 , 125.0 , 121.0 , 117.6 , 14.8 ; b nmr ( 128.38 mhz , cdcl3 ) 37.3 ; ir ( neat ) 3364 , 2925 , 1611 , 1560 , 1454 cm ; hrms ( ci ) m / z [ m ] calcd for c12h14bn 183.1219 , found 183.1213 . the title compound was obtained as a yellow oil in 69% yield ( 126.2 mg ) . h nmr ( 500 mhz , cdcl3 ) 7.80 ( s , 1h ) , 7.68 ( s , 1h ) , 7.62 ( d , j = 7.7 hz , 1h ) , 7.397.36 ( m , 1h ) , 7.197.15 ( m , 2h ) , 5.215.11 ( m , 1h ) , 5.07 ( d , j = 1.9 hz , 1h ) , 2.16 ( s , 3h ) , 0.87 ( s , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 148.4 , 139.6 , 139.2 , 129.6 , 128.0 , 125.1 , 121.0 , 117.4 , 113.0 , 23.9 ; b nmr ( 128.38 mhz , cdcl3 ) 36.9 ; ir ( neat ) 3368 , 2933 , 1567 , 1462 , 1453 cm ; hrms ( ci ) m / z [ m ] calcd for c12h14bn 183.1219 , found 183.1220 . the title compound was obtained as a yellow oil in 82% yield ( 184.5 mg ) . h nmr ( 500 mhz , cdcl3 ) 7.75 ( s , 1h ) , 7.73 ( s , 1h ) , 7.59 ( d , j = 7.8 hz , 1h ) , 7.347.38 ( m , 1h ) , 7.18 ( d , j = 8.1 hz , 1h ) , 7.167.13 ( m , 1h ) , 5.835.82 ( m , 1h ) , 4.364.35 ( m , 2h ) , 3.97 ( t , j = 5.4 hz , 2h ) , 2.522.49 ( m , 2h ) , 0.84 ( s , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 138.6 , 137.6 , 137.5 , 128.5 , 127.0 , 124.1 , 121.9 , 120.1 , 116.4 , 65.2 , 63.9 , 28.1 ; b nmr ( 128.38 mhz , cdcl3 ) 36.7 ; ir ( neat ) 3325 , 2923 , 1572 , 1458 , 1032 cm ; hrms ( ci ) m / z [ m ] calcd for c14h16bno 225.1325 , found 225.1336 . the title compound was obtained as a white solid in 81% yield ( 227.6 mg ) : mp 8388 c ; h nmr ( 500 mhz , cdcl3 ) 7.73 ( s , 1h ) , 7.69 ( s , 1h ) , 7.547.56 ( m , 1h ) , 7.327.35 ( m , 1h ) , 7.16 ( d , j = 8 hz , 1h ) , 7.137.10 ( m , 1h ) , 5.665.65 ( m , 1h ) , 4.054.02 ( m , 4h ) , 2.632.59 ( m , 2h ) , 2.502.49 ( m , 2h ) , 1.93 ( t , j = 6.5 hz , 2h ) , 0.81 ( s , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 140.9 , 139.5 , 138.7 , 129.4 , 127.8 , 125.2 , 121.1 , 120.9 , 117.3 , 108.3 , 64.7 , 36.5 , 31.8 , 28.7 ; b nmr ( 128.38 mhz , cdcl3 ) 37.0 ; ir ( neat ) 3308 , 2927 , 1609 , 1460 , 1047 cm ; hrms ( ci ) m / z [ m ] calcd for c17h20bno2 281.1587 , found 281.1593 . the title compound was obtained as a light yellow solid in 70% yield ( 118.3 mg ) : mp 5256 c ; h nmr ( 500 mhz , cdcl3 ) 7.70 ( s , 1h ) , 7.64 ( s , 1h ) , 7.55 ( d , j = 7.8 hz , 1h ) , 7.337.30 ( m , 1h ) , 7.15 ( d , j = 8.0 hz , 1h ) , 7.107.08 ( m , 1h ) , 6.736.66 ( m , 1h ) , 5.875.85 ( m , 1h ) , 5.745.71 , ( m , 1h ) , 0.70 ( s , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 140.7 , 140.3 , 139.9 , 129.6 , 128.2 , 125.3 , 121.2 , 117.5 , 114.8 ; b nmr ( 128.38 mhz , cdcl3 ) 37.2 ; ir ( neat ) 3365 , 2927 , 1610 , 1457 cm ; hrms ( ci ) the title compound was obtained as a white solid in 90% yield ( 233.1 mg ) : mp 7276 c ; h nmr ( 500 mhz , cdcl3 ) 7.73 ( s , 1h ) , 7.69 ( s , 1h ) , 7.60 ( d , j = 8.0 hz , 1h ) , 7.417.37 ( m , 1h ) , 7.207.15 ( m , 2h ) , 5.595.56 ( m , 1h ) , 2.782.72 ( m , 2h ) , 2.672.64 ( m , 2h ) , 2.252.17 ( m , 2h ) , 0.82 ( s , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 140.8 , 139.5 , 139.1 , 129.4 , 128.0 , 124.9 , 123.3 ( t , j = 239.6 hz ) , 121.0 , 118.2 ( t , j = 4.9 hz ) , 117.3 , 35.3 ( t , j = 26.4 hz ) , 31.0 ( t , j = 24.4 hz ) , 27.8 ( t , j = 5.2 hz ) ; b nmr ( 128.38 mhz , cdcl3 ) 36.2 ; ir ( neat ) 3397 , 2936 , 1613 , 1560 , 1425 , 1058 , cm ; hrms ( ci ) the title compound was obtained as a yellow solid in 75% yield ( 210.7 mg ) : mp 5862 c ; h nmr ( 500 mhz , cdcl3 ) 7.79 ( s , 1h ) , 7.65 ( s , 1h ) , 7.57 ( d , j = 7.75 hz , 1h ) , 7.347.31 ( m , 1h ) , 7.167.11 ( m , 2h ) , 6.58 ( d , j = 15.5 hz , 1h ) , 6.186.12 ( m , 1h ) , 2.262.22 ( m , 2h ) , 1.531.47 ( m , 2h ) , 1.401.28 ( m , 10h ) , 0.92 ( t , j = 6.9 hz , 3h ) , 0.86 ( s , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 139.5 , 138.8 , 133.4 , 132.2 , 129.3 , 127.6 , 125.6 , 121.0 , 117.4 , 34.0 , 32.2 , 30.0 , 29.9 , 29.7 , 29.6 , 23.0 , 14.4 ; b nmr ( 128.38 mhz , cdcl3 ) 37.1 ; ir ( neat ) 3366 , 2921 , 1556 , 1463 , 977 cm ; hrms ( ci ) m / z [ m ] calcd for c19h28bn 281.2315 , found 281.2328 . the title compound was obtained as a white solid in 65% yield ( 210.6 mg ) : mp 8387 c ; h nmr ( 500 mhz , cdcl3 ) 7.75 ( s , 1h ) , 7.69 ( s , 1h ) , 7.57 ( d , j = 7.7 hz , 1h ) , 7.367.33 ( m , 1h ) , 7.17 ( d , j = 8.0 hz , 1h ) , 7.147.11 ( m , 1h ) , 5.71 ( br , 1h ) , 4.07 ( br , 2h ) , 3.64 ( t , j = 5.5 hz , 2h ) , 4.47 ( br , 2h ) , 1.52 ( s , 9h ) , 0.80 ( s , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 155.3 , 139.9 , 139.6 , 138.7 , 129.5 , 128.0 , 125.1 , 121.1 , 120.7 , 117.4 , 79.7 , 44.3 , 40.1 , 34.9 , 29.4 , 28.8 ; b nmr ( 128.38 mhz , cdcl3 ) 32.0 ; ir ( neat ) 3327 , 2975 , 1677 , 1563 , 1423 , 1163 cm ; hrms ( ci ) m / z [ m + h ] calcd for c19h26bn2o2 325.2087 , found 325.2080 . the title compound was obtained as a yellow solid in 83% yield ( 226.5 mg ) : mp 7477 c ; h nmr ( 500 mhz , cdcl3 ) 7.66 ( s , 1h ) , 7.59 ( s , 1h ) , 7.55 ( d , j = 7.7 hz , 1h ) , 7.387.36 ( m , 1h ) , 7.357.31 ( m , 2h ) , 7.227.13 ( m , 5h ) , 6.58 ( dd , j = 11.5 , 1.4 hz , 1h ) , 5.69 ( dt , j = 11.5 , 7.2 hz , 1h ) , 2.79 ( t , j = 7.7 hz , 2h ) , 2.702.45 ( m , 2h ) , 0.73 ( s , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 142.3 , 140.6 , 139.7 , 132.1 , 129.9 , 129.5 , 128.9 , 128.6 , 127.9 , 126.1 , 125.3 , 121.0 , 117.5 , 36.7 , 30.6 ; b nmr ( 128.38 mhz , cdcl3 ) 37.4 ; ir ( neat ) 3373 , 2925 , 1556 , 1452 , 746 , 703 cm ; hrms ( ci ) m / z [ m ] calcd for c19h20bn 273.1689 , found 273.1683 . the title compound was obtained as a white oil in 73% yield ( 250.3 mg ) . h nmr ( 500 mhz , cdcl3 ) 8.05 ( s , 1h ) , 7.91 ( s , 1h ) , 7.777.75 ( m , 2h ) , 7.69 ( d , j = 8.0 hz , 1h ) , 7.517.47 ( m , 3h ) , 7.427.39 ( s , 1h ) , 7.26 ( d , j = 8.0 hz , 1h ) , 7.22 ( t , j = 7.5 hz , 1h ) , 6.70 ( d , j = 15.5 hz , 1h ) , 6.07 ( dt , j = 15.4 , 7.0 hz , 1h ) , 2.21 ( q , j = 7.0 hz , 2h ) , 1.501.45 ( m , 2h ) , 1.401.35 ( m , 10h ) , 0.96 ( t , j = 7.0 hz , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 139.8 , 139.6 , 133.2 , 132.7 , 132.3 , 129.5 , 128.8 , 128.2 , 128.0 , 125.9 , 121.0 , 118.0 , 33.7 , 32.2 , 29.9 , 29.8 , 29.7 , 29.6 , 23.0 , 14.4 ; b nmr ( 128.38 mhz , cdcl3 ) 34.4 ; ir ( neat ) 3370 , 2923 , 2852 , 1421 , 749 cm ; hrms ( ci ) m / z [ m + na ] calcd for c24h30bnna 366.2369 , found 366.2375 . the title compound was obtained as a brown oil in 90% yield ( 288.9 mg ) . h nmr ( 500 mhz , cdcl3 ) 8.03 ( s , 1h ) , 7.92 ( s , 1h ) , 7.72 ( dd , j = 7.25 , 1.5 hz , 2h ) , 7.68 ( d , j = 7.5 hz , 1h ) , 7.507.46 ( m , 3h ) , 7.427.39 ( m , 1h ) , 7.26 ( d , j = 8 hz , 1h ) , 7.237.20 ( m , 1h ) , 6.68 ( d , j = 15.5 hz , 1h ) , 6.00 ( dt , j = 15.4 , 7.0 hz , 1h ) , 3.57 ( t , j = 6.5 hz , 2h ) , 2.232.19 ( m , 2h ) , 1.861.83 ( m , 2h ) , 1.621.58 ( m , 2h ) ; c nmr ( 125.8 mhz , cdcl3 ) 140.1 , 139.6 , 133.4 , 133.1 , 131.1 , 129.5 , 128.8 , 128.2 , 128.1 , 125.7 , 121.7 , 118.0 , 45.3 , 32.7 , 32.3 , 27.0 ; b nmr ( 128.38 mhz , cdcl3 ) 35.0 ; ir ( neat ) 3373 , 2933 , 1558 , 1421 , 969 , 752 cm ; hrms ( ci ) m / z [ m ] calcd for c20h21bncl 321.1456 , found 321.1463 . the title compound was obtained as a yellow solid in 79% yield ( 225.1 mg ) : mp 8892 c ; h nmr ( 500 mhz , cdcl3 ) 7.95 ( s , 1h ) , 7.85 ( s , 1h ) , 7.797.7.78 ( m , 2h ) , 7.66 ( d , j = 7.5 hz , 1h ) , 7.457.39 ( m , 4h ) , 7.28 ( d , j = 8.0 hz , 1h ) , 7.227.19 ( m , 1h ) , 5.735.71 ( m , 1h ) , 2.192.13 ( m , 4h ) , 1.661.65 ( m , 4h ) ; c nmr ( 125.8 mhz , cdcl3 ) 142.3 , 140.5 , 139.6 , 132.8 , 129.5 , 128.9 , 128.1 , 128.0 , 125.6 , 124.2 , 121.5 , 117.9 , 29.9 , 26.1 , 23.5 , 22.6 ; b nmr ( 128.38 mhz , cdcl3 ) 34.0 ; ir ( neat ) 3375 , 2920 , 1558 , 1451 , 751 cm ; hrms ( ci ) m / z [ m ] calcd for c20h20bn 285.1689 , found 285.1705 . the title compound was obtained as a white solid in 86% yield ( 246.8 mg ) : mp 112115 c ; h nmr ( 500 mhz , cdcl3 ) 8.02 ( s , 1h ) , 7.91 ( s , 1h ) , 7.767.68 ( m , 2h ) , 7.69 ( d , j = 7.5 hz , 1h ) , 7.477.42 ( m , 4h ) , 7.29 ( d , j = 8.1 hz , 1h ) , 7.247.21 ( m , 1h ) , 5.755.73 ( m , 1h ) , 4.314.29 ( m , 2h ) , 3.84 ( t , j = 5.5 hz , 2h ) , 2.30 ( td , j = 5.3 , 2.7 hz , 2h ) ; c nmr ( 125.8 mhz , cdcl3 ) 140.9 , 139.7 , 139.4 , 132.7 , 129.6 , 128.9 , 128.4 , 128.2 , 125.3 , 122.8 , 121.7 , 118.0 , 66.2 , 64.9 , 29.5 ; b nmr ( 128.38 mhz , cdcl3 ) 34.0 ; ir ( neat ) 3287 , 2930 , 1564 , 1455 , 1229 , 758 cm ; hrms ( ci ) m / z [ m ] calcd for c19h18bno 287.1481 , found 287.1487 . the title compound was obtained as a brown oil in 76% yield ( 186.2 mg ) . h nmr ( 500 mhz , cdcl3 ) 7.96 ( br , 2h ) , 7.927.81 ( m , 2h ) , 7.70 ( d , j = 7.5 hz , 1h ) , 7.477.43 ( m , 4h ) , 7.29 ( d , j = 8.0 hz , 1h ) , 7.257.22 ( m , 1h ) , 5.065.02 ( m , 2h ) , 1.99 ( s , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 149.2 , 140.9 , 139.5 , 132.6 , 129.5 , 128.7 , 128.2 , 128.0 , 125.2 , 121.5 , 117.8 , 113.0 , 24.1 ; b nmr ( 128.38 mhz , cdcl3 ) 33.6 ; ir ( neat ) 3367 , 2925 , 1573 , 1463 , 757 cm ; hrms ( ci ) m / z [ m ] calcd for c17h16bn 245.1376 , found 245.1377 . the title compound was obtained as a white solid in 81% yield ( 277.8 mg ) : mp 108112 c ; h nmr ( 500 mhz , cdcl3 ) 7.98 ( s , 1h ) , 7.90 ( s , 1h ) , 7.807.78 ( m , 2h ) , 7.65 ( d , j = 7.5 hz , 1h ) , 7.457.39 ( m , 4h ) , 7.28 ( d , j = 8.0 hz , 1h ) , 7.217.18 ( m , 1h ) , 6.625.60 ( m , 1h ) , 4.053.98 ( m , 4h ) , 2.472.46 ( m , 2h ) , 2.38 ( td , j = 6.3 , 1.6 hz , 2h ) , 1.79 ( t , j = 6.5 hz , 2h ) ; c nmr ( 125.8 mhz , cdcl3 ) 141.9 , 141.1 , 139.6 , 132.7 , 129.4 , 128.9 , 128.1 , 128.1 , 125.3 , 121.5 , 121.1 , 117.8 , 108.2 , 64.5 , 36.4 , 31.6 , 29.0 ; b nmr ( 128.38 mhz , cdcl3 ) 34.4 ; ir ( neat ) 3326 , 1560 , 1421 , 1112 , 755 cm ; hrms ( ci ) m / z [ m ] calcd for c22h22bno2 343.1744 , found 343.1756 . the title compound was obtained as a colorless oil in 81% yield ( 247.8 mg ) . h nmr ( 500 mhz , cdcl3 ) 7.92 ( s , 1h ) , 7.67 ( s , 1h ) , 7.62 ( d , j = 7.8 hz , 1h ) , 7.397.35 ( m , 1h ) , 7.24 ( d , j = 8.0 hz , 1h ) , 7.187.15 ( m , 1h ) , 6.69 ( dd , j = 15.6 , 0.4 hz , 1h ) , 6.136.18 ( m , 1h ) , 2.312.26 ( m , 2h ) , 1.961.92 ( m , 1h ) , 1.571.54 ( m , 2h ) , 1.431.35 ( m , 10h ) , 1.23 ( d , j = 7.3 hz , 6h ) , 0.97 ( t , j = 6.9 hz , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 139.6 , 138.4 , 132.2 , 131.1 , 129.3 , 127.6 , 125.7 , 121.2 , 117.7 , 33.8 , 32.2 , 30.0 , 29.9 , 29.7 , 29.6 , 23.0 , 19.6 , 14.4 ; b nmr ( 128.38 mhz , cdcl3 ) 38.8 ; ir ( neat ) 3424 , 2924 , 1560 , 1427 , 969 cm ; hrms ( ci ) m / z [ m ] calcd for c21h32bn 309.2628 , found 309.2633 . the title compound was obtained as a yellow solid in 90% yield ( 276.3 mg ) : mp 5155 c ; h nmr ( 500 mhz , cdcl3 ) 7.92 ( s , 1h ) , 7.56 ( d , j = 7.7 hz , 1h ) , 7.327.29 ( m , 1h ) , 7.18 ( s , 1h ) , 7.11 ( t , j = 7.9 hz , 2h ) , 6.69 ( d , j = 15.6 hz , 1h ) , 6.386.24 ( m , 1h ) , 2.25 ( q , j = 7.2 hz , 2h ) , 1.521.48 ( m , 2h ) , 1.391.28 ( m , 10h ) , 0.920.89 ( m , 5h ) , 0.660.55 ( m , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 139.5 , 138.5 , 132.8 , 132.4 , 129.3 , 127.6 , 125.5 , 121.0 , 117.4 , 34.0 , 32.2 , 30.0 , 29.9 , 29.7 , 29.6 , 23.0 , 14.4 , 6.0 ; b nmr ( 128.38 mhz , cdcl3 ) 37.1 ; ir ( neat ) 3374 , 3010 , 2920 , 1557 , 1428 , 1105 cm ; hrms ( ci ) m / z [ m ] calcd for c21h30bn 307.2471 , found 307.2484 . the title compound was obtained as a yellow oil in 88% yield ( 317.6 mg ) . h nmr ( 500 mhz , cdcl3 ) 8.01 ( s , 1h ) , 7.85 ( s , 1h ) , 7.717.65 ( m , 3h ) , 7.417.37 ( m , 1h ) , 7.25 ( d , j = 8.5 hz , 1h ) , 7.227.19 ( m , 1h ) , 7.177.14 ( m , 2h ) , 6.62 ( dd , j = 15.6 , 0.7 hz , 1h ) , 6.01 ( dt , j = 15.5 , 7.0 hz , 1h ) , 2.17 ( qd , j = 7.3 , 1.3 hz , 2h ) , 1.481.49 ( m , 2h ) , 1.361.29 ( m , 10h ) , 0.92 ( t , j = 7.0 hz , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 163.6 ( d , j = 252 hz ) , 139.8 , 139.3 , 134.9 ( d , j = 7.4 hz ) , 132.4 , 132.3 , 129.3 , 127.9 , 125.7 , 121.6 , 117.8 , 115.1 ( d , j = 19.6 hz ) , 33.5 , 32.0 , 29.7 , 29.6 , 29.5 , 29.4 , 22.8 , 14.2 ; b nmr ( 128.38 mhz , cdcl3 ) 33.9 ; ir ( neat ) 3375 , 2923 , 1595 , 1224 , 832 cm ; hrms ( ci ) m / z [ m ] calcd for c24h29bnf 361.2377 , found 361.2368 . the title compound was obtained as a colorless oil in 81% yield ( 302.1 mg ) . h nmr ( 500 mhz , cdcl3 ) 8.04 ( s , 1h ) , 7.94 ( s , 1h ) , 7.68 ( d , j = 7.7 hz , 1h ) , 7.437.40 ( m , 2h ) , 7.32 ( d , j = 7.2 hz , 1h ) , 7.297.27 ( m , 2h ) , 7.22 ( t , j = 7.5 hz , 1h ) , 7.01 ( dd , j = 8.2 , 2.7 hz , 1h ) , 6.68 ( d , j = 15.6 hz , 1h ) , 6.106.04 ( m , 1h ) , 3.89 ( s , 3h ) , 2.212.16 ( m , 2h ) , 1.471.43 ( m , 2h ) , 1.361.28 ( m , 10h ) , 0.93 ( t , j = 6.9 hz , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 159.4 , 139.8 , 139.5 , 132.5 , 132.3 , 129.5 , 129.4 , 128.0 , 125.9 , 125.5 , 121.7 , 118.3 , 118.0 , 114.4 , 55.4 , 33.7 , 32.2 , 29.9 , 29.8 , 29.6 , 29.6 , 23.0 , 14.4 ; b nmr ( 128.38 mhz , cdcl3 ) 34.8 ; ir ( neat ) 3366 , 2924 , 1558 , 1451 , 1236 , 971 , 761 cm ; hrms ( ci ) m / z [ m + h ] calcd for c25h33bno 374.2655 , found 374.2661 . the title compound was obtained as a light brown oil in 83% yield ( 341.3 mg ) . h nmr ( 500 mhz , cdcl3 ) 8.03 ( s , 1h ) , 7.91 ( s , 1h ) , 7.80 ( d , j = 7.7 hz , 2h ) , 7.707.68 ( m , 3h ) , 7.427.39 ( m , 1h ) , 7.27 ( d , j = 8.1 hz , 1h ) , 7.227.21 ( m , 1h ) , 6.56 ( dd , j = 15.6 , 1.0 hz , 1h ) , 5.605.94 ( m , 1h ) , 2.14 ( m , 2h ) , 1.421.39 ( m , 2h ) , 1.271.32 ( m , 10h ) , 0.90 ( t , j = 5.1 hz , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 140.1 , 139.1 , 133.1 , 132.8 , 131.9 , 130.4 ( d , j = 32.3 hz ) , 129.4 , 128.1 , 126.3 ( q , j = 272.2 hz ) , 125.8 , 124.6 ( q , j = 3.7 hz ) , 121.9 , 117.9 , 33.5 , 32.0 , 29.6 , 29.5 , 29.3 , 22.8 , 14.2 ; b nmr ( 128.38 mhz , cdcl3 ) 34.5 ; ir ( neat ) 3395 , 2924 , 1559 , 1323 , 969 , 836 cm ; hrms ( ci ) m / z [ m ] calcd for c25h29bnf3 411.2345 , found 411.2353 . the title compound was obtained as a light yellow oil in 62% yield ( 199.0 mg ) . h nmr ( 500 mhz , cdcl3 ) 7.84 ( s , 1h ) , 7.61 ( d , j = 7.0 hz , 1h ) , 7.39 ( dd , j = 4.7 , 1.2 hz , 2h ) , 7.177.13 ( m , 1h ) , 6.66 ( dd , j = 15.5 , 1.0 hz , 1h ) , 6.106.01 ( m , 2h ) , 5.15 ( dd , j = 10.5 , 1.4 hz , 1h ) , 4.99 ( dd , j = 17.2 , 1.3 hz , 1h ) , 4.72 ( dd , j = 4.1 , 2.0 hz , 2h ) , 2.272.22 ( m , 2h ) , 1.541.49 ( m , 2h ) , 1.411.27 ( m , 10h ) , 0.930.89 ( m , 6h ) ; c nmr ( 125.8 mhz , cdcl3 ) 141.2 , 137.7 , 134.7 , 132.7 , 131.3 , 130.2 , 127.8 , 126.7 , 120.8 , 115.9 , 115.5 , 49.8 , 33.8 , 32.2 , 30.1 , 29.9 , 29.7 , 29.6 , 23.0 , 14.4 ; b nmr ( 128.38 mhz , cdcl3 ) 38.7 ; ir ( neat ) 2923 , 1556 , 1405 , 1212 , 968 cm ; hrms ( ci ) m / z [ m ] calcd for c22h32bn 321.2628 , found 321.2633 . the title compound was obtained as a light brown oil in 90% yield ( 333.9 mg ) . h nmr ( 500 mhz , cdcl3 ) 7.97 ( s , 1h ) , 7.68 ( d , j = 7.0 hz , 1h ) , 7.357.27 ( m , 5h ) , 7.197.16 ( m , 3h ) , 6.78 ( dd , j = 15.6 , 0.6 hz , 1h ) , 6.19 ( dt , j = 15.5 , 6.9 hz , 1h ) , 5.38 ( s , 2h ) , 2.362.37 ( m , 2h ) , 1.631.57 ( m , 2h ) , 1.501.39 ( m , 10h ) , 1.000.98 ( m , 6h ) ; c nmr ( 125.8 mhz , cdcl3 ) 141.3 , 138.8 , 138.02 , 132.7 , 131.4 , 130.2 , 129.0 , 127.9 , 127.1 , 126.9 , 126.1 , 120.9 , 115.9 , 51.4 , 33.8 , 32.2 , 30.1 , 29.9 , 29.8 , 29.7 , 23.0 , 14.4 ; b nmr ( 128.38 mhz , cdcl3 ) 39.0 ; ir ( neat ) 2924 , 2853 , 1610 , 1492 , 1402 , 1360 , 967 cm ; hrms ( ci ) m / z [ m ] calcd for c26h34bn 371.2784 , found 371.2791 . the title compound was obtained as a light yellow oil in 80% yield ( 285.6 mg ) . h nmr ( 500 mhz , cdcl3 ) 8.04 ( s , 1h ) , 7.91 ( s , 1h ) , 7.68 ( d , j = 7.5 hz , 1h ) , 7.577.52 ( m , 2h ) , 7.427.37 ( m , 2h ) , 7.297.25 ( m , 2h ) , 7.21 ( t , j = 7.5 hz , 1h ) , 6.68 ( d , j = 15.5 hz , 1h ) , 6.07 ( dt , j = 15.4 , 7.0 hz , 1h ) , 2.47 ( s , 3h ) , 2.20 ( q , j = 6.9 hz , 2h ) , 1.491.46 ( m , 2h ) , 1.391.33 ( m , 10h ) , 0.94 ( t , j = 6.9 hz , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 139.7 , 137.5 , 133.9 , 132.7 , 132.2 , 130.3 , 129.5 , 128.1 , 128.0 , 125.9 , 121.6 , 117.9 , 33.7 , 32.2 , 30.0 , 29.9 , 29.7 , 29.6 , 23.0 , 21.9 , 14.4 ; b nmr ( 128.38 mhz , cdcl3 ) 34.7 ; ir ( neat ) 3369 , 2923 , 2852 , 1612 , 1557 , 1450 , 969 cm ; hrms ( ci ) m / z [ m ] calcd for c25h32bn 357.2628 , found 357.2632 . the title compound was obtained as a brown oil in 83% yield ( 296.3 mg ) . h nmr ( 500 mhz , cdcl3 ) 7.85 ( s , 1h ) , 7.77 ( s , 1h ) , 7.57 ( d , j = 1.5 hz , 1h ) , 7.477.41 ( m , 5h ) , 7.337.31 ( m , 1h ) , 7.16 ( d , j = 8.5 hz , 1h ) , 6.46 ( d , j = 16.0 hz , 1h ) , 6.23 ( dt , j = 15.7 , 6.9 hz , 1h ) , 2.272.22 ( m , 2h ) , 1.521.48 ( m , 2h ) , 1.391.28 ( m , 10h ) , 0.92 ( t , j = 7.0 hz , 3h ) , 0.83 ( s , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 145.1 , 141.8 , 139.0 , 131.3 , 130.0 , 129.6 , 128.5 , 128.4 , 127.2 , 126.3 , 126.0 , 125.3 , 117.7 , 33.4 , 32.2 , 29.8 , 29.8 , 29.6 , 29.6 , 23.0 , 14.4 ; b nmr ( 128.38 mhz , cdcl3 ) 36.4 ; ir ( neat ) 3372 , 2924 , 2853 , 1617 , 1577 , 1456 , 962 , 762 cm ; hrms ( ci ) m / z [ m ] calcd for c25h32bn 357.2628 , found 357.2621 . the title compound was obtained as a yellow solid in 76% yield ( 169.4 mg ) : mp 106110 c ; h nmr ( 500 mhz , cdcl3 ) 7.75 ( s , 1h ) , 7.60 ( s , 1h ) , 7.48 ( d , j = 1.5 hz , 1h ) , 7.38 ( dd , j = 8.3 , 1.9 hz , 1h ) , 7.07 ( d , j = 8.4 hz , 1h ) , 6.62 ( dd , j = 15.7 , 0.7 hz , 1h ) , 6.48 ( dd , j = 15.7 , 1.5 hz , 1h ) , 6.266.14 ( m , 2h ) , 1.951.93 ( m , 6h ) , 0.86 ( s , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 137.4 , 137.6 , 133.9 , 130.1 , 130.0 , 125.7 , 125.4 , 124.5 , 124.3 , 123.1 , 116.6 , 18.3 , 17.8 ; b nmr ( 128.38 mhz , cdcl3 ) 37.2 ; ir ( neat ) 3371 , 2957 , 2910 , 1610 , 1557 , 1436 cm ; hrms ( ci ) m / z [ m ] calcd for c15h18bn 223.1532 , found 223.1532 .

conditions have been developed for the palladium - catalyzed cross - coupling of 3-bromo-2,1-borazaronaphthalenes with potassium alkenyltrifluoroborates . twenty - seven alkenyl - substituted azaborines have been synthesized through this method , providing access to a family of 2,1-borazaronaphthalenes with alkenyl substitution at the c3 position .

Introduction Results and Discussion Experimental Section

2-alkenylnaphthalenes can be synthesized through a variety of transition - metal - catalyzed transformations , including heck , mizoroki heck , hiyama , and suzuki miyaura reactions . however , installation of an alkenyl substituent on a functionalized naphthalene is more challenging and often requires four or five steps . one method to afford alkenyl - substituted naphthalenes is to perform free - radical bromination of a methyl - substituted derivative followed by a wittig olefination . aside from the generation of a stoichiometric amount of difficult - to - remove phosphine oxide waste , additional substitution on similarly , titanium - mediated carbonylation with dichloromethyl methyl ether followed by wittig olefination afforded 2-alkyl-3-alkenylnaphthalene in moderate yield over two steps . further , there is only one example demonstrating the installation of an alkenyl substituent on a 2-arylnapthalene through the cross - coupling of naphthyl iodide and alkenylboronic acid , which provides 2-aryl-3-alkenylnaphthalene in good yield . the naphthyl iodide in this case was synthesized through gold - catalyzed cyclization / iodination , which required preparation of the requisite starting material ( 1-arylalka-2,3-dienyl acetate ) . additionally , to the best of our knowledge , installation of an alkenyl substituent at the c3 position of a 1,2-disubstituted naphthalene has not been reported . accessing 2,1-borazaronaphthalenes , a class of azaborines that are bn - isosteres of naphthalene , with alkenyl substituents at the c3 position under mild reaction conditions would therefore serve as an example of how functionalized azaborines could be prepared more efficiently than their corresponding all - carbon analogues . recently , we reported the bromination of 2,1-borazaronaphthalenes in high yield under mild reaction conditions with complete regiochemical control . because of the inherent reactivity of azaborine , bromination occurs selectively at the c3 position of the azaborine in the presence of alkyl / aryl substituents on boron and nitrogen . miyaura cross - coupling reactions with potassium alkenyltrifluoroborates as a route to c3-alkenyl - substituted 2,1-borazaronaphthalenes , a compound class that has not been previously reported . optimization of the reaction of 3-bromo-2-methyl-2,1-borazaronaphthalene 1 with functionalized alkenyltrifluoroborate 2 was carried out by screening palladium sources , solvents , and bases . conditions for the cross - coupling of potassium aryltrifluoroborates with brominated 2,1-borazaronaphthalenes did not afford the desired product in appreciable yield ( table 1 , entry 1 ) . several solvents / ratios were tested for these ligands , and the best result was obtained using toluene as the cosolvent and pd(dppf)cl2 as the palladium source ( entry 7 ) . a representative set of potassium alkenyltrifluoroborates were subjected to the developed reaction conditions with 3-bromo-2-methyl-2,1-borazaronaphthalene as the electrophilic partner ( table 2 ) . vinyltrifluoroborate was a suitable nucleophile for the reaction because the azaborine was obtained in 70% yield ( entry 7 ) . the scalable nature of the cross - coupling reaction was demonstrated by performing the coupling on a 4.5 mmol scale ( 1 g of azaborine ) with one - third palladium loading ( 2 mol % ) , which provided the desired product in similar yield ( entry 8) . reaction conditions ( unless otherwise noted ) : 1.0 equiv of 3-bromo-2-methyl-2,1-borazaronaphthalene , 1.1 equiv of potassium alkenyltrifluoroborate , 6.0 mol % pd(dppf)cl2 , 3.0 equiv of base , 1:1 toluene / h2o , and 60 c for 18 h. the developed reaction conditions were extended to cross - coupling of 3-bromo-2-phenyl-2,1-borazaronaphthalene with an array of potassium alkenyltrifluoroborates to demonstrate that azaborine can also be substituted with an aryl group on the boron . the mild reaction conditions of the coupling were demonstrated in the successful coupling of an alkenyltrifluoroborate with an alkyl chloride substituent , affording the desired product in 90% yield ( table 3 , entry 2 ) . reaction conditions ( unless otherwise noted ) : 1.0 equiv of 3-bromo-2-phenyl-2,1-borazaronaphthalene , 1.1 equiv of potassium alkenyltrifluoroborate , 6.0 mol % pd(dppf)cl2 , 3.0 equiv of base , 1:1 toluene / h2o , and 60 c for 18 h. to demonstrate the versatility of this method , an array of brominated 2,1-borazaronaphthalenes were synthesized and subjected to the developed reaction conditions with 1-decenyltrifluoroborate as the nucleophile in the reaction . the cross - coupled products were obtained with secondary cyclic and acyclic groups on boron in yields up to 90% ( table 4 , entries 1 and 2 ) . azaborines with various substitution patterns on the arene of boron were suitable electrophiles for coupling as the cross - coupled products were isolated in high yield ( entries 35 ) . substitution on the nitrogen of the 2,1-borazaronaphthalenes did not affect the coupling as reactions with both n - allyl and n - benzyl substituents provided the desired product in high yields ( entries 6 and 7 ) . the desired product was obtained in 83% yield to afford c6-alkenyl - substituted 2,1-borazaronaphthalenes , examples of which are absent in the literature.1 the reaction conditions were then extended to cross - coupling of 3,6-dibrominated 2,1-borazaronaphthalene . the addition of 2.2 equiv of 1-propenyltrifluoroborate provides a doubly cross - coupled product in 76% yield ( eq 2).2 in conclusion , a general method for suzuki miyaura cross - coupling of brominated 2,1-borazaronaphthalenes with potassium alkenyltrifluoroborates has been developed . azaborines with alkyl and aryl groups on boron and with or without substitution on nitrogen are suitable reagents for the coupling . through this route , b nmr spectra were obtained on a spectrometer equipped with the appropriate decoupling accessories . hrms data were obtained by either esi or ci using a tof mass spectrometer . to a biotage 10 ml microwave vial equipped with a magnetic stir bar were added 3-bromo-2,1-alkenyltrifluoroborate ( 1.1 mmol , 1.1 equiv ) , cs2co3 ( 3.0 mmol , 977 mg ) , pd(dppf)cl2 ( 6.0 mol % , 42 mg ) , and potassium alkenyltrifluoroborate ( 1.0 mmol , 1.0 equiv ) . after being concentrated in vacuo , the product was isolated by flash column chromatography and eluted with a gradient of etoac in hexanes ( 0 to 10% etoac ) . h nmr ( 500 mhz , cdcl3 ) 7.80 ( s , 1h ) , 7.68 ( s , 1h ) , 7.62 ( d , j = 7.7 hz , 1h ) , 7.397.36 ( m , 1h ) , 7.197.15 ( m , 2h ) , 5.215.11 ( m , 1h ) , 5.07 ( d , j = 1.9 hz , 1h ) , 2.16 ( s , 3h ) , 0.87 ( s , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 148.4 , 139.6 , 139.2 , 129.6 , 128.0 , 125.1 , 121.0 , 117.4 , 113.0 , 23.9 ; b nmr ( 128.38 mhz , cdcl3 ) 36.9 ; ir ( neat ) 3368 , 2933 , 1567 , 1462 , 1453 cm ; hrms ( ci ) m / z [ m ] calcd for c12h14bn 183.1219 , found 183.1220 . the title compound was obtained as a white solid in 65% yield ( 210.6 mg ) : mp 8387 c ; h nmr ( 500 mhz , cdcl3 ) 7.75 ( s , 1h ) , 7.69 ( s , 1h ) , 7.57 ( d , j = 7.7 hz , 1h ) , 7.367.33 ( m , 1h ) , 7.17 ( d , j = 8.0 hz , 1h ) , 7.147.11 ( m , 1h ) , 5.71 ( br , 1h ) , 4.07 ( br , 2h ) , 3.64 ( t , j = 5.5 hz , 2h ) , 4.47 ( br , 2h ) , 1.52 ( s , 9h ) , 0.80 ( s , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 155.3 , 139.9 , 139.6 , 138.7 , 129.5 , 128.0 , 125.1 , 121.1 , 120.7 , 117.4 , 79.7 , 44.3 , 40.1 , 34.9 , 29.4 , 28.8 ; b nmr ( 128.38 mhz , cdcl3 ) 32.0 ; ir ( neat ) 3327 , 2975 , 1677 , 1563 , 1423 , 1163 cm ; hrms ( ci ) m / z [ m + h ] calcd for c19h26bn2o2 325.2087 , found 325.2080 . h nmr ( 500 mhz , cdcl3 ) 8.05 ( s , 1h ) , 7.91 ( s , 1h ) , 7.777.75 ( m , 2h ) , 7.69 ( d , j = 8.0 hz , 1h ) , 7.517.47 ( m , 3h ) , 7.427.39 ( s , 1h ) , 7.26 ( d , j = 8.0 hz , 1h ) , 7.22 ( t , j = 7.5 hz , 1h ) , 6.70 ( d , j = 15.5 hz , 1h ) , 6.07 ( dt , j = 15.4 , 7.0 hz , 1h ) , 2.21 ( q , j = 7.0 hz , 2h ) , 1.501.45 ( m , 2h ) , 1.401.35 ( m , 10h ) , 0.96 ( t , j = 7.0 hz , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 139.8 , 139.6 , 133.2 , 132.7 , 132.3 , 129.5 , 128.8 , 128.2 , 128.0 , 125.9 , 121.0 , 118.0 , 33.7 , 32.2 , 29.9 , 29.8 , 29.7 , 29.6 , 23.0 , 14.4 ; b nmr ( 128.38 mhz , cdcl3 ) 34.4 ; ir ( neat ) 3370 , 2923 , 2852 , 1421 , 749 cm ; hrms ( ci ) m / z [ m + na ] calcd for c24h30bnna 366.2369 , found 366.2375 . the title compound was obtained as a brown oil in 76% yield ( 186.2 mg ) . the title compound was obtained as a colorless oil in 81% yield ( 247.8 mg ) . h nmr ( 500 mhz , cdcl3 ) 8.01 ( s , 1h ) , 7.85 ( s , 1h ) , 7.717.65 ( m , 3h ) , 7.417.37 ( m , 1h ) , 7.25 ( d , j = 8.5 hz , 1h ) , 7.227.19 ( m , 1h ) , 7.177.14 ( m , 2h ) , 6.62 ( dd , j = 15.6 , 0.7 hz , 1h ) , 6.01 ( dt , j = 15.5 , 7.0 hz , 1h ) , 2.17 ( qd , j = 7.3 , 1.3 hz , 2h ) , 1.481.49 ( m , 2h ) , 1.361.29 ( m , 10h ) , 0.92 ( t , j = 7.0 hz , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 163.6 ( d , j = 252 hz ) , 139.8 , 139.3 , 134.9 ( d , j = 7.4 hz ) , 132.4 , 132.3 , 129.3 , 127.9 , 125.7 , 121.6 , 117.8 , 115.1 ( d , j = 19.6 hz ) , 33.5 , 32.0 , 29.7 , 29.6 , 29.5 , 29.4 , 22.8 , 14.2 ; b nmr ( 128.38 mhz , cdcl3 ) 33.9 ; ir ( neat ) 3375 , 2923 , 1595 , 1224 , 832 cm ; hrms ( ci ) m / z [ m ] calcd for c24h29bnf 361.2377 , found 361.2368 . h nmr ( 500 mhz , cdcl3 ) 7.97 ( s , 1h ) , 7.68 ( d , j = 7.0 hz , 1h ) , 7.357.27 ( m , 5h ) , 7.197.16 ( m , 3h ) , 6.78 ( dd , j = 15.6 , 0.6 hz , 1h ) , 6.19 ( dt , j = 15.5 , 6.9 hz , 1h ) , 5.38 ( s , 2h ) , 2.362.37 ( m , 2h ) , 1.631.57 ( m , 2h ) , 1.501.39 ( m , 10h ) , 1.000.98 ( m , 6h ) ; c nmr ( 125.8 mhz , cdcl3 ) 141.3 , 138.8 , 138.02 , 132.7 , 131.4 , 130.2 , 129.0 , 127.9 , 127.1 , 126.9 , 126.1 , 120.9 , 115.9 , 51.4 , 33.8 , 32.2 , 30.1 , 29.9 , 29.8 , 29.7 , 23.0 , 14.4 ; b nmr ( 128.38 mhz , cdcl3 ) 39.0 ; ir ( neat ) 2924 , 2853 , 1610 , 1492 , 1402 , 1360 , 967 cm ; hrms ( ci ) m / z [ m ] calcd for c26h34bn 371.2784 , found 371.2791 . h nmr ( 500 mhz , cdcl3 ) 8.04 ( s , 1h ) , 7.91 ( s , 1h ) , 7.68 ( d , j = 7.5 hz , 1h ) , 7.577.52 ( m , 2h ) , 7.427.37 ( m , 2h ) , 7.297.25 ( m , 2h ) , 7.21 ( t , j = 7.5 hz , 1h ) , 6.68 ( d , j = 15.5 hz , 1h ) , 6.07 ( dt , j = 15.4 , 7.0 hz , 1h ) , 2.47 ( s , 3h ) , 2.20 ( q , j = 6.9 hz , 2h ) , 1.491.46 ( m , 2h ) , 1.391.33 ( m , 10h ) , 0.94 ( t , j = 6.9 hz , 3h ) ; c nmr ( 125.8 mhz , cdcl3 ) 139.7 , 137.5 , 133.9 , 132.7 , 132.2 , 130.3 , 129.5 , 128.1 , 128.0 , 125.9 , 121.6 , 117.9 , 33.7 , 32.2 , 30.0 , 29.9 , 29.7 , 29.6 , 23.0 , 21.9 , 14.4 ; b nmr ( 128.38 mhz , cdcl3 ) 34.7 ; ir ( neat ) 3369 , 2923 , 2852 , 1612 , 1557 , 1450 , 969 cm ; hrms ( ci ) m / z [ m ] calcd for c25h32bn 357.2628 , found 357.2632 .

mass spectrometric analysis of proteome samples from eukaryotic cell lines , tissues or biological fluids is still hampered by the vast complexity of samples and concentration differences of proteins within making comparisons of proteomics and transcriptomics data difficult . online and off - line two - dimensional ( 2d ) or multidimensional separation to reduce sample complexity is therefore still of high importance . the most widely used methods involve two subsequent steps , an off - line fractionation step followed by ( low ph ) reversed phase liquid chromatography ( rp - lc ) directly coupled to mass spectrometry . ideally , the separation in the two - dimensions is highly complementary and uses different mechanisms , that is , they separate according to different properties of the analyte such as size , charge and hydrophobicity . many approaches have shown decent orthogonality such as hydrophilic interaction liquid chromatography ( hilic ) , electrostatic repulsion hydrophilic interaction chromatography ( erlic ) , strong cation exchange ( scx ) , isoelectric focusing ( such as offgel ) , scx - weak - anion exchange ( wax ) 3d and high - ph rp . particularly , high ph rp chromatography has shown great potential as it is highly resolving and partly orthogonal to low ph rp . however , this setup requires the samples to be dried down after the first dimension resulting in sample loss , unless another , third dimension such as sax is added , which increases the number of fractions and thus instrument time and reduces robustness of the system . furthermore , the high ph of the first rp dimension is corroding most of the silica - based stationary phase , leading to reproducibility issues and short column lifetime . while high - ph rp can never achieve full orthogonality to low - ph rp as they both separate according to hydrophobicity , ion exchange chromatography such as scx or strong anion exchange ( sax ) chromatography at least theoretically can . but while scx has been widely used , sax has been not widely considered for proteomic analysis . this is surprising as it was observed that the majority of tryptic peptides cluster between pi of 3 and 5 , partly because many post - translational modifications such as phosphorylation , pyro - glutamination or acetylation decrease the pi . this suggests that the majority of the tryptic peptides in a total cell digest are likely acidic , thus negatively charged and should be well separable by sax . some groups reported studies involving sax fractionation , mostly for phospho - proteomics and glycomics . on the proteome level , an online mixed mode reversed phase anion exchange ( mm , rp - ax ) , a sax - scx mixed bed ion exchange chromatography , a sax microreactor or a sax stage - tip approach were successfully used ; however , the use of sax for large - scale proteome analyses is still underdeveloped . here , we present a sax column with a hyperbranched architecture , quaternary ammonium ion functionality and an ultralow hydrophobicity . while the column was originally designed for the separation of small organic and haloacetic acids , we show its remarkable separation power for proteome research . this column exhibits high reproducibility , high capacity , robustness and very high orthogonality when coupled to low ph rp , allowing the identification of more than 9000 proteins from a raw264.7 macrophage cell lysate on an orbitrap velos pro mass spectrometer within only one week of instrument time . we show that both hydrophilicity and the ion exchange properties of the column are important for the high orthogonality of this approach , which we term hydrophilic strong anion exchange ( hsax ) chromatography . the standard proteins mixture consisted in bovine thyroglobulin , bovine serum albumin , chicken ovalbumin , bovine beta - casein , cytochrome c from horse heart , lysozyme from chicken eggs . all proteins were purchased from sigma aldrich ( st . louis , mo ) . ( walkerburn , scotland ) , while those for online system were purchased from merck kgaa ( darmstadt , germany ) . the mouse macrophage cell line raw264.7 was obtained from atcc and grown in dmem , 10% heat - inactivated fetal bovine serum ( fbs , sigma ) , 2 mm l - glutamine , 5000 u / ml penicillin and 5000 g / ml streptomycin ( invitrogen ) . raw 264.7 cells were harvested , washed with ice - cold pbs and lysed in 8 m urea/50 mm tris - hcl ( ph 8.0)/10 mm dtt plus a phosphatase inhibitor cocktail containing 1.15 mm sodium molybdate , 1 mm sodium orthovanadate , 4 mm sodium tartrate dehydrate , 5 mm glycerophosphate ( all sigma aldrich ) and 1 mm sodium fluoride ( analar normapur , west sussex , uk ) ; finally , 1 l of benzonase ( merck ) was added and the lysate passed through a 26.5 g needle . the solution was left to reduce for 60 min at 30 c under shaking . afterward , 15 mm iodoacetamide ( iaa ) was added and the sample was left to alkylate for 40 min at room temperature ( rt ) in the dark . the iaa was then deactivated with 20 mm dtt for 40 min at rt and at this point the protein concentration was assessed by rc / dc protein assay according to the manufacturer s instructions ( biorad ) . prior to digestion , the protein mixture was diluted 8 times with 50 mm tris - hcl ph 8.0 to 1 m urea . trypsin tpck ( worthington ) was methylated as described previously , added to the protein mixture ( 1:100 , trypsin : sample ) and the digestion was performed at rt overnight . another 1:100 was added the day after for 3 h followed by addition of 1% tfa to stop trypsin activity . finally , the sample was desalted by seppack oasis solid phase extraction cartridges ( waters ) , dried down and stored at 80 c . the same procedure was applied to the standard protein mixture ( 6-mix ) . an off - line thermo hplc ultimate 3000 system equipped with the wps-3000t(b ) fc autosampler , the dgp-3600bm pump system including the srd-3600 system for solvent autodegasser , the vwd-3400rs uv / vis photometer and the tcc-3000sd thermo - controlled column compartment were used . in this work , we compared three different strong anion exchange ( sax ) columns : the as24 , the as11-hc and the as15 ( ionpac series , thermo - fisher scientific ) that were chosen according to their chemical / physical characteristics . the as24 ( 2 250 mm , 2000 pore size ) is a low bleed column primarily designed for the separation of environmental ions as well as for ion chromatography separation coupled with mass spectrometry . as24 is a high capacity ( 140 eq ) analytical column and it is compatible with ph 014 eluents and samples ( table 1 ) . it consists in a supermacroporous resin with alkanol quaternary ammonium ions as functional groups , which are ultralow hydrophobic . it uses hyperbranched chemistry with extremely hydrophilic architecture whereas as11-hc and as15 used aromatic monomers and are thus significantly more hydrophobic . finally , the as25 is very similar to as24 but it is the most hydrophilic member of the product family ( ionpac as25 , 2 250 mm , 2000 , 87.5 eq of capacity ) but has a lower capacity . it was tested to confirm that the hydrophilic architecture of as24 improves the orthogonal separation of tryptic peptides . to compare the results of sax / rp with ( high ph ) rp/ ( low ph ) rp , two reversed - phase columns were tested off - line in the first dimension , which differ in matrix composition . in this work , acclaim 120 c18 ( a conventional rp , 3 m , 120 , 2.1 150 mm , thermo - fisher scientific ) and acclaim rslc polar advantage ii -pa2- ( a polar - embedded rp , 2.2 m , 120 , 2.1 100 mm , thermo - fisher scientific ) columns were used to separate tryptic digested proteins from raw264.7 cell lysate . while both c18 columns are based on ultrapure silica , acclaim pa2 has the advantage to have specially designed amide - embedded ligands . those features make the column compatible with 100% aqueous environments over a wide range of ph ( 1.510 ) , exhibiting high polarity for selectivity complementary to conventional rp columns . the acclaim pa2 column provided higher efficiencies than the acclaim 120 c18 column ( supporting information , figure s1 ) and therefore it was chosen for the comparison with the as24 . for the off - line 2d lc , a highly resolving c18 column was used ( gemini - c18 , 3 m , 110 , 3 250 mm , phenomenex ) . this column uses ultrapure spherical silica particles and can operate at wide ph range ( 112 ) increasing method development flexibility . peptide separation on the off - line 1d separation system employing strong anion exchange was achieved with a flow rate of 0.25 ml / min ( solvent a : 20 mm tris - hcl ph 8.0 ; solvent b : 20 mm tris - hcl ph 8.0 , 1 m nacl ) . a gradient ( slope 7 for 6-mix , 8 for raw264.7 cells ) spanning 0100% mobile phase b over 35 min was used . in case of high ph rp , separation was based on an acetonitrile ( acn ) gradient ( solvent a : 20 mm nh4oh / h2o , ph 10.0 ; solvent b : 20 mm nh4oh / acn , ph 10.0 ) at the same flow rate ( 0.250 ml / min , 080% b in 35 min ) . when the acclaim 120 was tested , the acn gradient was set at 580% b for 35 min . the off - line 2d was commonly performed at ph 2.5 ( solvent a : 0.1% tfa / h2o ; solvent b : 0.08% tfa / acn ) at a flow rate of 0.4 ml / min and a multistep gradient of 35 min : 515% b in 5 min , following by 30 min up to 90% b. all chromatograms were recorded by a uv detector at 214 nm . off - line 1d - fractions ( either sax without further handling or high ph rp , after drying down and resuspension in 0.1% tfa ) from the raw264.7 cell lysate were run online on an orbitrap velos pro ( thermo - fisher scientific ) . all nanoflow chromatography was performed on an ultimate 3000 nano lc system ( thermo - fisher scientific ) , using an acclaim pepmap 100 ( 75 m i d 500 mm , 3 m c18 ) in conjunction with an acclaim c18 pepmap trapping column ( 100 m i d 20 mm , 5 m c18 ) ( thermo - fisher scientific ) . linear gradient elution was performed using buffer a ( 0.1% formic acid ) and buffer b ( 0.08% formic acid , 80% acn ) starting from 5% buffer b to 35% over 277 min at a flow rate of 300 nl / min . a coated silica tip ( new objective , woburn , ma ) was used for electrospraying the sample into the mass spectrometer , at an ion spray voltage of 1.45 kv . ms analysis was operated in data dependent mode , such that the top 20 most abundant precursors in each ms scan were subjected to ms / ms ( cid in the linear trap , normalized collision energy = 35% , precursor isolation width = 2 da , intensity threshold for precursor selection = 2000 ) . as global parameters , precursor ions between m / z 4002000 were selected , at resolution of 60000 collected in profile mode . dynamic exclusion was enabled with a repeat count of 1 and exclusion duration set to 60 s. the lock mass feature was enabled for m / z = 445.120025 ( [ si(ch3)2o]6 ) as the internal calibrant ion . the data analysis of all ms / ms proteomics data sets was performed through the trans - proteomic pipeline ( tpp ) as previously described . briefly , mass spectrometry raw output files were first converted to mzxml by readw and afterward searched through the x!tandem ( cyclone 2011.12.01 ) search engine with k - score plug - in . data was searched against a randomized forward and reverse ipi mouse database ( version 3.87 ) and x!tandem parameters included cysteine carbamylation as fixed modifications , methionine oxidation and asparagine / glutamine deamidation were set as variable modifications . cyclization of glutamine / glutamic acid at the n - termini ( pyroglutamate ) was automatically searched , since tandem automatically checks for formation of pyroglutamic acid , that is , the loss of water ( e ) or ammonia ( q ) , respectively , when a peptide starts with e or q. this modification is considered to be an n - terminal modification only , so it does not affect any potential modifications specified for q , e or c. search parameters specified a precursor mass tolerance of 25 ppm ( required by tpp , typical mass accuracy was < 2 ppm ) , a ms / ms tolerance at 0.4 da and full trypsin specificity allowing for up to 2 missed cleavages . peptideprophet was then used to validate the search engine results and to assign accurate probabilities to peptide results were filtered at a calculated 1% fdr on the peptide level and then protein level . data were imported into r ( v2.11.0 ) , an open source platform for statistical analysis ( http://www.r-project.org ) and graphs were plotted using the ggplot2 library . for identification via andromeda , raw - files were loaded into maxquant 1.3.0.5 and searched against the same ipi mouse database ( version 3.87 ) . variable and fixed modifications were the same as above ; precursor mass accuracy was set to 6 ppm , ms / ms tolerance to 0.4 da and trypsin specificity allowing for up to 2 missed cleavages . false - discovery rate was set to 0.05 for peptides and 0.01 for proteins . from a range of 12 high - capacity hydroxide - selective anion - exchange columns ( ionpac series , thermo - fisher scientific ) which were initially developed for the separation of small organic and haloacetic acids and inorganic anions , we tested three members ( as24 , as11-hc and as15 ) for the use in off - line fractionation in a proteomics setting . these columns all contain the same functionality of alkanol quaternary ammonium ions , but differ in backbone hydrophobicity , with as24 exhibiting ultralow hydrophobicity , as11-hc medium hydrophobicity and as15 high hydrophobicity . in initial experiments we injected 180 g of a tryptic digest of six proteins fractions were automatically collected and reinjected onto a c18 rp column using an ultimate 3000 hplc ( thermo - fisher scientific ) and data acquired on a uv detector . with the use of chromeleon software ( v6.8 ) , 2d retention maps were created by which the evaluation of the peptide distribution over the two dimensions was possible . retention maps showed a highly orthogonal separation by as24as shown by the wide distribution over the two - dimensional space some separation by as11-hc and almost full retention of peptides on as15 even when eluted with 1 m nacl ( figure 1a ) . as the functionality in all three materials is identical , the difference of separation efficiency must depend on their differences in hydrophobicity . indeed , addition of at least 25% acn to the mobile phase allowed elution of tryptic peptides from as15 and improved elution from as11-hc ( figure 1b ) , showing that high hydrophobicity of the chemical backbones of these columns hampers the use for separation of peptides . furthermore , the most hydrophilic member of the ion exchange materials in this line ( as25 ) performs similarly well as as24 ( supporting information , figure s2 ) which led us to conclude that both the hydrophilicity in conjunction with the ion exchange functionality are important for the high orthogonality of this type of chromatography that we termed hydrophilic strong anion exchange ( hsax ) chromatography . ( a ) 2d retention maps of peptides of a 6-protein mix digest show that elution with up to 1 m nacl in absence of organic solvent leads to high orthogonality for as24 ( ultralow hydrophobicity ) , but part and full retention of peptides by as11-hc ( medium hydrophobicity ) and as15 ( high hydrophobicity ) , respectively . ( b ) addition of 25% acetonitrile ( acn ) into the elution buffer enhances elution from as11-hc and as15 . ( c ) three replicate injections on as24 over the course of 48 h show high reproducibility . ( d ) increasing amounts of protein digest loaded on as24 show good linear behavior without peak broadening . as the functional groups are primarily quaternary amines , changes of ph have no to little effect on column chemistry , but will change the available negative charge on peptides . when the mobile phase was adjusted to ph 3 , a major breakthrough was observed in 2d retention maps , which was absent at ph 8 . furthermore , acidic ph in sample solution , while keeping the mobile phase at ph 8 , or changing the mobile phase to ph 10 reduced retention of peptides or had no beneficial effect ( supporting information , figure s3 ) . we therefore performed all following experiments in 20 mm tris - hcl , at ph 8 . this has actually the benefit that tryptic digestion of the cell lysate can be performed at 20 mm tris - hcl , ph 8 and directly injected on the column , avoiding solid phase extraction or lyophilization and , therefore , minimizing sample loss . now that running conditions were optimized , we calculated the column efficiency of the as24 on a digested peptide mixture from bsa ( supporting information , figure s4 ) and we analyzed the run - to - run reproducibility and linearity which is required for quantitative proteomics experiments , using replicate injections of digested 6-mix samples over a period of 48 h ( figure 1c ) and injections of increasing material ( figure 1d ) . in both cases , as24 showed very high reproducibility ( supporting information , figure s5 ) and good linearity ( supporting information , figure s6 ) . after optimized parameters were established , the method was applied to a very complex mixture , a total cell lysate of the mouse macrophage cell line raw264.7 . tryptic peptides of 200 g of cell lysate were separated on the as24 column in a 35 min gradient from 0 to 1 m nacl ( 34 fractions ) which were automatically collected and injected onto a gemini c18 column ( phenomenex ) . 2d retention maps show a very high orthogonality of separation between the two chromatographic dimensions ( figure 2a ) , displayed by the elution of peptides over the entire 2d map . separation of total cell lysate digests by hsax and a high ph / low ph reversed phase approach show differences in orthogonality . tryptic raw264.7 macrophage cell lysate digests were separated on an as24 sax column ( a ) or an acclaim pa2 rp column at ph 10 ( b ) . thirty - four fractions of both first dimensions were separated in the second dimension by reversed phase chromatography at ph 3 . furthermore , we separated the same cell lysate digest on two different columns using high ph ( ph 10)/ low ph ( ph 3 ) rp / rp chromatography ( supporting information , figure s1 ) which has shown good orthogonality in the past and is currently seen as the gold standard in two - dimensional separation due to its high resolution . we then optimized elution parameters and chose the acclaim pa2 ( thermo - fisher scientific ) as it performed best , likely due to its higher polarity than the conventional c18 ( acclaim 120 , thermo - fisher scientific ) . then we compared its orthogonality to the as24 ion exchange column using 2d peptide maps using identical run time and numbers of fractions ( figure 2b ) . 2d retention maps show that the hsax / rp approach performs considerably better than the rp / rp approach , as a much greater area in the 2d maps is covered with peaks , while peptides in the rp / rp approach are scattered around the diagonal . this provides further evidence that high ph rp / low ph rp can not be fully orthogonal , as both dimensions separate according to hydrophobicity . recent approaches to concatenate early and late fractions of high ph rp to increase orthogonality have improved results ; however , concatenation involves user interference and might provide problems in quantitative proteomics experiments when the same peptides are then present in non - neighboring fractions as quantitative proteomics software such as maxquant requires peptides to be in neighboring fractions for accurate quantitation . in order to test both hsax and high ph rp approaches for their performance in a proteomics experiment , we injected aliquots ( 36% , corresponding to 300400 ng of protein digest ; 1 10 base peak chromatogram ) of each of the 34 fractions of the raw264.7 cell lysate separated by hsax or high ph rp onto an orbitrap velos pro mass spectrometer , using 300 min gradients on a 75 m 50 cm pepmap c18 column ( thermo - fisher scientific ) , corresponding to one week of instrument time each . online separation of hsax and high ph rp fractions showed a similar degree of orthogonality comparable to the offline 2d chromatography ( supporting information , figure s7a and b ) . ms / ms spectra were searched against the mouse ipi database v3.87 using x!tandem . results were filtered at peptide- and proteinprophet estimated 1% fdr . while we identified with the rp / rp approach 99110 peptides and 8627 proteins ( 1% fdr ) , the hsax approach resulted in identification of 126318 unique peptides and 9469 proteins , representing 28% and 10% more identifications respectively ( table 2 and supporting information , table s1 ) , while keeping the average sequence coverage equal ( 31% respectively 32% ) ( supporting information , figure s8 ) . combining both approaches , we identified 159847 peptides and 9871 proteins showing that we could increase the number of peptides and proteins identified by only 20% and 4% respectively compared to hsax alone ( table 2 ) . in addition , we analyzed the data using the andromeda search engine included in maxquant which identified overall 68% less proteins and peptides in each experiment , but confirmed the superiority of our sax approach ( table 2 and supporting information , table s1 ) . this data compares well to two recent publications in which about similar number of proteins were identified from a mammalian cell line using 3 different proteases and 2 weeks of instrument time or in which samples were analyzed repeatedly with inclusion lists . unique proteins ( fdr 1% ) according to proteinprophet ( x!tandem ) and maxquant ( andromeda ) . unique nonstripped peptides ( fdr 1% ) according to peptideprophet ( x!tandem ) and maxquant ( andromeda ) . number differing from figure 4a due to fdr recalculation on combined pep.xml ( peptideprophet ) . the high orthogonality of the hsax approach is not only shown by the broad distribution in the 2d peptide maps , but also exemplified by the fact that we identified more than 3000 proteins in 16 out of 34 fractions , while the rp / rp approach achieved this for only 6 fractions ( figure 3a ) . data also indicated that we could have saved instrument time by combining the last 5 fractions , which could have reduced run time by almost another day . the hsax approach also showed a considerably higher number of proteins ( 7292 vs 6315 in rp / rp , + 15% ) that were identified with 3 and more peptides which considerably improves identification and quantitation in proteomics experiments . comparison of protein and peptide identifications from off - line separation by either hsax / rp or rp / rp and lc ms experiments . ( a ) the high orthogonality of the hsax approach is exemplified by the identification of more than 3000 proteins in 16 out of 34 fractions , outperforming rp / rp approach ( 6 out of 34 fractions ) . ( b ) moreover , the hsax was comparable to the rp / rp approach in separating peptides in a single fraction ( 69% of rp peptides were present in one fraction compared to 55% for the hsax approach ) , which is beneficial for quantitative proteomics experiments . as quantitative proteomics performs best when peptides are only present in one fraction , we tested how many peptides were only identified in a single fraction . here , the rp / rp approach expectedly outperformed the hsax approach , showing a higher resolution as 69% of peptides were present in one fraction compared to 55% for the hsax approach ( figure 3b ) . only 18.5% ( hsax ) and 11.6% ( rp ) of all peptides respectively were identified in 3 or more fractions . in addition , separate experiments with dimethyl - labeled proteome samples indicated that deuterated samples do not change retention time in hsax chromatography as they can do in rp experiments ( data not shown ) , which can potentially separate light and heavy forms of isotopically labeled peptides into neighboring fractions complicating peptide quantitation . interestingly , the two techniques seem to favor certain subsets of peptides as only 58679 peptides ( 46% for hsax and 60% for rp ) are shared among the two approaches ( figure 4a ) . to further identify the characteristics of these subsets , we analyzed which amino acids were over - represented in the data sets unique to each approach ( supporting information , figure s9 ) and identified peptides with a higher number of acidic residues to be the major contributor to the difference ( figure 4b ) . furthermore , the peptides identified by the hsax approach matched much better to an in silico digest of all proteins of the mouse ipi database ( figure 4b ) , suggesting that the hsax approach allows for the identification of more representative peptides of the total proteome than the rp / rp approach . hsax / rp and rp / rp lead to the identification of different peptide subsets . ( a ) both hsax and rp approaches lead to the identification of different subsets of peptides with only about half the peptides identified in the two approaches ( please note : number of combined peptides higher than in table 2 due to fdr recalculation ) ( proportional venn diagram has been produced using bioinforx ) . ( b ) in - depth analysis of the amino acid distribution in the subset of unique peptides revealed that hsax favored more acidic peptides than rp , a trend that is more representative to the in - silico tryptic digestion of the whole ipi database . ( c ) moreover , both hsax and rp approaches showed similar peptide charge state distribution over the whole gradient , favoring doubly and triply charged peptide ions . finally , we tested how separation via hsax affected the charge - state distribution of peptide ions in mass spectrometry over the gradient . unlike scx , peptides fractionated by sax should not show a charge - distribution as the negative charges by which the peptides were separated in the first dimension at ph 8 do not affect electrospray ionization ( esi ) after separation at ph 3 in the second dimension , where basic residues are critical . evaluation of peptide charge states showed that peptides separated by both hsax and rp / rp showed a relatively even charge state distribution over the whole gradient ( figure 4c ) . this is of great advantage as the identification of doubly and triply charged peptide ions is highly favored in esi ms / ms compared to singly and highly charged ions which populate , for example , early and late fractions of scx experiments . in this work we demonstrate that hydrophilic strong anion exchange chromatography provides a robust and reproducible separation of complex protein digest samples for proteomics analysis . its very high orthogonality proved to be superior to an optimized high ph rp / low ph rp approach and enabled us to identify > 9,000 proteins from raw264.7 mouse macrophage cell lysate in just one week of mass spectrometry instrument time which will allow a better comparison of proteomics and transcriptomics data . in the future , we will test the applicability of the hsax material for online 2d lc - ms and the separation of complex mammalian proteomes for quantitative proteomics as well as phosphopeptide analysis .

due to its compatibility and orthogonality to reversed phase ( rp ) liquid chromatography ( lc ) separation , ion exchange chromatography , and mainly strong cation exchange ( scx ) , has often been the first choice in multidimensional lc experiments in proteomics . here , we have tested the ability of three strong anion exchanger ( sax ) columns differing in their hydrophobicity to fractionate raw264.7 macrophage cell lysate . ionpac as24 , a strong anion exchange material with ultralow hydrophobicity , demonstrated to be superior to other materials by fractionation and separation of tryptic peptides from both a mixture of 6 proteins as well as mouse cell lysate . the chromatography displayed very high orthogonality and high robustness depending on the hydrophilicity of column chemistry , which we termed hydrophilic strong anion exchange ( hsax ) . mass spectrometry analysis of 34 sax fractions from raw264.7 macrophage cell lysate digest resulted in an identification of 9469 unique proteins and 126318 distinct peptides in one week of instrument time . moreover , when compared to an optimized high ph / low ph rp separation approach , the method presented here raised the identification of proteins and peptides by 10 and 28% , respectively . this novel hsax approach provides robust , reproducible , and highly orthogonal separation of complex protein digest samples for deep coverage proteome analysis .

Introduction Experimental Section Results and Discussion Conclusion

mass spectrometric analysis of proteome samples from eukaryotic cell lines , tissues or biological fluids is still hampered by the vast complexity of samples and concentration differences of proteins within making comparisons of proteomics and transcriptomics data difficult . the most widely used methods involve two subsequent steps , an off - line fractionation step followed by ( low ph ) reversed phase liquid chromatography ( rp - lc ) directly coupled to mass spectrometry . many approaches have shown decent orthogonality such as hydrophilic interaction liquid chromatography ( hilic ) , electrostatic repulsion hydrophilic interaction chromatography ( erlic ) , strong cation exchange ( scx ) , isoelectric focusing ( such as offgel ) , scx - weak - anion exchange ( wax ) 3d and high - ph rp . particularly , high ph rp chromatography has shown great potential as it is highly resolving and partly orthogonal to low ph rp . however , this setup requires the samples to be dried down after the first dimension resulting in sample loss , unless another , third dimension such as sax is added , which increases the number of fractions and thus instrument time and reduces robustness of the system . furthermore , the high ph of the first rp dimension is corroding most of the silica - based stationary phase , leading to reproducibility issues and short column lifetime . while high - ph rp can never achieve full orthogonality to low - ph rp as they both separate according to hydrophobicity , ion exchange chromatography such as scx or strong anion exchange ( sax ) chromatography at least theoretically can . this is surprising as it was observed that the majority of tryptic peptides cluster between pi of 3 and 5 , partly because many post - translational modifications such as phosphorylation , pyro - glutamination or acetylation decrease the pi . on the proteome level , an online mixed mode reversed phase anion exchange ( mm , rp - ax ) , a sax - scx mixed bed ion exchange chromatography , a sax microreactor or a sax stage - tip approach were successfully used ; however , the use of sax for large - scale proteome analyses is still underdeveloped . here , we present a sax column with a hyperbranched architecture , quaternary ammonium ion functionality and an ultralow hydrophobicity . while the column was originally designed for the separation of small organic and haloacetic acids , we show its remarkable separation power for proteome research . this column exhibits high reproducibility , high capacity , robustness and very high orthogonality when coupled to low ph rp , allowing the identification of more than 9000 proteins from a raw264.7 macrophage cell lysate on an orbitrap velos pro mass spectrometer within only one week of instrument time . we show that both hydrophilicity and the ion exchange properties of the column are important for the high orthogonality of this approach , which we term hydrophilic strong anion exchange ( hsax ) chromatography . the mouse macrophage cell line raw264.7 was obtained from atcc and grown in dmem , 10% heat - inactivated fetal bovine serum ( fbs , sigma ) , 2 mm l - glutamine , 5000 u / ml penicillin and 5000 g / ml streptomycin ( invitrogen ) . finally , the sample was desalted by seppack oasis solid phase extraction cartridges ( waters ) , dried down and stored at 80 c . in this work , we compared three different strong anion exchange ( sax ) columns : the as24 , the as11-hc and the as15 ( ionpac series , thermo - fisher scientific ) that were chosen according to their chemical / physical characteristics . the as24 ( 2 250 mm , 2000 pore size ) is a low bleed column primarily designed for the separation of environmental ions as well as for ion chromatography separation coupled with mass spectrometry . it was tested to confirm that the hydrophilic architecture of as24 improves the orthogonal separation of tryptic peptides . to compare the results of sax / rp with ( high ph ) rp/ ( low ph ) rp , two reversed - phase columns were tested off - line in the first dimension , which differ in matrix composition . in this work , acclaim 120 c18 ( a conventional rp , 3 m , 120 , 2.1 150 mm , thermo - fisher scientific ) and acclaim rslc polar advantage ii -pa2- ( a polar - embedded rp , 2.2 m , 120 , 2.1 100 mm , thermo - fisher scientific ) columns were used to separate tryptic digested proteins from raw264.7 cell lysate . peptide separation on the off - line 1d separation system employing strong anion exchange was achieved with a flow rate of 0.25 ml / min ( solvent a : 20 mm tris - hcl ph 8.0 ; solvent b : 20 mm tris - hcl ph 8.0 , 1 m nacl ) . in case of high ph rp , separation was based on an acetonitrile ( acn ) gradient ( solvent a : 20 mm nh4oh / h2o , ph 10.0 ; solvent b : 20 mm nh4oh / acn , ph 10.0 ) at the same flow rate ( 0.250 ml / min , 080% b in 35 min ) . off - line 1d - fractions ( either sax without further handling or high ph rp , after drying down and resuspension in 0.1% tfa ) from the raw264.7 cell lysate were run online on an orbitrap velos pro ( thermo - fisher scientific ) . cyclization of glutamine / glutamic acid at the n - termini ( pyroglutamate ) was automatically searched , since tandem automatically checks for formation of pyroglutamic acid , that is , the loss of water ( e ) or ammonia ( q ) , respectively , when a peptide starts with e or q. this modification is considered to be an n - terminal modification only , so it does not affect any potential modifications specified for q , e or c. search parameters specified a precursor mass tolerance of 25 ppm ( required by tpp , typical mass accuracy was < 2 ppm ) , a ms / ms tolerance at 0.4 da and full trypsin specificity allowing for up to 2 missed cleavages . from a range of 12 high - capacity hydroxide - selective anion - exchange columns ( ionpac series , thermo - fisher scientific ) which were initially developed for the separation of small organic and haloacetic acids and inorganic anions , we tested three members ( as24 , as11-hc and as15 ) for the use in off - line fractionation in a proteomics setting . these columns all contain the same functionality of alkanol quaternary ammonium ions , but differ in backbone hydrophobicity , with as24 exhibiting ultralow hydrophobicity , as11-hc medium hydrophobicity and as15 high hydrophobicity . retention maps showed a highly orthogonal separation by as24as shown by the wide distribution over the two - dimensional space some separation by as11-hc and almost full retention of peptides on as15 even when eluted with 1 m nacl ( figure 1a ) . indeed , addition of at least 25% acn to the mobile phase allowed elution of tryptic peptides from as15 and improved elution from as11-hc ( figure 1b ) , showing that high hydrophobicity of the chemical backbones of these columns hampers the use for separation of peptides . furthermore , the most hydrophilic member of the ion exchange materials in this line ( as25 ) performs similarly well as as24 ( supporting information , figure s2 ) which led us to conclude that both the hydrophilicity in conjunction with the ion exchange functionality are important for the high orthogonality of this type of chromatography that we termed hydrophilic strong anion exchange ( hsax ) chromatography . ( a ) 2d retention maps of peptides of a 6-protein mix digest show that elution with up to 1 m nacl in absence of organic solvent leads to high orthogonality for as24 ( ultralow hydrophobicity ) , but part and full retention of peptides by as11-hc ( medium hydrophobicity ) and as15 ( high hydrophobicity ) , respectively . as the functional groups are primarily quaternary amines , changes of ph have no to little effect on column chemistry , but will change the available negative charge on peptides . when the mobile phase was adjusted to ph 3 , a major breakthrough was observed in 2d retention maps , which was absent at ph 8 . this has actually the benefit that tryptic digestion of the cell lysate can be performed at 20 mm tris - hcl , ph 8 and directly injected on the column , avoiding solid phase extraction or lyophilization and , therefore , minimizing sample loss . after optimized parameters were established , the method was applied to a very complex mixture , a total cell lysate of the mouse macrophage cell line raw264.7 . tryptic peptides of 200 g of cell lysate were separated on the as24 column in a 35 min gradient from 0 to 1 m nacl ( 34 fractions ) which were automatically collected and injected onto a gemini c18 column ( phenomenex ) . 2d retention maps show a very high orthogonality of separation between the two chromatographic dimensions ( figure 2a ) , displayed by the elution of peptides over the entire 2d map . separation of total cell lysate digests by hsax and a high ph / low ph reversed phase approach show differences in orthogonality . tryptic raw264.7 macrophage cell lysate digests were separated on an as24 sax column ( a ) or an acclaim pa2 rp column at ph 10 ( b ) . furthermore , we separated the same cell lysate digest on two different columns using high ph ( ph 10)/ low ph ( ph 3 ) rp / rp chromatography ( supporting information , figure s1 ) which has shown good orthogonality in the past and is currently seen as the gold standard in two - dimensional separation due to its high resolution . we then optimized elution parameters and chose the acclaim pa2 ( thermo - fisher scientific ) as it performed best , likely due to its higher polarity than the conventional c18 ( acclaim 120 , thermo - fisher scientific ) . 2d retention maps show that the hsax / rp approach performs considerably better than the rp / rp approach , as a much greater area in the 2d maps is covered with peaks , while peptides in the rp / rp approach are scattered around the diagonal . this provides further evidence that high ph rp / low ph rp can not be fully orthogonal , as both dimensions separate according to hydrophobicity . recent approaches to concatenate early and late fractions of high ph rp to increase orthogonality have improved results ; however , concatenation involves user interference and might provide problems in quantitative proteomics experiments when the same peptides are then present in non - neighboring fractions as quantitative proteomics software such as maxquant requires peptides to be in neighboring fractions for accurate quantitation . in order to test both hsax and high ph rp approaches for their performance in a proteomics experiment , we injected aliquots ( 36% , corresponding to 300400 ng of protein digest ; 1 10 base peak chromatogram ) of each of the 34 fractions of the raw264.7 cell lysate separated by hsax or high ph rp onto an orbitrap velos pro mass spectrometer , using 300 min gradients on a 75 m 50 cm pepmap c18 column ( thermo - fisher scientific ) , corresponding to one week of instrument time each . online separation of hsax and high ph rp fractions showed a similar degree of orthogonality comparable to the offline 2d chromatography ( supporting information , figure s7a and b ) . while we identified with the rp / rp approach 99110 peptides and 8627 proteins ( 1% fdr ) , the hsax approach resulted in identification of 126318 unique peptides and 9469 proteins , representing 28% and 10% more identifications respectively ( table 2 and supporting information , table s1 ) , while keeping the average sequence coverage equal ( 31% respectively 32% ) ( supporting information , figure s8 ) . in addition , we analyzed the data using the andromeda search engine included in maxquant which identified overall 68% less proteins and peptides in each experiment , but confirmed the superiority of our sax approach ( table 2 and supporting information , table s1 ) . this data compares well to two recent publications in which about similar number of proteins were identified from a mammalian cell line using 3 different proteases and 2 weeks of instrument time or in which samples were analyzed repeatedly with inclusion lists . the high orthogonality of the hsax approach is not only shown by the broad distribution in the 2d peptide maps , but also exemplified by the fact that we identified more than 3000 proteins in 16 out of 34 fractions , while the rp / rp approach achieved this for only 6 fractions ( figure 3a ) . data also indicated that we could have saved instrument time by combining the last 5 fractions , which could have reduced run time by almost another day . the hsax approach also showed a considerably higher number of proteins ( 7292 vs 6315 in rp / rp , + 15% ) that were identified with 3 and more peptides which considerably improves identification and quantitation in proteomics experiments . ( a ) the high orthogonality of the hsax approach is exemplified by the identification of more than 3000 proteins in 16 out of 34 fractions , outperforming rp / rp approach ( 6 out of 34 fractions ) . ( b ) moreover , the hsax was comparable to the rp / rp approach in separating peptides in a single fraction ( 69% of rp peptides were present in one fraction compared to 55% for the hsax approach ) , which is beneficial for quantitative proteomics experiments . as quantitative proteomics performs best when peptides are only present in one fraction , we tested how many peptides were only identified in a single fraction . here , the rp / rp approach expectedly outperformed the hsax approach , showing a higher resolution as 69% of peptides were present in one fraction compared to 55% for the hsax approach ( figure 3b ) . only 18.5% ( hsax ) and 11.6% ( rp ) of all peptides respectively were identified in 3 or more fractions . in addition , separate experiments with dimethyl - labeled proteome samples indicated that deuterated samples do not change retention time in hsax chromatography as they can do in rp experiments ( data not shown ) , which can potentially separate light and heavy forms of isotopically labeled peptides into neighboring fractions complicating peptide quantitation . to further identify the characteristics of these subsets , we analyzed which amino acids were over - represented in the data sets unique to each approach ( supporting information , figure s9 ) and identified peptides with a higher number of acidic residues to be the major contributor to the difference ( figure 4b ) . furthermore , the peptides identified by the hsax approach matched much better to an in silico digest of all proteins of the mouse ipi database ( figure 4b ) , suggesting that the hsax approach allows for the identification of more representative peptides of the total proteome than the rp / rp approach . ( a ) both hsax and rp approaches lead to the identification of different subsets of peptides with only about half the peptides identified in the two approaches ( please note : number of combined peptides higher than in table 2 due to fdr recalculation ) ( proportional venn diagram has been produced using bioinforx ) . ( b ) in - depth analysis of the amino acid distribution in the subset of unique peptides revealed that hsax favored more acidic peptides than rp , a trend that is more representative to the in - silico tryptic digestion of the whole ipi database . finally , we tested how separation via hsax affected the charge - state distribution of peptide ions in mass spectrometry over the gradient . this is of great advantage as the identification of doubly and triply charged peptide ions is highly favored in esi ms / ms compared to singly and highly charged ions which populate , for example , early and late fractions of scx experiments . in this work we demonstrate that hydrophilic strong anion exchange chromatography provides a robust and reproducible separation of complex protein digest samples for proteomics analysis . its very high orthogonality proved to be superior to an optimized high ph rp / low ph rp approach and enabled us to identify > 9,000 proteins from raw264.7 mouse macrophage cell lysate in just one week of mass spectrometry instrument time which will allow a better comparison of proteomics and transcriptomics data . in the future , we will test the applicability of the hsax material for online 2d lc - ms and the separation of complex mammalian proteomes for quantitative proteomics as well as phosphopeptide analysis .

the liver is target of several pathogens , including bacteria derived from the gastrointestinal tract , parasites like plasmodium spp . and hepatitis viruses , such as hepatitis a virus ( hav ) , hepatitis b virus ( hbv ) or hepatitis c virus ( hcv ) . bacteria derived from the gut lumen reach the liver via the portal vein that drains blood from the gastrointestinal tract . pathogenic bacteria can actively traverse the gut wall and enter the body , but also gut microbiota may translocate once integrity of the gut wall is impaired , for instance , during increased venous pressure or chronic gut inflammatory diseases , and gain access to the bloodstream . upon entering the bloodstream , bacteria are delivered via the portal vein to the liver where they encounter the liver 's macrophage defense system . parasites like plasmodium spp . gain access to the bloodstream through mosquito bites and reach the liver via the bloodstream . the infection process in the liver involves transit of plasmodium sporozoites through various liver cell populations , including kupffer cells ( kcs ) before infecting their final target cell , the hepatocyte . viruses targeting the liver like hav , hbv or hcv may reach the liver after crossing mucosal surfaces in the gastrointestinal or genitourinary tract , or by directly gaining access to the bloodstream . once circulating in the blood , hepatitis viruses show a remarkable liver tropism that is often mediated by high - jacking physiological transport pathways that converge in the liver . by this way , hepatitis viruses not only exit the bloodstream in the correct organ , but also efficiently achieve a tropism for hepatocytes . the high blood volume passing through the liver , that is , 20% of the total cardiac output , together with the slow blood flow and low shear forces in liver sinusoids together facilitate to hepatic clearance of the blood from molecules requiring metabolic degradation , but at the same time also allow pathogens to target the liver and establish infection of hepatocytes if they manage to escape immune - mediated destruction by sinusoidal cell populations . common to those viruses and parasites that target the liver and establish persistent infection , is the ability to circumvent the induction of strong innate immunity . rna viruses like hcv are detected by helicases like rig - i recognizing viral rna in the cytosol . rig - i activates the adapter molecule , mavs , which is localized in the outer mitochondrial membrane . activation of mavs induces several transcription factors leading ultimately to the production of type i interferons . the hcv - encoded protease ns3/4a cleaves mavs at cys508 preventing anchoring to mitochondria and therefore inhibiting rig - i signaling . a similar mechanism has been shown for hav , where the hav encoded serine protease 3 cleaves mavs at gln428 , thereby preventing rig - i signaling and type i interferon induction . as hav is always cleared by the immune response , further research is required to identify the molecular mechanisms that determine the failure of the immune response to eliminate hcv - infected hepatocytes . in contrast , hbv infection is characterized by an almost complete lack of innate immunity during the acute infection and the rapid release of large amounts of viral antigens after infection in the absence of inflammation . the combination of lack of inflammation and large amounts of circulating viral antigens has been shown to be involved in the development of t cells with an exhausted phenotype , and is believed to be responsible for the exhaustion of hbv - specific immunity that facilitates persistent infection . thus , infection in the liver often occurs in the absence of strong innate immunity , which impedes pathogen - specific effector t cells that express cxcr3 to relocate via chemokines to sites of infection . as the liver sinusoids are a maze - like structure , finding infected hepatocytes in the absence of an inflammation - dependent localizing signal is a difficult task and likely requires high numbers of pathogen - specific cd8 t cells . notwithstanding the ability of certain pathogens to evade innate immunity , the liver is an organ with predominant innate immunity that can mount appropriate inflammatory responses upon infection . the initiation of protective immunity against infectious pathogens that reside within cells , such as viruses , intracellular parasites and intracellular bacteria , requires cytotoxic cd8 t - cell responses . for their activation , cd8 t cells are stimulated by peptides presented on major histocompatibility complex ( mhc ) i molecules . such peptide - loaded mhc i molecules are typically derived from proteins endogenously expressed in cells , cleaved by the proteasome and followed by tap - mediated transport of peptides into the endoplasmic reticulum for chaperone - assisted loading onto mhc i molecules . this pathway , however , does not allow to present exogenous antigens on mhc i molecules , which is required for professional antigen - presenting cells to activate a cd8 t - cell response . the process of presentation of exogenous antigens to naive cd8 t cells has been termed cross - priming and is required for induction of protective immunity against those pathogens that evade expression of their antigens in professional antigen - presenting cells . cross - priming is a complex process that almost exclusively occurs within secondary lymphoid organs . among the specialized immune cells , a subpopulation of dendritic cells ( dcs ) is capable of cross - priming naive cd8 t cells . this dc population is characterized in mice by expression of cd24 , cd8 , cd103 and xcr1 in mice , and is dependent on the transcription factors irf8 and batf3 . in humans , cross - priming capacity is also restricted to cd11ccd141 dc population with xcr1 expression . within secondary lymphoid tissues , cross - priming - competent dcs that have received appropriate activation signals and matured into immunogenic dcs , interact with cd8 and cd4 t cells in a spatiotemporally controlled manner to generate long - lasting and potent antiviral immunity . the relevance of cross - priming for successful immune surveillance has been shown for numerous infectious microorganisms . as cross - priming does not require antigen expression , it also allows to circumvent immune evasion strategies of pathogens that aim at limiting antigen presentation . as the highly organized microarchitecture of lymphoid tissues is optimized to facilitate interaction of antigen ( cross)-presenting cells with the low number of antigen - specific t cells , cross - priming in lymphoid tissues appears to be most suited to generate effector as well as memory cd8 t cells that recognize and control infectious microorganisms . after generation of pathogen - specific cd8 t - cell immunity within lymphoid tissues , such cd8 t cells need to relocate to peripheral organs and recognize their target cells within infected tissue . as immune evasion from antigen presentation may also occur in infected cells , cross - presentation of pathogen - derived antigens during the effector phase in infected organs may help the immune response to achieve immune surveillance . the liver is recognized as an organ that bears unique immune functions and can skew immune responses . apart from the liver microenvironment that constitutes a tolerogenic milieu rich in immune - regulatory mediators such as prostaglandins , transforming growth factor ( tgf) or interleukin ( il)-10 , the liver harbors many antigen - presenting cell populations that have been suggested to engage in priming of naive t cells and thereby contribute to skewing of immune responses . hepatocytes not only function as metabolic units , but participate also in shaping of immune responses by presenting antigens on mhc i to cd8 cells . hepatocytes can prime naive cd8 t cells , which results in clonal t - cell deletion in a bim - dependent manner leading to t - cell apoptosis , thus establishing antigen - specific tolerance . hepatocytes also engulf and remove naive cd8 t cells following priming by a process called suicidal emperipolesis . the relevance of hepatocyte - mediated deletion of cd8 t cells for liver tolerance became clear through the discovery that initial ( auto-)antigen presentation by hepatocytes leads to clonal deletion , whereas initial antigen presentation in lymphoid tissues causes autoimmune hepatitis , once cd8 t cells recognize again antigen presented on hepatocytes . the relevance of hepatocyte - restricted priming of virus - specific cd8 t cells during viral infection of the liver has not been thoroughly addressed so far . liver sinusoidal endothelial cells ( lsecs ) comprise the most prominent non - parenchymal cell population in the liver that line the liver sinusoids . the endocytosed antigens are used for antigen ( cross)-presentation on mhc class i and ii molecules to cd8 and cd4 t cells , respectively . as lsec do not provide co - stimulatory signals via cd80/86 or il-12 during priming of naive t cells , these cells do not undergo maturation into effector cd4 or cd8 t cells . priming of naive cd4 t cells by lsecs rather leads to generation of regulatory t cells that can suppress organ - specific autoimmunity even in the central nervous system demonstrating the relevance of hepatic immune functions for systemic immune responses . priming by immature dcs in the absence of inflammatory signals leads to a nonresponsive , anergic state or clonal deletion . in contrast , cd8 t - cell priming by cross - presenting lsecs leads to development into a distinct memory - like state where cd8 t cells can be reactivated by combinatorial stimuli involving t - cell receptor ( tcr ) signaling , cd28 signaling and signaling through the il-12 receptor . induction of this memory - like phenotype does not require an inflammatory stimulation and maturation step of lsec . rather , this unique memory t - cell programing occurs in the absence of cd28 or il-12 signals , but involves il-6 trans - signaling , which identifies a for unknown direct t cell adjuvant critical for induction of memory t cells . thus , antigen - presenting lsecs cross - prime memory cd8 t cells with proliferative potential or induce differentiation of regulatory cd4 t cells , which shows a unique functional diversity of this liver - resident antigen - presenting cell population in skewing of immune responses . strategically located within the sinusoids , kcs remove particulate antigens from the blood circulating through the liver . simultaneously , kcs come in close contact with circulating t cells as well as liver - resident nk cells and nkt cells . under steady - state , noninflammatory conditions , kcs possess a tolerogenic phenotype and influence the liver micromilieu by secretion of il-10 . however , under inflammatory circumstances , kcs may activate t cells and nkt cells leading to immunity against bacterial infection and causing liver immunopathology . liver dcs compared with dcs from lymphoid tissues show a tolerogenic phenotype . upon stimulation with toll like receptor ( tlr ) ligands , liver myeloid dcs produce less il-12 , but produce more il-10 and il-27 maintaining the livers unique tolerogenic milieu . also liver plasmacytoid dcs are characterized by reduced expression of mhc ii and impaired type i interferon expression upon stimulation . both , the tolerogenic phenotype of kcs and dcs may result from the continuous presence of pathogen - associated molecular patterns derived from bacteria and antigens from the gut . the close link between the gut and the liver where gut - derived bacterial degradation products or even entire bacteria at low concentrations reach the liver via the portal vein , necessitates a containment of innate immunity toward these inflammatory signals . notwithstanding the tolerance in responding to continuous exposure to low dose of pathogen - associated molecular patterns , the liver can mount innate immunity in response to infection , thereby demonstrating that innate defense mechanisms are intact . hepatic stellate cells ( hsc ) are located in the space of diss and control the sinusoidal diameter through contraction , thereby achieving dynamic regulation of blood flow . hscs express mhc class i and ii molecules as well as the lipid - presenting cd1 molecule together with co - stimulatory molecules , like cd86 , and co - inhibitory molecules like b7h1 . although well positioned to interact with cd8 t cells , their participation in local t - cell responses by antigen cross - presentation several reports indicate hscs capable for cross - presentation to cd8 t cells . yet , facs - sorted hscs devoid of any contaminating lsec are not able to cross - present exogenous antigen to cd8 t cells . together , liver antigen - presenting cells contribute to the liver 's unique immune functions through local activation and differentiation of naive t cells . however , little is known on the relevance of local antigen ( cross)-presentation for the local induction of effector functions , that is , immune surveillance in the liver . infectious microorganisms reaching the liver have first contact with sinusoidal cell populations , but hepatitis viruses and plasmodia spp . target the hepatocyte and use this cell population for massive expansion , as well as for establishing a state of persistence . although innate immune responses generated by infected cells , that is , hepatocytes , or by immune cells detecting microbial - associated molecular patterns may help to contain infection and hinder the replication of pathogens , cd8 t - cell immunity is required to achieve control of infection in the liver , exemplified for control of hbv infection through cd8 t cells in chimpanzees . the sentinel function of sinusoidal cell populations allows them to mount innate immunity and inflammation , which may help through expression of interferons to contain pathogen infection in the liver . the induction of effector functions from pathogen - specific cd8 t cells through their local activation in the infected organ is necessary to achieve control of infection . such local activation of effector cd8 t cells is believed to occur mainly by direct recognition of pathogen - derived peptides on mhc i molecules expressed by the infected cell itself , but may also result from mhc i - restricted cross - presentation of pathogen - specific antigens on noninfected cells or by non - mhc - restricted activation of innate - like lymphocytes resident to the liver . immune surveillance in the liver is believed to occur mainly through recruitment of circulating pathogen - specific effector cd8 t cells . adhesion of circulating cd8 t cells to sinusoidal cell populations in the liver is facilitated by the narrow sinusoidal diameter that together with the low perfusion pressure leads to low shear forces . as a consequence , lymphocyte adhesion to sinusoidal cells does not require expression of selectins to slow down circulating lymphocytes and narrow diameter within the sinusoids . as sinusoidal lining lsec possess the so - called fenestrae , holes within the sinusoidal endothelial barrier of about 200 nm in size , and a lack of a basal membrane separating hepatocytes from the sinusoidal lumen , circulating cd8 t cells may establish direct contact with hepatocytes . such direct contact with t cells may result from hepatocytes extending their protrusions through endothelial fenestrae or t cells passing through endothelial fenestrae . thereby , t cells can recognize virus - derived antigens presented by mhc class i molecules directly on hepatocytes without the need for transmigration . remarkably , adhesion of cd8 t cells within the liver can occur without inflammation in the infected area . in this case , during adhesion , t cells do not adhere directly to the endothelial cell layer , but rather to platelet aggregations within the sinusoids . these platelets in turn adhere to lsec even under steady - state conditions creating a platform for cd8 t - cell adhesion even under under noninflammatory conditions . this is of interest as hbv acts as a stealth virus and does not induce inflammation in the liver . interestingly , hbv - specific cd8 t cells do not arrest in the liver upon antigen - specific contact with hepatocytes expressing hbv antigens , but are randomly recruited to platelet aggregates forming on lsec . the molecular mechanisms governing this stochastic interaction of t cells with platelets still await clarification . such stochastic recruitment of cd8 t cells from the circulation may not be very efficient and may further add to the necessity for large numbers of t cd8 t cells to control infection in the liver . once arrested in the sinusoid , t cells do not need to transmigrate across lsecs , but rather establish firm contact with hbv - expressing hepatocytes through fenestrae . this suggests that hepatic immune surveillance functions as intravascular immune surveillance that is facilitated by the unique characteristics of sinusoidal structure and blood flow . the establishment of persistent viral infections of the liver , in particular , chronic hepatitis b or chronic hepatitis c , suggests that antiviral immune surveillance in the liver can be circumvented by these hepatotropic viruses . various mechanisms have been identified that contribute to the development of persistent hbv or hcv infection . yet , control of hbv - infected hepatocytes occurs to a significant extent through non - cytolytic mechanisms . in a hbv - transgenic mouse model , it was shown that tumor necrosis factor ( tnf ) and interferons control hbv gene expression and replication in a non - cytolytic manner . these results were confirmed in experiments using hbv - infected chimpanzees . mechanistically , cytokine - induced activation of nucleic acid - degrading enzymes , that is , apobecs , were responsible for this non - cytolytic cytokine effect on hbv replication . however , hbv - specific cd8 t cells were eventually required for elimination of hbv - infected hepatocytes through cytotoxic effector mechanisms . notwithstanding of the cytokine - induced control of hbv - replication , hbv - specific cd8 t cells are required for elimination of hbv - infected hepatocytes to achieve control of hbv infection . for efficient immune surveillance against plasmodium - infected hepatocytes , a strong initial priming period is required to generate high numbers of pathogen - specific cd8 t cells . direct recognition of plasmodium - infected hepatocytes by pathogen - specific cd8 t cells is then required to induce death of the infected hepatocytes . recently , a further mechanism has been found that identifies the need for several cd8 t cells recognizing and attacking infected epithelial target cells to elicit killing . recognition of an infected cell by a single cd8 t cell may not be sufficient to induce apoptosis , but rather two or more cd8 t cells are required to cooperatively achieve killing of target cells . within a pool of cd8 t cells , killing capacity of a single cd8 t cell may range from 2 to 16 infected cells per day . the relevance of this swarm hunting behavior for clearance of virus - infected hepatocytes has not been addressed yet . in general , killing of hepatocytes requires delivery of death - inducing signals via perforin / granzyme b and fasl . although the role of cross - priming for generation of virus - specific immunity is widely recognized , the role of cross - presentation of viral antigens to virus - specific cd8 t cells during the effector phase is much less characterized . obviously , immune escape from mhc class i - restricted antigen presentation in infected cells poses a hurdle to cd8 t - cell immune surveillance and may result in the failure to control viral infection despite the presence of virus - specific cd8 t cells . in the liver with its particular intravascular immune surveillance , cross - presentation may therefore benefit the recruitment of circulating t cells . indeed , cross - presentation of soluble antigens by lsecs leads to antigen - specific recruitment of cd8 t cells to the liver even in the absence of inflammation . using bioluminescence imaging allows for longitudinal detection of antiviral immunity in the liver within the same animals after infection with hepatotropic adenoviruses encoding for immunological relevant antigens combined with the marker protein luciferase . detection of a decrease in luciferase expression , by reduced bioluminescence signal , in adenovirus - infected hepatocytes is superior to serological biomarkers of antiviral immunity , the hepatocellular enzymes alt or ast , as it reflects in real time the number of living infected hepatocytes . this experimental setup allowed to investigate the link between the extraordinary scavenger function , and the ability to cross - present circulating antigens by lsecs and hepatic immune surveillance . infection of hepatocytes with recombinant adenoviruses coding for ovalbumin , which shows high hepatocyte tropism , led to ovalbumin cross - presentation to ovalbumin - specific cd8 t cells . such cross - presentation led to stimulation of circulating ovalbumin - specific cd8 t cells and to secretion of tnf by such activated cd8 t cells . surprisingly , in transgenic mouse models where mhc class i - restricted interaction of virus - infected hepatocytes with cd8 t cells was excluded , virus - specific cd8 t cells still induced viral hepatitis , indicating that other mechanisms that do not rely on direct target - cell recognition allow locally activated cd8 t cells to achieve immune surveillance in the liver . conversely , exclusive mhc class i - restricted presentation of virus - derived antigens on hepatocytes led only to 4050% of antiviral cd8 t - cell effector function , suggesting that cross - presentation of viral antigens released from infected hepatocytes was operative in at least 50% of the total antiviral cd8 t - cell activity . this was termed noncanonical cd8 t - cell effector function , as it does not require direct mhc class i - restricted recognition of virus - infected target cells with virus - specific cd8 t cells to elicit antiviral effector functions ( figure 1noncanonical cd8 t - cell effector function ) . mechanistically , this noncanonical cd8 t - cell effector function is initiated by lsecs cross - presenting hepatocyte - derived viral antigens to cd8 t cells . tnf released from such cd8 t cells activated through cross - presenting lsecs than acts on hepatocytes to initiate a tnfr1-restricted induction of caspase - mediated apoptosis . neither ifn nor type i interferons contribute to this noncanonical cd8 t - cell effector function against virus - infected hepatocytes , which demonstrates a unique role for tnf in the local immune surveillance in the liver . the noncanonical cd8 t - cell effector function was important to obtain rapid control over viral infection of the liver , which is particularly important to rapidly gain control over the spread of viral infection among hepatocytes in the liver . it is of interest to note that lsecs fail to cross - present hbv - derived peptides to hbv - specific cd8 t cells , indicating that hbv may escape from this noncanonical cd8 t - cell effector function , and that this may have a role in the establishment of persistent infection or even in the failure to terminate persistent infection . taken together , mhc class i - restricted antigen ( cross)-presentation is operational in immune surveillance against viral infection of the liver . furthermore , sinusoidal liver cells engage in mutual exchange of mhc class i molecules to improve local immune surveillance . transfer of mhc class i molecules from hscs resulted in improved cross - presentation by lsecs . such trogocytosis of mhc class i molecules is distinct from cross - dressing of peptide - loaded mhc class i molecules that has been shown to be involved in cross - priming . continuous supply of mhc class i molecules from other liver cell populations may ensure optimal performance of lsecs in cross - presentation and local intrahepatic immune surveillance . in addition to cross - presentation of peptides generated by proteasomal digestion or endosomal degradation from viral proteins on mhc class i molecules and subsequent recognition by cd8 t cells bearing / t - cell receptors , other antigen - presentation pathways are increasingly recognized in their importance for local , organ - specific immune surveillance . the cd1 and mhc - like protein 1 ( mr1 ) function to present lipid antigens or bacterial metabolites to nkt cells or mucosal - associated invariant t cells ( mait cells ) , respectively , and thereby cause their activation as well as effector function . mait cells have been first described in 1999 and gained increasing attention over the last years for their prominent role in antibacterial defense . in humans , mait cell represent the most abundant innate - like t - cell population with about 5% of the total t - cell repertoire and that can reach up to 45% of the total liver lymphocytes . they express a semi - invariant t - cell receptor ( v7.2-j33/12/20 ) recognizing antigen presented by the mhc - like protein 1 ( mr1 ) . mr1 is highly conserved molecule across many species , including mouse and human , sharing > 90% homology at the protein level . the identity of antigens presented in the context of mr1 remain largely unknown , but structural analysis of the mr1 antigen - binding cleft revealed that bacterial metabolites are presented by mr1 . kjer - nielsen et al . showed that a photodegradable product of folic acid ( vitamin b9 ) stabilized the mr1/2-microglobulin complex , and that this complex sufficed to activate mait cells . a derivative of riboflavin ( vitamin b2 ) that binds to mr1 is responsible for mait - cell activation , and as these vitamins are selectively produced by bacteria , this supports the notion that mait cells have an important role in antibacterial defense . under physiological conditions , the mr1 protein is almost absent from the cell surface owing to lack of stabilization by its ligand . upon infection of cells by bacteria producing riboflavin , however , the mr1 complex is stabilized and transported to the surface . yet , infection is not an absolute prerequisite for mr1-dependent activation of mait cells , suggesting that uptake of bacterial constituents also suffice for mr1-mediated mait - cell activation . in particular , biliary epithelial cells but very likely also liver sinusoidal cells are involved in mait - cell activation upon contact with bacteria ( figure 2mr1-restricted activation of mait cells ) . besides direct antigen recognition , mait cells can also be activated in an mr1-independent manner . it has been shown that the cytokines il-12 and il-18 activate mait cells , and lead to expression and release of ifn. thereby , mait cells may contribute to antiviral defense in the liver , where secreted ifn participate in the non - cytolytic elimination of hbv . along this line , tcr - independent stimulation of mait cells leads to ifn-dependent reduction of hcv replication in infected hepatocytes , suggesting that mait cell - derived ifn operates in antiviral defense in the liver . nkt cells belong to the so - called innate - like t cells and can be divided into two subpopulations : the type i or invariant nkt cells ( inkt ) or the type ii or diverse nkt cells . inkt cells express like mait cells an invariant t - cell receptor chain ( v14-j18 in mice , v24-j18 in humans ) with a limited number of t - cell receptor chains . this invariant tcr recognizes antigens in the context of the mhc class i like molecule cd1 that presents glycolipids . unlike for mr1 , glycolipid antigens presented by cd1 molecules are not restricted , but rather a broad variety of endogenous and exogenous lipids can be presented on cd1 . therefore , nkt cells acts as interface bridging innate and adaptive immunity . nkt cells comprise only about 0.1% of the peripheral t - cell pool , but are specifically enriched in the liver making up to 40% of all lymphocytes in the murine liver and up to 25% of lymphocytes in the human liver . besides being stimulated through the cd1 molecule presenting glycolipids , inkt cells are also stimulated by activation of toll - like receptors , or by the cytokines il-12 , il-18 and type i interferons . the outcome of nkt cell stimulation varies on the type of stimulation , the localization of stimulation and the antigen - presenting cell . upon stimulation with the lipid galcer presented by cd1 molecules , inkt cells secrete ifn , il-4 and il-17 , but produce ifn only by stimulation through il-12 and il-18 , or by following ischemia or toxin - induced liver injury . a prominent role of cd1 and inkt cells in control of bacterial infection of the liver has been described ( figure 2cd1-restricted activation of nkt cells ) . however , reports indicated that inkt cells inhibit viral replication of hcv in hepatocytes via the secretion of ifn. during early hbv infection , high numbers of activated inkt are found that may contribute to viral defense by secreting ifn inhibiting hbv replication . a recent report shows that nkt cells contribute to the antiviral response during hbv infection . hepatocytes infected with hbv present endoplasmic reticulum - associated endogenous lipids generated by hbv - induced secretory phospholipases leading to activation of nkt cells . absence of nkt cells , cd1 molecules or a defect in transferring endoplasmic reticulum - associated lipids to cd1 molecules leads to failure of inducing a strong t- and b - cell immunity and control of viral infection . thus , liver nkt cells appear to contribute to both , antibacterial as well as antiviral immunity . immune surveillance in the liver is believed to occur mainly through recruitment of circulating pathogen - specific effector cd8 t cells . adhesion of circulating cd8 t cells to sinusoidal cell populations in the liver is facilitated by the narrow sinusoidal diameter that together with the low perfusion pressure leads to low shear forces . as a consequence , lymphocyte adhesion to sinusoidal cells does not require expression of selectins to slow down circulating lymphocytes and narrow diameter within the sinusoids . as sinusoidal lining lsec possess the so - called fenestrae , holes within the sinusoidal endothelial barrier of about 200 nm in size , and a lack of a basal membrane separating hepatocytes from the sinusoidal lumen , circulating cd8 t cells may establish direct contact with hepatocytes . such direct contact with t cells may result from hepatocytes extending their protrusions through endothelial fenestrae or t cells passing through endothelial fenestrae . thereby , t cells can recognize virus - derived antigens presented by mhc class i molecules directly on hepatocytes without the need for transmigration . remarkably , adhesion of cd8 t cells within the liver can occur without inflammation in the infected area . in this case , during adhesion , t cells do not adhere directly to the endothelial cell layer , but rather to platelet aggregations within the sinusoids . these platelets in turn adhere to lsec even under steady - state conditions creating a platform for cd8 t - cell adhesion even under under noninflammatory conditions . this is of interest as hbv acts as a stealth virus and does not induce inflammation in the liver . interestingly , hbv - specific cd8 t cells do not arrest in the liver upon antigen - specific contact with hepatocytes expressing hbv antigens , but are randomly recruited to platelet aggregates forming on lsec . the molecular mechanisms governing this stochastic interaction of t cells with platelets still await clarification . such stochastic recruitment of cd8 t cells from the circulation may not be very efficient and may further add to the necessity for large numbers of t cd8 t cells to control infection in the liver . once arrested in the sinusoid , t cells do not need to transmigrate across lsecs , but rather establish firm contact with hbv - expressing hepatocytes through fenestrae . this suggests that hepatic immune surveillance functions as intravascular immune surveillance that is facilitated by the unique characteristics of sinusoidal structure and blood flow . the establishment of persistent viral infections of the liver , in particular , chronic hepatitis b or chronic hepatitis c , suggests that antiviral immune surveillance in the liver can be circumvented by these hepatotropic viruses . various mechanisms have been identified that contribute to the development of persistent hbv or hcv infection . yet , control of hbv - infected hepatocytes occurs to a significant extent through non - cytolytic mechanisms . in a hbv - transgenic mouse model , it was shown that tumor necrosis factor ( tnf ) and interferons control hbv gene expression and replication in a non - cytolytic manner . mechanistically , cytokine - induced activation of nucleic acid - degrading enzymes , that is , apobecs , were responsible for this non - cytolytic cytokine effect on hbv replication . however , hbv - specific cd8 t cells were eventually required for elimination of hbv - infected hepatocytes through cytotoxic effector mechanisms . notwithstanding of the cytokine - induced control of hbv - replication , hbv - specific cd8 t cells are required for elimination of hbv - infected hepatocytes to achieve control of hbv infection . for efficient immune surveillance against plasmodium - infected hepatocytes , a strong initial priming period is required to generate high numbers of pathogen - specific cd8 t cells . direct recognition of plasmodium - infected hepatocytes by pathogen - specific cd8 t cells is then required to induce death of the infected hepatocytes . recently , a further mechanism has been found that identifies the need for several cd8 t cells recognizing and attacking infected epithelial target cells to elicit killing . recognition of an infected cell by a single cd8 t cell may not be sufficient to induce apoptosis , but rather two or more cd8 t cells are required to cooperatively achieve killing of target cells . within a pool of cd8 t cells , killing capacity of a single cd8 t cell may range from 2 to 16 infected cells per day . the relevance of this swarm hunting behavior for clearance of virus - infected hepatocytes has not been addressed yet . in general , killing of hepatocytes requires delivery of death - inducing signals via perforin / granzyme b and fasl . although the role of cross - priming for generation of virus - specific immunity is widely recognized , the role of cross - presentation of viral antigens to virus - specific cd8 t cells during the effector phase is much less characterized . obviously , immune escape from mhc class i - restricted antigen presentation in infected cells poses a hurdle to cd8 t - cell immune surveillance and may result in the failure to control viral infection despite the presence of virus - specific cd8 t cells . in the liver with its particular intravascular immune surveillance , cross - presentation may therefore benefit the recruitment of circulating t cells . indeed , cross - presentation of soluble antigens by lsecs leads to antigen - specific recruitment of cd8 t cells to the liver even in the absence of inflammation . using bioluminescence imaging allows for longitudinal detection of antiviral immunity in the liver within the same animals after infection with hepatotropic adenoviruses encoding for immunological relevant antigens combined with the marker protein luciferase . detection of a decrease in luciferase expression , by reduced bioluminescence signal , in adenovirus - infected hepatocytes is superior to serological biomarkers of antiviral immunity , the hepatocellular enzymes alt or ast , as it reflects in real time the number of living infected hepatocytes . this experimental setup allowed to investigate the link between the extraordinary scavenger function , and the ability to cross - present circulating antigens by lsecs and hepatic immune surveillance . infection of hepatocytes with recombinant adenoviruses coding for ovalbumin , which shows high hepatocyte tropism , led to ovalbumin cross - presentation to ovalbumin - specific cd8 t cells . such cross - presentation led to stimulation of circulating ovalbumin - specific cd8 t cells and to secretion of tnf by such activated cd8 t cells . surprisingly , in transgenic mouse models where mhc class i - restricted interaction of virus - infected hepatocytes with cd8 t cells was excluded , virus - specific cd8 t cells still induced viral hepatitis , indicating that other mechanisms that do not rely on direct target - cell recognition allow locally activated cd8 t cells to achieve immune surveillance in the liver . conversely , exclusive mhc class i - restricted presentation of virus - derived antigens on hepatocytes led only to 4050% of antiviral cd8 t - cell effector function , suggesting that cross - presentation of viral antigens released from infected hepatocytes was operative in at least 50% of the total antiviral cd8 t - cell activity . this was termed noncanonical cd8 t - cell effector function , as it does not require direct mhc class i - restricted recognition of virus - infected target cells with virus - specific cd8 t cells to elicit antiviral effector functions ( figure 1noncanonical cd8 t - cell effector function ) . mechanistically , this noncanonical cd8 t - cell effector function is initiated by lsecs cross - presenting hepatocyte - derived viral antigens to cd8 t cells . tnf released from such cd8 t cells activated through cross - presenting lsecs than acts on hepatocytes to initiate a tnfr1-restricted induction of caspase - mediated apoptosis . neither ifn nor type i interferons contribute to this noncanonical cd8 t - cell effector function against virus - infected hepatocytes , which demonstrates a unique role for tnf in the local immune surveillance in the liver . the noncanonical cd8 t - cell effector function was important to obtain rapid control over viral infection of the liver , which is particularly important to rapidly gain control over the spread of viral infection among hepatocytes in the liver . it is of interest to note that lsecs fail to cross - present hbv - derived peptides to hbv - specific cd8 t cells , indicating that hbv may escape from this noncanonical cd8 t - cell effector function , and that this may have a role in the establishment of persistent infection or even in the failure to terminate persistent infection . taken together , mhc class i - restricted antigen ( cross)-presentation is operational in immune surveillance against viral infection of the liver . furthermore , sinusoidal liver cells engage in mutual exchange of mhc class i molecules to improve local immune surveillance . transfer of mhc class i molecules from hscs resulted in improved cross - presentation by lsecs . such trogocytosis of mhc class i molecules is distinct from cross - dressing of peptide - loaded mhc class i molecules that has been shown to be involved in cross - priming . continuous supply of mhc class i molecules from other liver cell populations may ensure optimal performance of lsecs in cross - presentation and local intrahepatic immune surveillance . in addition to cross - presentation of peptides generated by proteasomal digestion or endosomal degradation from viral proteins on mhc class i molecules and subsequent recognition by cd8 t cells bearing / t - cell receptors , other antigen - presentation pathways are increasingly recognized in their importance for local , organ - specific immune surveillance . the cd1 and mhc - like protein 1 ( mr1 ) function to present lipid antigens or bacterial metabolites to nkt cells or mucosal - associated invariant t cells ( mait cells ) , respectively , and thereby cause their activation as well as effector function . mait cells have been first described in 1999 and gained increasing attention over the last years for their prominent role in antibacterial defense . in humans , mait cell represent the most abundant innate - like t - cell population with about 5% of the total t - cell repertoire and that can reach up to 45% of the total liver lymphocytes . they express a semi - invariant t - cell receptor ( v7.2-j33/12/20 ) recognizing antigen presented by the mhc - like protein 1 ( mr1 ) . mr1 is highly conserved molecule across many species , including mouse and human , sharing > 90% homology at the protein level . the identity of antigens presented in the context of mr1 remain largely unknown , but structural analysis of the mr1 antigen - binding cleft revealed that bacterial metabolites are presented by mr1 . kjer - nielsen et al . showed that a photodegradable product of folic acid ( vitamin b9 ) stabilized the mr1/2-microglobulin complex , and that this complex sufficed to activate mait cells . a derivative of riboflavin ( vitamin b2 ) that binds to mr1 is responsible for mait - cell activation , and as these vitamins are selectively produced by bacteria , this supports the notion that mait cells have an important role in antibacterial defense . under physiological conditions , the mr1 protein is almost absent from the cell surface owing to lack of stabilization by its ligand . upon infection of cells by bacteria producing riboflavin , however , the mr1 complex is stabilized and transported to the surface . yet , infection is not an absolute prerequisite for mr1-dependent activation of mait cells , suggesting that uptake of bacterial constituents also suffice for mr1-mediated mait - cell activation . in particular , biliary epithelial cells but very likely also liver sinusoidal cells are involved in mait - cell activation upon contact with bacteria ( figure 2mr1-restricted activation of mait cells ) . besides direct antigen recognition , mait cells can also be activated in an mr1-independent manner . it has been shown that the cytokines il-12 and il-18 activate mait cells , and lead to expression and release of ifn. thereby , mait cells may contribute to antiviral defense in the liver , where secreted ifn participate in the non - cytolytic elimination of hbv . along this line , tcr - independent stimulation of mait cells leads to ifn-dependent reduction of hcv replication in infected hepatocytes , suggesting that mait cell - derived ifn operates in antiviral defense in the liver . nkt cells belong to the so - called innate - like t cells and can be divided into two subpopulations : the type i or invariant nkt cells ( inkt ) or the type ii or diverse nkt cells . inkt cells express like mait cells an invariant t - cell receptor chain ( v14-j18 in mice , v24-j18 in humans ) with a limited number of t - cell receptor chains . this invariant tcr recognizes antigens in the context of the mhc class i like molecule cd1 that presents glycolipids . unlike for mr1 , glycolipid antigens presented by cd1 molecules are not restricted , but rather a broad variety of endogenous and exogenous lipids can be presented on cd1 . therefore , nkt cells acts as interface bridging innate and adaptive immunity . nkt cells comprise only about 0.1% of the peripheral t - cell pool , but are specifically enriched in the liver making up to 40% of all lymphocytes in the murine liver and up to 25% of lymphocytes in the human liver . besides being stimulated through the cd1 molecule presenting glycolipids , inkt cells are also stimulated by activation of toll - like receptors , or by the cytokines il-12 , il-18 and type i interferons . the outcome of nkt cell stimulation varies on the type of stimulation , the localization of stimulation and the antigen - presenting cell . upon stimulation with the lipid galcer presented by cd1 molecules , inkt cells secrete ifn , il-4 and il-17 , but produce ifn only by stimulation through il-12 and il-18 , or by following ischemia or toxin - induced liver injury . a prominent role of cd1 and inkt cells in control of bacterial infection of the liver has been described ( figure 2cd1-restricted activation of nkt cells ) . however , reports indicated that inkt cells inhibit viral replication of hcv in hepatocytes via the secretion of ifn. during early hbv infection , high numbers of activated inkt are found that may contribute to viral defense by secreting ifn inhibiting hbv replication . a recent report shows that nkt cells contribute to the antiviral response during hbv infection . hepatocytes infected with hbv present endoplasmic reticulum - associated endogenous lipids generated by hbv - induced secretory phospholipases leading to activation of nkt cells . absence of nkt cells , cd1 molecules or a defect in transferring endoplasmic reticulum - associated lipids to cd1 molecules leads to failure of inducing a strong t- and b - cell immunity and control of viral infection . thus , liver nkt cells appear to contribute to both , antibacterial as well as antiviral immunity . taken together , immune surveillance in the liver is mainly triggered by sinusoidal liver cell populations , which establishes an intravascular immune surveillance paradigm for the liver . mhc class i - restricted antigen ( cross)-presentation is not only important for cross - priming of cd8 t - cell immunity , but is also instrumental for efficient virus - specific immune surveillance in the liver through lsec - dependent activation of noncanonical cd8 t - cell effector functions that eliminate virus - infected hepatocytes via tnf . complemented is antiviral immune surveillance by antibacterial immune surveillance in the liver , which may similarly be initiated by sinusoidal liver cell populations , yet involves cd1- and mr1-restricted activation of nkt and mait cells rather than mhc class i - restricted activation of cd8 t cells . thus , separate antigen - presentation pathways and distinct immune effector cell populations in the liver may serve to respond to viral and bacterial infections , and very likely cooperate to control infections locally in the liver .

although the liver 's function as unique immune organ regulating immunity has received a lot of attention over the last years , the mechanisms determining hepatic immune surveillance against infected hepatocytes remain less well defined . liver sinusoidal cells , in particular , liver sinusoidal endothelial cells ( lsecs ) and kupffer cells ( kcs ) , serve as physical platform for recruitment and anchoring of blood - borne immune cells in the liver . liver sinusoidal cells also function as portal of entry for infectious microorganisms targeting the liver such as hepatotropic viruses , bacteria or parasites . at the same time , liver sinusoidal cells actively contribute to achieve immune surveillance against bacterial and viral infections . kcs function as adhesion hubs for cd8 t cells from the circulation , which initiates the interaction of virus - specific cd8 t cells with infected hepatocytes . through their phagocytic function , kcs contribute to removal of bacteria from the circulation and engage in cross talk with sinusoidal lymphocyte populations to achieve elimination of phagocytosed bacteria . lsecs contribute to local immune surveillance through cross - presentation of viral antigens that causes antigen - specific retention of cd8 t cells from the circulation . such cross - presentation of viral antigens activates cd8 t cells to release tnf that in turn triggers selective killing of virus - infected hepatocytes . beyond major histocompatibility complex ( mhc)-restricted t - cell immunity , cd1- and mr1-restricted innate - like lymphocytes are found in liver sinusoids whose roles in local immune surveillance against infection need to be defined . thus , liver sinusoidal cell populations bear key functions for hepatic recruitment and for local activation of immune cells , which are both required for efficient immune surveillance against infection in the liver .

Infectious microorganisms targeting the liver The role of cross-priming and cross-presentation for immune surveillance The liver as immune organ Immune surveillance in the liver Direct MHC I-restricted recognition of virus-infected hepatocytes by CD8 T cells Cross-presentation and non-canonical MHC class I-mediated CD8 T-cell effector function Non-MHC-mediated immune surveillance through CD1 and MR1 Conclusion

the infection process in the liver involves transit of plasmodium sporozoites through various liver cell populations , including kupffer cells ( kcs ) before infecting their final target cell , the hepatocyte . the high blood volume passing through the liver , that is , 20% of the total cardiac output , together with the slow blood flow and low shear forces in liver sinusoids together facilitate to hepatic clearance of the blood from molecules requiring metabolic degradation , but at the same time also allow pathogens to target the liver and establish infection of hepatocytes if they manage to escape immune - mediated destruction by sinusoidal cell populations . the combination of lack of inflammation and large amounts of circulating viral antigens has been shown to be involved in the development of t cells with an exhausted phenotype , and is believed to be responsible for the exhaustion of hbv - specific immunity that facilitates persistent infection . thus , infection in the liver often occurs in the absence of strong innate immunity , which impedes pathogen - specific effector t cells that express cxcr3 to relocate via chemokines to sites of infection . as the liver sinusoids are a maze - like structure , finding infected hepatocytes in the absence of an inflammation - dependent localizing signal is a difficult task and likely requires high numbers of pathogen - specific cd8 t cells . the initiation of protective immunity against infectious pathogens that reside within cells , such as viruses , intracellular parasites and intracellular bacteria , requires cytotoxic cd8 t - cell responses . for their activation , cd8 t cells are stimulated by peptides presented on major histocompatibility complex ( mhc ) i molecules . this pathway , however , does not allow to present exogenous antigens on mhc i molecules , which is required for professional antigen - presenting cells to activate a cd8 t - cell response . the process of presentation of exogenous antigens to naive cd8 t cells has been termed cross - priming and is required for induction of protective immunity against those pathogens that evade expression of their antigens in professional antigen - presenting cells . among the specialized immune cells , a subpopulation of dendritic cells ( dcs ) is capable of cross - priming naive cd8 t cells . as the highly organized microarchitecture of lymphoid tissues is optimized to facilitate interaction of antigen ( cross)-presenting cells with the low number of antigen - specific t cells , cross - priming in lymphoid tissues appears to be most suited to generate effector as well as memory cd8 t cells that recognize and control infectious microorganisms . after generation of pathogen - specific cd8 t - cell immunity within lymphoid tissues , such cd8 t cells need to relocate to peripheral organs and recognize their target cells within infected tissue . as immune evasion from antigen presentation may also occur in infected cells , cross - presentation of pathogen - derived antigens during the effector phase in infected organs may help the immune response to achieve immune surveillance . apart from the liver microenvironment that constitutes a tolerogenic milieu rich in immune - regulatory mediators such as prostaglandins , transforming growth factor ( tgf) or interleukin ( il)-10 , the liver harbors many antigen - presenting cell populations that have been suggested to engage in priming of naive t cells and thereby contribute to skewing of immune responses . hepatocytes can prime naive cd8 t cells , which results in clonal t - cell deletion in a bim - dependent manner leading to t - cell apoptosis , thus establishing antigen - specific tolerance . the relevance of hepatocyte - restricted priming of virus - specific cd8 t cells during viral infection of the liver has not been thoroughly addressed so far . liver sinusoidal endothelial cells ( lsecs ) comprise the most prominent non - parenchymal cell population in the liver that line the liver sinusoids . priming of naive cd4 t cells by lsecs rather leads to generation of regulatory t cells that can suppress organ - specific autoimmunity even in the central nervous system demonstrating the relevance of hepatic immune functions for systemic immune responses . in contrast , cd8 t - cell priming by cross - presenting lsecs leads to development into a distinct memory - like state where cd8 t cells can be reactivated by combinatorial stimuli involving t - cell receptor ( tcr ) signaling , cd28 signaling and signaling through the il-12 receptor . rather , this unique memory t - cell programing occurs in the absence of cd28 or il-12 signals , but involves il-6 trans - signaling , which identifies a for unknown direct t cell adjuvant critical for induction of memory t cells . thus , antigen - presenting lsecs cross - prime memory cd8 t cells with proliferative potential or induce differentiation of regulatory cd4 t cells , which shows a unique functional diversity of this liver - resident antigen - presenting cell population in skewing of immune responses . although well positioned to interact with cd8 t cells , their participation in local t - cell responses by antigen cross - presentation several reports indicate hscs capable for cross - presentation to cd8 t cells . together , liver antigen - presenting cells contribute to the liver 's unique immune functions through local activation and differentiation of naive t cells . infectious microorganisms reaching the liver have first contact with sinusoidal cell populations , but hepatitis viruses and plasmodia spp . although innate immune responses generated by infected cells , that is , hepatocytes , or by immune cells detecting microbial - associated molecular patterns may help to contain infection and hinder the replication of pathogens , cd8 t - cell immunity is required to achieve control of infection in the liver , exemplified for control of hbv infection through cd8 t cells in chimpanzees . the sentinel function of sinusoidal cell populations allows them to mount innate immunity and inflammation , which may help through expression of interferons to contain pathogen infection in the liver . the induction of effector functions from pathogen - specific cd8 t cells through their local activation in the infected organ is necessary to achieve control of infection . such local activation of effector cd8 t cells is believed to occur mainly by direct recognition of pathogen - derived peptides on mhc i molecules expressed by the infected cell itself , but may also result from mhc i - restricted cross - presentation of pathogen - specific antigens on noninfected cells or by non - mhc - restricted activation of innate - like lymphocytes resident to the liver . immune surveillance in the liver is believed to occur mainly through recruitment of circulating pathogen - specific effector cd8 t cells . adhesion of circulating cd8 t cells to sinusoidal cell populations in the liver is facilitated by the narrow sinusoidal diameter that together with the low perfusion pressure leads to low shear forces . remarkably , adhesion of cd8 t cells within the liver can occur without inflammation in the infected area . these platelets in turn adhere to lsec even under steady - state conditions creating a platform for cd8 t - cell adhesion even under under noninflammatory conditions . interestingly , hbv - specific cd8 t cells do not arrest in the liver upon antigen - specific contact with hepatocytes expressing hbv antigens , but are randomly recruited to platelet aggregates forming on lsec . such stochastic recruitment of cd8 t cells from the circulation may not be very efficient and may further add to the necessity for large numbers of t cd8 t cells to control infection in the liver . the establishment of persistent viral infections of the liver , in particular , chronic hepatitis b or chronic hepatitis c , suggests that antiviral immune surveillance in the liver can be circumvented by these hepatotropic viruses . however , hbv - specific cd8 t cells were eventually required for elimination of hbv - infected hepatocytes through cytotoxic effector mechanisms . notwithstanding of the cytokine - induced control of hbv - replication , hbv - specific cd8 t cells are required for elimination of hbv - infected hepatocytes to achieve control of hbv infection . for efficient immune surveillance against plasmodium - infected hepatocytes , a strong initial priming period is required to generate high numbers of pathogen - specific cd8 t cells . direct recognition of plasmodium - infected hepatocytes by pathogen - specific cd8 t cells is then required to induce death of the infected hepatocytes . although the role of cross - priming for generation of virus - specific immunity is widely recognized , the role of cross - presentation of viral antigens to virus - specific cd8 t cells during the effector phase is much less characterized . obviously , immune escape from mhc class i - restricted antigen presentation in infected cells poses a hurdle to cd8 t - cell immune surveillance and may result in the failure to control viral infection despite the presence of virus - specific cd8 t cells . in the liver with its particular intravascular immune surveillance , cross - presentation may therefore benefit the recruitment of circulating t cells . indeed , cross - presentation of soluble antigens by lsecs leads to antigen - specific recruitment of cd8 t cells to the liver even in the absence of inflammation . detection of a decrease in luciferase expression , by reduced bioluminescence signal , in adenovirus - infected hepatocytes is superior to serological biomarkers of antiviral immunity , the hepatocellular enzymes alt or ast , as it reflects in real time the number of living infected hepatocytes . infection of hepatocytes with recombinant adenoviruses coding for ovalbumin , which shows high hepatocyte tropism , led to ovalbumin cross - presentation to ovalbumin - specific cd8 t cells . such cross - presentation led to stimulation of circulating ovalbumin - specific cd8 t cells and to secretion of tnf by such activated cd8 t cells . surprisingly , in transgenic mouse models where mhc class i - restricted interaction of virus - infected hepatocytes with cd8 t cells was excluded , virus - specific cd8 t cells still induced viral hepatitis , indicating that other mechanisms that do not rely on direct target - cell recognition allow locally activated cd8 t cells to achieve immune surveillance in the liver . conversely , exclusive mhc class i - restricted presentation of virus - derived antigens on hepatocytes led only to 4050% of antiviral cd8 t - cell effector function , suggesting that cross - presentation of viral antigens released from infected hepatocytes was operative in at least 50% of the total antiviral cd8 t - cell activity . this was termed noncanonical cd8 t - cell effector function , as it does not require direct mhc class i - restricted recognition of virus - infected target cells with virus - specific cd8 t cells to elicit antiviral effector functions ( figure 1noncanonical cd8 t - cell effector function ) . mechanistically , this noncanonical cd8 t - cell effector function is initiated by lsecs cross - presenting hepatocyte - derived viral antigens to cd8 t cells . neither ifn nor type i interferons contribute to this noncanonical cd8 t - cell effector function against virus - infected hepatocytes , which demonstrates a unique role for tnf in the local immune surveillance in the liver . the noncanonical cd8 t - cell effector function was important to obtain rapid control over viral infection of the liver , which is particularly important to rapidly gain control over the spread of viral infection among hepatocytes in the liver . it is of interest to note that lsecs fail to cross - present hbv - derived peptides to hbv - specific cd8 t cells , indicating that hbv may escape from this noncanonical cd8 t - cell effector function , and that this may have a role in the establishment of persistent infection or even in the failure to terminate persistent infection . continuous supply of mhc class i molecules from other liver cell populations may ensure optimal performance of lsecs in cross - presentation and local intrahepatic immune surveillance . in addition to cross - presentation of peptides generated by proteasomal digestion or endosomal degradation from viral proteins on mhc class i molecules and subsequent recognition by cd8 t cells bearing / t - cell receptors , other antigen - presentation pathways are increasingly recognized in their importance for local , organ - specific immune surveillance . in humans , mait cell represent the most abundant innate - like t - cell population with about 5% of the total t - cell repertoire and that can reach up to 45% of the total liver lymphocytes . in particular , biliary epithelial cells but very likely also liver sinusoidal cells are involved in mait - cell activation upon contact with bacteria ( figure 2mr1-restricted activation of mait cells ) . immune surveillance in the liver is believed to occur mainly through recruitment of circulating pathogen - specific effector cd8 t cells . adhesion of circulating cd8 t cells to sinusoidal cell populations in the liver is facilitated by the narrow sinusoidal diameter that together with the low perfusion pressure leads to low shear forces . remarkably , adhesion of cd8 t cells within the liver can occur without inflammation in the infected area . these platelets in turn adhere to lsec even under steady - state conditions creating a platform for cd8 t - cell adhesion even under under noninflammatory conditions . interestingly , hbv - specific cd8 t cells do not arrest in the liver upon antigen - specific contact with hepatocytes expressing hbv antigens , but are randomly recruited to platelet aggregates forming on lsec . such stochastic recruitment of cd8 t cells from the circulation may not be very efficient and may further add to the necessity for large numbers of t cd8 t cells to control infection in the liver . the establishment of persistent viral infections of the liver , in particular , chronic hepatitis b or chronic hepatitis c , suggests that antiviral immune surveillance in the liver can be circumvented by these hepatotropic viruses . however , hbv - specific cd8 t cells were eventually required for elimination of hbv - infected hepatocytes through cytotoxic effector mechanisms . notwithstanding of the cytokine - induced control of hbv - replication , hbv - specific cd8 t cells are required for elimination of hbv - infected hepatocytes to achieve control of hbv infection . for efficient immune surveillance against plasmodium - infected hepatocytes , a strong initial priming period is required to generate high numbers of pathogen - specific cd8 t cells . direct recognition of plasmodium - infected hepatocytes by pathogen - specific cd8 t cells is then required to induce death of the infected hepatocytes . although the role of cross - priming for generation of virus - specific immunity is widely recognized , the role of cross - presentation of viral antigens to virus - specific cd8 t cells during the effector phase is much less characterized . obviously , immune escape from mhc class i - restricted antigen presentation in infected cells poses a hurdle to cd8 t - cell immune surveillance and may result in the failure to control viral infection despite the presence of virus - specific cd8 t cells . in the liver with its particular intravascular immune surveillance , cross - presentation may therefore benefit the recruitment of circulating t cells . indeed , cross - presentation of soluble antigens by lsecs leads to antigen - specific recruitment of cd8 t cells to the liver even in the absence of inflammation . detection of a decrease in luciferase expression , by reduced bioluminescence signal , in adenovirus - infected hepatocytes is superior to serological biomarkers of antiviral immunity , the hepatocellular enzymes alt or ast , as it reflects in real time the number of living infected hepatocytes . infection of hepatocytes with recombinant adenoviruses coding for ovalbumin , which shows high hepatocyte tropism , led to ovalbumin cross - presentation to ovalbumin - specific cd8 t cells . such cross - presentation led to stimulation of circulating ovalbumin - specific cd8 t cells and to secretion of tnf by such activated cd8 t cells . surprisingly , in transgenic mouse models where mhc class i - restricted interaction of virus - infected hepatocytes with cd8 t cells was excluded , virus - specific cd8 t cells still induced viral hepatitis , indicating that other mechanisms that do not rely on direct target - cell recognition allow locally activated cd8 t cells to achieve immune surveillance in the liver . conversely , exclusive mhc class i - restricted presentation of virus - derived antigens on hepatocytes led only to 4050% of antiviral cd8 t - cell effector function , suggesting that cross - presentation of viral antigens released from infected hepatocytes was operative in at least 50% of the total antiviral cd8 t - cell activity . this was termed noncanonical cd8 t - cell effector function , as it does not require direct mhc class i - restricted recognition of virus - infected target cells with virus - specific cd8 t cells to elicit antiviral effector functions ( figure 1noncanonical cd8 t - cell effector function ) . mechanistically , this noncanonical cd8 t - cell effector function is initiated by lsecs cross - presenting hepatocyte - derived viral antigens to cd8 t cells . neither ifn nor type i interferons contribute to this noncanonical cd8 t - cell effector function against virus - infected hepatocytes , which demonstrates a unique role for tnf in the local immune surveillance in the liver . the noncanonical cd8 t - cell effector function was important to obtain rapid control over viral infection of the liver , which is particularly important to rapidly gain control over the spread of viral infection among hepatocytes in the liver . it is of interest to note that lsecs fail to cross - present hbv - derived peptides to hbv - specific cd8 t cells , indicating that hbv may escape from this noncanonical cd8 t - cell effector function , and that this may have a role in the establishment of persistent infection or even in the failure to terminate persistent infection . continuous supply of mhc class i molecules from other liver cell populations may ensure optimal performance of lsecs in cross - presentation and local intrahepatic immune surveillance . in addition to cross - presentation of peptides generated by proteasomal digestion or endosomal degradation from viral proteins on mhc class i molecules and subsequent recognition by cd8 t cells bearing / t - cell receptors , other antigen - presentation pathways are increasingly recognized in their importance for local , organ - specific immune surveillance . in particular , biliary epithelial cells but very likely also liver sinusoidal cells are involved in mait - cell activation upon contact with bacteria ( figure 2mr1-restricted activation of mait cells ) . taken together , immune surveillance in the liver is mainly triggered by sinusoidal liver cell populations , which establishes an intravascular immune surveillance paradigm for the liver . mhc class i - restricted antigen ( cross)-presentation is not only important for cross - priming of cd8 t - cell immunity , but is also instrumental for efficient virus - specific immune surveillance in the liver through lsec - dependent activation of noncanonical cd8 t - cell effector functions that eliminate virus - infected hepatocytes via tnf . complemented is antiviral immune surveillance by antibacterial immune surveillance in the liver , which may similarly be initiated by sinusoidal liver cell populations , yet involves cd1- and mr1-restricted activation of nkt and mait cells rather than mhc class i - restricted activation of cd8 t cells . thus , separate antigen - presentation pathways and distinct immune effector cell populations in the liver may serve to respond to viral and bacterial infections , and very likely cooperate to control infections locally in the liver .

a cost - effective analysis is important in all public health decision - making , and critical in low- and middle - income countries where health care resources are severely limited and rationing decisions are necessary . although some of the hospitals in these low - income countries have intensive care capabilities , many more do not have these advanced capabilities . in hospitals without intensive care units ( icus ) , survival of patients with respiratory failure is dismal as no rescue mechanical ventilation is possible . the only option available for these moribund patients is transfer to a hospital with an icu , if one is even available . because of financial constraints , it may be important for low - income countries to consider less traditional mechanisms to treat respiratory failure patients . mechanical ventilation is expensive , with cost - effectiveness estimates of 29,000110,000 $ /qaly , ( 1994 usd ) . however , these studies estimating cost effectiveness were performed in developed countries where the cost may be substantially inflated compared to the cost of critical care and mechanical ventilation in low - income countries . newer treatments for respiratory failure include noninvasive positive pressure ventilation ( niv ) initially followed by mechanical ventilation for niv failures . this approach has been shown to reduce the need for endotracheal intubation , and is associated with lower mortality , less complications , and reduced length of stay in conditions such as chronic obstructive pulmonary disease ( copd ) . additionally , several studies have documented niv as cost - effective . as noninvasive ventilation can occur safely outside the icu , this modality may be a potential option in hospitals without icu facilities . in this approach , although rescue mechanical ventilation for noninvasive ventilation failures would not be available , it may still offer a survival advantage at a reasonable cost for many patients . the purpose of this study is to compare the cost - effectiveness of the use of ward - based niv plus standard treatment to standard treatment alone in copd - related respiratory failure patients in india . this study was submitted to the investigational review board of st . john hospital and medical center and was determined to be exempt from review . following recommendations from the us public health service panel on cost - effectiveness in health and medicine and the american thoracic society ( ats ) workgroup , the analysis was conducted from the societal perspective , with a lifetime time horizon and the assumption of a discount rate of 3% for costs and health effects . the indian consumer price index ( cpi ) was used to inflate historical cost estimates to 2012 . the analysis was a decision tree with one - way sensitivity analysis , two - way sensitivity analysis , and probabilistic ( monte carlo ) analysis . estimates and sources used in the model as it was assumed that mechanical ventilation was not available , mortality estimates in both groups ( niv and standard treatment ) were estimated from the proportion of individuals requiring rescue mechanical intubation in the studies comparing niv ( with standard care ) and standard care alone patients . several authors have performed meta - analysis of niv and standard treatment in copd respiratory failure patients , and reported the rescue intubation rate . although the estimates were all similar , as we needed to choose one , we used the intubation rate from the most recent meta - analysis , which also had the largest number of patients and included all the studies from the previous meta - analysis . for sensitivity analysis the mean and sd were also used in the probabilistic analysis , with a beta distribution . all studies comparing niv with the standard treatment found that the niv group had a shorter total los . data from indian references were preferred as local practices are likely to be representative of true indian los . prasad ( 2007 , randomized , n = 19 , los standard treatment = 13.33 4.69 days and niv treatment = 9.63 1.41 days ) and khilnani ( 2010 , randomized , n = 40 , los standard treatment = 17.8 2.6 days and niv treatment , los = 9.4 4.3 days ) were both reviewed . no data were provided on los for standard or niv treatment groups in patients not needing rescue intubation , and both indian studies had > 50% intubation rate ( which increases los ) . one older study ( brochard , 1995 , randomized , n = 85 , france based ) noted total los for standard treatment not needing intubation of 20 ( 6 ) days and niv treatment not requiring intubation of 17 ( 9 ) days ( difference of 3 days ) . estimates from both the indian studies were very close regarding the total los of the niv treatment group ( 9.4 vs 9.6 days ) , and the study that showed the smallest difference in los between the two groups ( the prasad study with a difference was 3.7 days compared to the khilnani study with a difference of 8.4 days ) was utilized to prevent any bias in the study against niv treatment . this was additionally supported by the older non - indian study , which noted a difference in los of 3 days , and the large recent meta - analysis , which found that the difference in los for all the 11 studies included was 2.68 days . for sensitivity analysis regarding the los from all studies conducted so far , comparison of niv to standard treatment has revealed that the niv treatment group always had a lower los . because of larger sd in standard treatment los , it may be possible in an iteration of the probabilistic analysis for the value chosen in los standard treatment to be less than the value selected for los niv treatment . as this is not realistic , the if function was used in the probabilistic analysis such that if the value chosen for los in the standard treatment group was less than value chosen for los in the niv group , then the value for los in the niv group used in that iteration of the probabilistic analysis would be the los of the standard treatment group ( making the los equivalent in both groups for that iteration of the probabilistic analysis ) . only one study was found that provided data on the number of days of niv in copd respiratory failure patients ( that did not need rescue intubation ) . in this study , patients were on niv for a mean of 4 days ( sd = 4 ) . for sensitivity analysis , data from indian references were preferred as local practices are likely to be representative of the actual number of days before death . as described earlier , only two indian randomized trials of copd getting niv or standard treatment were found . the number of days till death was assumed to be the number of days before mechanical ventilation . although not specifically reported , both studies noted either rapid improvement with treatment ( both standard and niv treatment groups ) or rapid decline so that by 24 h , the majority needing intubation had been intubated . one study also noted a more rapid deterioration in the standard treatment patients needing intubation and less rapid deterioration in niv patients . other studies were also reviewed but no specific data were provided on days before intubation . in this analysis , 1 day ( range of 03 ) was chosen for the standard treatment group and 2 days ( range 04 ) was chosen for the niv treatment group . to test the impact of this assumption , two - way sensitivity was performed . the cost of health care in india , even hospital - based health care , is much less expensive than in the us and other developed countries . there was a cost comparison study for ward noninvasive ventilation versus ward standard treatment that was performed in england . in this english study , the noninvasive ventilation group in the wards had 30% higher costs than standard care in the wards . as this was our best estimate , for this study , we added 30% to the indian cost of standard treatment in the wards to estimate the cost for noninvasive ventilation in the wards in india . in all of the cost sensitivities and probabilistic analyses , we assumed the cost could vary 50% . to determine the chronic costs for moderate to severe copd , an indian study was used ( 1320 rupees in 2001 ) and the indian consumer price indicator ( cpi ) calculator was used to inflate to 2012 rupees ( inr ) . currency converter used to convert from inr to usd . for sensitivity and probabilistic analysis , function was used to discount into the future the yearly costs of copd treatment and qaly 's with a 3% discount rate , with a second analysis done at 5% discount rate . as recommended by the reference case , a discount rate of 3% was used , with a second analysis at 5% . willingness to pay ( wtp ) estimate was based on 3x gross domestic product ( gdp ) per capita , as suggested by the world health organization ( who ) . we made the assumption that the qaly 's were equivalent in standard and niv treatment groups after discharge . this assumption is supported by a study evaluating standard and non - invasive ventilation for copd patients in india that found no difference at 46 weeks in blood gas parameters and pulmonary function variables . it is also intuitive that once discharged alive , there is no difference between the two groups as there is no evidence showing that using niv or not using niv causes any long - term negative consequences . qaly estimates came from a study that measured qaly and survival years in 105 copd respiratory failure patients treated only with niv or standard therapy . as the cost of treatment for copd was discounted in the future ( using the annuity function in treeage ) , the qaly was also discounted in the future . for sensitivity analysis and probabilistic analysis cost - effectiveness analysis , one - way sensitivity [ incremental cost effectiveness ratio ( icer ) ] , and two - way sensitivity analysis ( mortality rates , days till death , and los in both strategies ) were performed . analyses were conducted using software ( treeage pro 2014 ; williamstown , ma , usa ) . all costs were inflated using indian consumer price index and discounted in the future using 3% discount rate . the model used in the analysis was a decision tree with one - way sensitivity analysis , two - way sensitivity analysis , and probabilistic ( monte carlo ) analysis . as it was assumed that mechanical ventilation was not available , mortality estimates in both groups ( niv and standard treatment ) were estimated from the proportion of individuals requiring rescue mechanical intubation in the studies comparing niv ( with standard care ) and standard care alone patients . several authors have performed meta - analysis of niv and standard treatment in copd respiratory failure patients , and reported the rescue intubation rate . although the estimates were all similar , as we needed to choose one , we used the intubation rate from the most recent meta - analysis , which also had the largest number of patients and included all the studies from the previous meta - analysis . for sensitivity analysis the mean and sd were also used in the probabilistic analysis , with a beta distribution . all studies comparing niv with the standard treatment found that the niv group had a shorter total los . data from indian references were preferred as local practices are likely to be representative of true indian los . prasad ( 2007 , randomized , n = 19 , los standard treatment = 13.33 4.69 days and niv treatment = 9.63 1.41 days ) and khilnani ( 2010 , randomized , n = 40 , los standard treatment = 17.8 2.6 days and niv treatment , los = 9.4 4.3 days ) were both reviewed . no data were provided on los for standard or niv treatment groups in patients not needing rescue intubation , and both indian studies had > 50% intubation rate ( which increases los ) . n = 85 , france based ) noted total los for standard treatment not needing intubation of 20 ( 6 ) days and niv treatment not requiring intubation of 17 ( 9 ) days ( difference of 3 days ) . estimates from both the indian studies were very close regarding the total los of the niv treatment group ( 9.4 vs 9.6 days ) , and the study that showed the smallest difference in los between the two groups ( the prasad study with a difference was 3.7 days compared to the khilnani study with a difference of 8.4 days ) was utilized to prevent any bias in the study against niv treatment . this was additionally supported by the older non - indian study , which noted a difference in los of 3 days , and the large recent meta - analysis , which found that the difference in los for all the 11 studies included was 2.68 days . for sensitivity analysis , in probabilistic analysis , the means and sds were used with a normal distribution . regarding the los from all studies conducted so far , comparison of niv to standard treatment has revealed that the niv treatment group always had a lower los . because of larger sd in standard treatment los , it may be possible in an iteration of the probabilistic analysis for the value chosen in los standard treatment to be less than the value selected for los niv treatment . as this is not realistic , the if function was used in the probabilistic analysis such that if the value chosen for los in the standard treatment group was less than value chosen for los in the niv group , then the value for los in the niv group used in that iteration of the probabilistic analysis would be the los of the standard treatment group ( making the los equivalent in both groups for that iteration of the probabilistic analysis ) . only one study was found that provided data on the number of days of niv in copd respiratory failure patients ( that did not need rescue intubation ) . in this study , patients were on niv for a mean of 4 days ( sd = 4 ) . for sensitivity analysis , data from indian references were preferred as local practices are likely to be representative of the actual number of days before death . as described earlier , only two indian randomized trials of copd getting niv or standard treatment were found . the number of days till death was assumed to be the number of days before mechanical ventilation . although not specifically reported , both studies noted either rapid improvement with treatment ( both standard and niv treatment groups ) or rapid decline so that by 24 h , the majority needing intubation had been intubated . one study also noted a more rapid deterioration in the standard treatment patients needing intubation and less rapid deterioration in niv patients . other studies were also reviewed but no specific data were provided on days before intubation . in this analysis , 1 day ( range of 03 ) was chosen for the standard treatment group and 2 days ( range 04 ) was chosen for the niv treatment group . to test the impact of this assumption , two - way sensitivity was performed . the cost of health care in india , even hospital - based health care , is much less expensive than in the us and other developed countries . there was a cost comparison study for ward noninvasive ventilation versus ward standard treatment that was performed in england . in this english study , the noninvasive ventilation group in the wards had 30% higher costs than standard care in the wards . as this was our best estimate , for this study , we added 30% to the indian cost of standard treatment in the wards to estimate the cost for noninvasive ventilation in the wards in india . in all of the cost sensitivities and probabilistic analyses , we assumed the cost could vary 50% . to determine the chronic costs for moderate to severe copd , an indian study was used ( 1320 rupees in 2001 ) and the indian consumer price indicator ( cpi ) calculator was used to inflate to 2012 rupees ( inr ) . currency converter used to convert from inr to usd . for sensitivity and probabilistic analysis , function was used to discount into the future the yearly costs of copd treatment and qaly 's with a 3% discount rate , with a second analysis done at 5% discount rate . as recommended by the reference case , a discount rate of 3% was used , with a second analysis at 5% . willingness to pay ( wtp ) estimate was based on 3x gross domestic product ( gdp ) per capita , as suggested by the world health organization ( who ) . we made the assumption that the qaly 's were equivalent in standard and niv treatment groups after discharge . this assumption is supported by a study evaluating standard and non - invasive ventilation for copd patients in india that found no difference at 46 weeks in blood gas parameters and pulmonary function variables . it is also intuitive that once discharged alive , there is no difference between the two groups as there is no evidence showing that using niv or not using niv causes any long - term negative consequences . qaly estimates came from a study that measured qaly and survival years in 105 copd respiratory failure patients treated only with niv or standard therapy . as the cost of treatment for copd was discounted in the future ( using the annuity function in treeage ) , the qaly was also discounted in the future . for sensitivity analysis and probabilistic analysis , 25% was used . cost - effectiveness analysis , one - way sensitivity [ incremental cost effectiveness ratio ( icer ) ] , and two - way sensitivity analysis ( mortality rates , days till death , and los in both strategies ) were performed . analyses were conducted using software ( treeage pro 2014 ; williamstown , ma , usa ) . all costs were inflated using indian consumer price index and discounted in the future using 3% discount rate . the mortality rate in these strategies ( i.e. the rescue intubation rate from the largest meta - analysis ) was 0.101 for the niv group and 0.278 for the standard treatment group . the cost ( usd 2012)/qaly for the standard treatment and the niv treatment were $ 78/qaly ( $ 535.02/6.82 ) and $ 75/qaly ( $ 636.33/8.49 ) , respectively , and did not change much in the probabilistic analysis [ table 3 ] . the niv group was slightly more expensive ( by approximately 100 usd ) but resulted in increased effectiveness ( qaly increased by 1.67 qaly ) . this was substantially lower than the gdp per capita for india ( 1489 usd ) , suggesting the niv strategy was very cost - effective . using a 5% discount rate resulted in only minimally different results . icer report wtp*=4467 results of the probabalistic analysis ( 50,000 iterations ) results of the one - way sensitivity analysis on icer are revealed in figure 2 . it represents how the expected value for the icer would vary as each variable was processed through its range of possibilities ( while all other variables were held constant ) . notably , icer remains very low and is always positive ( implying that through the range of values for each variable , the niv strategy is always cost - effective ) . figure 3 reveals the results of the cost - effectiveness analysis displaying the increased cost but also increased effectiveness in the niv strategy . qaly = qaly after hospitalization , dstdlive = days of standard treatment in survivors , cdaywrd = cost of day on ward , cdaynivwrd = cost of niv treatment on ward per day , ccopdyr = cost of health care for copd per year , totadayniv = total los in days of niv treatment in survivors , daysstddie = days of standard treatment before death , daysnivdie = days of niv treatment before death , pdieniv = probability of death in the niv group cost - effectiveness analysis with 3% discount rate a two - way sensitivity analysis varying the probability of death in both groups at the same time ( while holding all other variables constant ) is shown in figure 4 . notably , the range of the niv strategy was preferred throughout the entire range of the standard treatment probabilities . however , to determine what death probabilities would cause the standard treatment strategy to be preferred , the ranges for possible death were made equivalent when this was done , it was clear that the strategy preferred was always the niv strategy until the death rate in the niv group was equal to the death rate in the standard treatment [ figure 4 ] . as all studies conducted to date have found a decreased intubation rate ( which is the marker for death in this model that assumes no icu facilities available so that those needing intubation would die ) in the niv group , this again supports the niv treatment . additionally , this is intuitive as the cost of niv treatment is more than the cost of standard treatment ; hence , when the mortality rates become equal , it would be the point that would make the standard treatment more cost - effective ( same probability of death but less expensive ) . two - way sensitivity analysis for probability of death in the niv group and the standard treatment group.pdieniv = probability of death in niv group , pdiestd = probability of death in standard treatment group , wtp = willingness to pay two - way sensitivity analysis evaluating the impact of varying the days hospitalized in the two groups found that the niv strategy was preferred through the entire range , even when the total days hospitalized for niv was more than for standard treatment . two - way sensitivity analysis on the number of days before death in both the groups also found the niv strategy to be the preferred strategy through the ranges . figure 6 reveals the incremental cost - effectiveness results from the probabilistic analysis ( the ellipse is the 95% confidence interval ) . niv was the preferred strategy and in some of the iterations , was not only cost - effective ( results located below 0 cost where the niv strategy was less costly ) but more effective as well . this cost - effectiveness analysis pertains to the specific population of copd patients suffering from respiratory failure who were hypothetically treated in an indian hospital without access to any icu facilities . using mortality information alone ( when mortality is assumed to occur at the point of rescue intubation in published data ) , the noninvasive ventilation strategy was slightly more costly in comparison to standard treatment but had improved effectiveness ( more qalys ) . according to the who choosing interventions that are cost - effective ( choice ) initiative , interventions are considered very cost - effective if the cost - effectiveness per year is less than the gdp per capita , which for india is approximately 1,489 ( usd 2012 ) . the niv strategy had cost - effectiveness of only 61 ( usd 2012)/qaly ( 636.33/8.49 ) , which is only 4% of the indian gdp / capita . although it may not be appropriate to extrapolate the cost - effectiveness of niv to every low - income country , the fact that the cost estimate / qaly was so low suggests that there may be additional opportunities for noninvasive ventilation in other low - income countries when mechanical ventilation is not available . in india , there is still a critical shortage of icu beds ; there is an estimated need of 5 million icu beds annually but only 70,000 are available . one - analysis of indian icus found that medical professionals in small hospitals managed 40% of the inpatient beds with limited facilities . some studies reveal lower mortality , less complications ( pneumonia , sepsis , etc . ) , and lower los in niv treated patients . some other conditions [ acute respiratory distress syndrome ( ards ) , acute respiratory infections ] may or may not benefit from noninvasive ventilation . one recent study estimated the cost for hospitalization for acute respiratory infections was substantial at $ 54.00355.00 ( usd ) . the addition of niv for respiratory failure in these patients may positively impact this expenditure . however , additional evaluations are necessary before the cost - effectiveness of noninvasive ventilation can be extrapolated in other conditions . this cost analysis assumed that care would be provided in the ward , as several studies ( including one india - based study ) have suggested this is safe and effective . ward - based noninvasive ventilation is becoming more accepted in india , which makes this strategy more realistic . economic consequences of this strategy that contribute to increased cost in the niv group include less mortality and therefore , more cost after hospitalization in the higher proportion of survivors . however , even with increased survival , the incremental cost - effectiveness was only $ 61 ( 2012 usd ) . one limitation in this study is that the cost - effectiveness analysis is based on estimates drawn from the literature . as suggested by the icer tornado diagram [ figure 3 ] , several parameters where estimations were based on assumptions ( days of niv before death , days of standard treatment before death , and days of niv ) were not critical to the cost analysis . additionally , even with the large variance , the icer never crossed 0 in the tornado diagram . in order to address some of the limitations arising out of using literature - based parameters , indian - based estimates were used when available . additionally , estimates varied widely in the probabilistic analysis and the noninvasive ventilation strategy was still preferred . while the best estimates for cost - effectiveness analysis would ideally come from a large randomized controlled trial from different hospitals in india , this analysis may provide the rationale for perusing that type of analysis . cost effectiveness analysis may prove a powerful tool in addressing the difficulty of traditional icu health care in low- and middle - income countries . this analysis establishes preliminary evidence for the cost - effectiveness of ward - based niv treatment for copd - induced respiratory failure in hospitals without icus in india . in this analysis , we found that noninvasive ventilation treatment was cost - effective at 61 ( usd , 2012)/qalys , substantially less than 1 xgdp / qaly , which in india is 1489 ( usd , 2012 ) . as 40% of the hospitals in india have no icu , ward - based noninvasive ventilation may become a crucial new option to provide some life - saving respiratory support . future research needs to assess the cost - effectiveness of noninvasive treatment in other low- and middle - income countries , as the potential benefits may be substantial .

introduction : the majority of indian hospitals do not provide intensive care unit ( icu ) care or ward - based noninvasive positive pressure ventilation ( niv ) . because no mechanical ventilation or niv is available in these hospitals , the majority of patients suffering from respiratory failure die.objective:to perform a cost - effective analysis of two strategies ( ward - based niv with concurrent standard treatment vs standard treatment alone ) in chronic obstructive pulmonary disease ( copd ) respiratory failure patients treated in indian hospitals without icu care.materials and methods : a decision - analytical model was created to compare the cost - effectiveness for the two strategies . estimates from the literature were used for parameters in the model . future costs were discounted at 3% . all costs were reported in usd ( 2012 ) . one - way , two - way , and probabilistic sensitivity analysis were performed . the time horizon was lifetime and perspective was societal.results:the niv strategy resulted in 17.7% more survival and was slightly more costly ( increased cost of $ 101 ( usd 2012 ) but resulted in increased quality - adjusted life - years ( qalys ) ( 1.67 qaly ) . the cost - effectiveness ( 2012 usd)/qaly in the standard and niv groups was $ 78/qaly ( $ 535.02/6.82 ) and $ 75/qaly ( $ 636.33/8.49 ) , respectively . incremental cost - effectiveness ratio ( icer ) was only $ 61 usd / qaly . this was substantially lower than the gross domestic product ( gdp ) per capita for india ( 1489 usd ) , suggesting the niv strategy was very cost effective . using a 5% discount rate resulted in only minimally different results . probabilistic analysis suggests that niv strategy was preferred 100% of the time when willingness to pay was > $ 250 2012 usd.conclusion:ward-based niv treatment is cost - effective in india , and may increase survival of patients with copd respiratory failure when icu is not available .

INTRODUCTION MATERIALS AND METHODS Model and estimates Mortality estimates Total length of stay (LOS) estimates Days on NIV Days before the death of paitents in the NIV and standard treated groups Cost estimates Discount rate and willingness to pay QALY estimates Statistical analysis RESULTS DISCUSSION CONCLUSION

a cost - effective analysis is important in all public health decision - making , and critical in low- and middle - income countries where health care resources are severely limited and rationing decisions are necessary . although some of the hospitals in these low - income countries have intensive care capabilities , many more do not have these advanced capabilities . in hospitals without intensive care units ( icus ) , survival of patients with respiratory failure is dismal as no rescue mechanical ventilation is possible . mechanical ventilation is expensive , with cost - effectiveness estimates of 29,000110,000 $ /qaly , ( 1994 usd ) . however , these studies estimating cost effectiveness were performed in developed countries where the cost may be substantially inflated compared to the cost of critical care and mechanical ventilation in low - income countries . newer treatments for respiratory failure include noninvasive positive pressure ventilation ( niv ) initially followed by mechanical ventilation for niv failures . this approach has been shown to reduce the need for endotracheal intubation , and is associated with lower mortality , less complications , and reduced length of stay in conditions such as chronic obstructive pulmonary disease ( copd ) . the purpose of this study is to compare the cost - effectiveness of the use of ward - based niv plus standard treatment to standard treatment alone in copd - related respiratory failure patients in india . following recommendations from the us public health service panel on cost - effectiveness in health and medicine and the american thoracic society ( ats ) workgroup , the analysis was conducted from the societal perspective , with a lifetime time horizon and the assumption of a discount rate of 3% for costs and health effects . the analysis was a decision tree with one - way sensitivity analysis , two - way sensitivity analysis , and probabilistic ( monte carlo ) analysis . estimates and sources used in the model as it was assumed that mechanical ventilation was not available , mortality estimates in both groups ( niv and standard treatment ) were estimated from the proportion of individuals requiring rescue mechanical intubation in the studies comparing niv ( with standard care ) and standard care alone patients . several authors have performed meta - analysis of niv and standard treatment in copd respiratory failure patients , and reported the rescue intubation rate . for sensitivity analysis the mean and sd were also used in the probabilistic analysis , with a beta distribution . all studies comparing niv with the standard treatment found that the niv group had a shorter total los . prasad ( 2007 , randomized , n = 19 , los standard treatment = 13.33 4.69 days and niv treatment = 9.63 1.41 days ) and khilnani ( 2010 , randomized , n = 40 , los standard treatment = 17.8 2.6 days and niv treatment , los = 9.4 4.3 days ) were both reviewed . no data were provided on los for standard or niv treatment groups in patients not needing rescue intubation , and both indian studies had > 50% intubation rate ( which increases los ) . one older study ( brochard , 1995 , randomized , n = 85 , france based ) noted total los for standard treatment not needing intubation of 20 ( 6 ) days and niv treatment not requiring intubation of 17 ( 9 ) days ( difference of 3 days ) . estimates from both the indian studies were very close regarding the total los of the niv treatment group ( 9.4 vs 9.6 days ) , and the study that showed the smallest difference in los between the two groups ( the prasad study with a difference was 3.7 days compared to the khilnani study with a difference of 8.4 days ) was utilized to prevent any bias in the study against niv treatment . for sensitivity analysis regarding the los from all studies conducted so far , comparison of niv to standard treatment has revealed that the niv treatment group always had a lower los . because of larger sd in standard treatment los , it may be possible in an iteration of the probabilistic analysis for the value chosen in los standard treatment to be less than the value selected for los niv treatment . as this is not realistic , the if function was used in the probabilistic analysis such that if the value chosen for los in the standard treatment group was less than value chosen for los in the niv group , then the value for los in the niv group used in that iteration of the probabilistic analysis would be the los of the standard treatment group ( making the los equivalent in both groups for that iteration of the probabilistic analysis ) . only one study was found that provided data on the number of days of niv in copd respiratory failure patients ( that did not need rescue intubation ) . although not specifically reported , both studies noted either rapid improvement with treatment ( both standard and niv treatment groups ) or rapid decline so that by 24 h , the majority needing intubation had been intubated . one study also noted a more rapid deterioration in the standard treatment patients needing intubation and less rapid deterioration in niv patients . in this analysis , 1 day ( range of 03 ) was chosen for the standard treatment group and 2 days ( range 04 ) was chosen for the niv treatment group . to test the impact of this assumption , two - way sensitivity was performed . the cost of health care in india , even hospital - based health care , is much less expensive than in the us and other developed countries . as this was our best estimate , for this study , we added 30% to the indian cost of standard treatment in the wards to estimate the cost for noninvasive ventilation in the wards in india . in all of the cost sensitivities and probabilistic analyses , we assumed the cost could vary 50% . for sensitivity and probabilistic analysis , function was used to discount into the future the yearly costs of copd treatment and qaly 's with a 3% discount rate , with a second analysis done at 5% discount rate . willingness to pay ( wtp ) estimate was based on 3x gross domestic product ( gdp ) per capita , as suggested by the world health organization ( who ) . we made the assumption that the qaly 's were equivalent in standard and niv treatment groups after discharge . qaly estimates came from a study that measured qaly and survival years in 105 copd respiratory failure patients treated only with niv or standard therapy . as the cost of treatment for copd was discounted in the future ( using the annuity function in treeage ) , the qaly was also discounted in the future . for sensitivity analysis and probabilistic analysis cost - effectiveness analysis , one - way sensitivity [ incremental cost effectiveness ratio ( icer ) ] , and two - way sensitivity analysis ( mortality rates , days till death , and los in both strategies ) were performed . all costs were inflated using indian consumer price index and discounted in the future using 3% discount rate . the model used in the analysis was a decision tree with one - way sensitivity analysis , two - way sensitivity analysis , and probabilistic ( monte carlo ) analysis . as it was assumed that mechanical ventilation was not available , mortality estimates in both groups ( niv and standard treatment ) were estimated from the proportion of individuals requiring rescue mechanical intubation in the studies comparing niv ( with standard care ) and standard care alone patients . several authors have performed meta - analysis of niv and standard treatment in copd respiratory failure patients , and reported the rescue intubation rate . for sensitivity analysis the mean and sd were also used in the probabilistic analysis , with a beta distribution . all studies comparing niv with the standard treatment found that the niv group had a shorter total los . prasad ( 2007 , randomized , n = 19 , los standard treatment = 13.33 4.69 days and niv treatment = 9.63 1.41 days ) and khilnani ( 2010 , randomized , n = 40 , los standard treatment = 17.8 2.6 days and niv treatment , los = 9.4 4.3 days ) were both reviewed . no data were provided on los for standard or niv treatment groups in patients not needing rescue intubation , and both indian studies had > 50% intubation rate ( which increases los ) . n = 85 , france based ) noted total los for standard treatment not needing intubation of 20 ( 6 ) days and niv treatment not requiring intubation of 17 ( 9 ) days ( difference of 3 days ) . estimates from both the indian studies were very close regarding the total los of the niv treatment group ( 9.4 vs 9.6 days ) , and the study that showed the smallest difference in los between the two groups ( the prasad study with a difference was 3.7 days compared to the khilnani study with a difference of 8.4 days ) was utilized to prevent any bias in the study against niv treatment . for sensitivity analysis , in probabilistic analysis , the means and sds were used with a normal distribution . regarding the los from all studies conducted so far , comparison of niv to standard treatment has revealed that the niv treatment group always had a lower los . because of larger sd in standard treatment los , it may be possible in an iteration of the probabilistic analysis for the value chosen in los standard treatment to be less than the value selected for los niv treatment . as this is not realistic , the if function was used in the probabilistic analysis such that if the value chosen for los in the standard treatment group was less than value chosen for los in the niv group , then the value for los in the niv group used in that iteration of the probabilistic analysis would be the los of the standard treatment group ( making the los equivalent in both groups for that iteration of the probabilistic analysis ) . only one study was found that provided data on the number of days of niv in copd respiratory failure patients ( that did not need rescue intubation ) . although not specifically reported , both studies noted either rapid improvement with treatment ( both standard and niv treatment groups ) or rapid decline so that by 24 h , the majority needing intubation had been intubated . one study also noted a more rapid deterioration in the standard treatment patients needing intubation and less rapid deterioration in niv patients . in this analysis , 1 day ( range of 03 ) was chosen for the standard treatment group and 2 days ( range 04 ) was chosen for the niv treatment group . to test the impact of this assumption , two - way sensitivity was performed . the cost of health care in india , even hospital - based health care , is much less expensive than in the us and other developed countries . as this was our best estimate , for this study , we added 30% to the indian cost of standard treatment in the wards to estimate the cost for noninvasive ventilation in the wards in india . in all of the cost sensitivities and probabilistic analyses , we assumed the cost could vary 50% . for sensitivity and probabilistic analysis , function was used to discount into the future the yearly costs of copd treatment and qaly 's with a 3% discount rate , with a second analysis done at 5% discount rate . willingness to pay ( wtp ) estimate was based on 3x gross domestic product ( gdp ) per capita , as suggested by the world health organization ( who ) . we made the assumption that the qaly 's were equivalent in standard and niv treatment groups after discharge . qaly estimates came from a study that measured qaly and survival years in 105 copd respiratory failure patients treated only with niv or standard therapy . as the cost of treatment for copd was discounted in the future ( using the annuity function in treeage ) , the qaly was also discounted in the future . for sensitivity analysis and probabilistic analysis , 25% was used . cost - effectiveness analysis , one - way sensitivity [ incremental cost effectiveness ratio ( icer ) ] , and two - way sensitivity analysis ( mortality rates , days till death , and los in both strategies ) were performed . all costs were inflated using indian consumer price index and discounted in the future using 3% discount rate . the rescue intubation rate from the largest meta - analysis ) was 0.101 for the niv group and 0.278 for the standard treatment group . the cost ( usd 2012)/qaly for the standard treatment and the niv treatment were $ 78/qaly ( $ 535.02/6.82 ) and $ 75/qaly ( $ 636.33/8.49 ) , respectively , and did not change much in the probabilistic analysis [ table 3 ] . the niv group was slightly more expensive ( by approximately 100 usd ) but resulted in increased effectiveness ( qaly increased by 1.67 qaly ) . this was substantially lower than the gdp per capita for india ( 1489 usd ) , suggesting the niv strategy was very cost - effective . using a 5% discount rate resulted in only minimally different results . icer report wtp*=4467 results of the probabalistic analysis ( 50,000 iterations ) results of the one - way sensitivity analysis on icer are revealed in figure 2 . notably , icer remains very low and is always positive ( implying that through the range of values for each variable , the niv strategy is always cost - effective ) . figure 3 reveals the results of the cost - effectiveness analysis displaying the increased cost but also increased effectiveness in the niv strategy . qaly = qaly after hospitalization , dstdlive = days of standard treatment in survivors , cdaywrd = cost of day on ward , cdaynivwrd = cost of niv treatment on ward per day , ccopdyr = cost of health care for copd per year , totadayniv = total los in days of niv treatment in survivors , daysstddie = days of standard treatment before death , daysnivdie = days of niv treatment before death , pdieniv = probability of death in the niv group cost - effectiveness analysis with 3% discount rate a two - way sensitivity analysis varying the probability of death in both groups at the same time ( while holding all other variables constant ) is shown in figure 4 . notably , the range of the niv strategy was preferred throughout the entire range of the standard treatment probabilities . however , to determine what death probabilities would cause the standard treatment strategy to be preferred , the ranges for possible death were made equivalent when this was done , it was clear that the strategy preferred was always the niv strategy until the death rate in the niv group was equal to the death rate in the standard treatment [ figure 4 ] . as all studies conducted to date have found a decreased intubation rate ( which is the marker for death in this model that assumes no icu facilities available so that those needing intubation would die ) in the niv group , this again supports the niv treatment . additionally , this is intuitive as the cost of niv treatment is more than the cost of standard treatment ; hence , when the mortality rates become equal , it would be the point that would make the standard treatment more cost - effective ( same probability of death but less expensive ) . two - way sensitivity analysis for probability of death in the niv group and the standard treatment group.pdieniv = probability of death in niv group , pdiestd = probability of death in standard treatment group , wtp = willingness to pay two - way sensitivity analysis evaluating the impact of varying the days hospitalized in the two groups found that the niv strategy was preferred through the entire range , even when the total days hospitalized for niv was more than for standard treatment . two - way sensitivity analysis on the number of days before death in both the groups also found the niv strategy to be the preferred strategy through the ranges . figure 6 reveals the incremental cost - effectiveness results from the probabilistic analysis ( the ellipse is the 95% confidence interval ) . niv was the preferred strategy and in some of the iterations , was not only cost - effective ( results located below 0 cost where the niv strategy was less costly ) but more effective as well . this cost - effectiveness analysis pertains to the specific population of copd patients suffering from respiratory failure who were hypothetically treated in an indian hospital without access to any icu facilities . using mortality information alone ( when mortality is assumed to occur at the point of rescue intubation in published data ) , the noninvasive ventilation strategy was slightly more costly in comparison to standard treatment but had improved effectiveness ( more qalys ) . according to the who choosing interventions that are cost - effective ( choice ) initiative , interventions are considered very cost - effective if the cost - effectiveness per year is less than the gdp per capita , which for india is approximately 1,489 ( usd 2012 ) . the niv strategy had cost - effectiveness of only 61 ( usd 2012)/qaly ( 636.33/8.49 ) , which is only 4% of the indian gdp / capita . although it may not be appropriate to extrapolate the cost - effectiveness of niv to every low - income country , the fact that the cost estimate / qaly was so low suggests that there may be additional opportunities for noninvasive ventilation in other low - income countries when mechanical ventilation is not available . one - analysis of indian icus found that medical professionals in small hospitals managed 40% of the inpatient beds with limited facilities . one recent study estimated the cost for hospitalization for acute respiratory infections was substantial at $ 54.00355.00 ( usd ) . however , additional evaluations are necessary before the cost - effectiveness of noninvasive ventilation can be extrapolated in other conditions . ward - based noninvasive ventilation is becoming more accepted in india , which makes this strategy more realistic . economic consequences of this strategy that contribute to increased cost in the niv group include less mortality and therefore , more cost after hospitalization in the higher proportion of survivors . however , even with increased survival , the incremental cost - effectiveness was only $ 61 ( 2012 usd ) . one limitation in this study is that the cost - effectiveness analysis is based on estimates drawn from the literature . as suggested by the icer tornado diagram [ figure 3 ] , several parameters where estimations were based on assumptions ( days of niv before death , days of standard treatment before death , and days of niv ) were not critical to the cost analysis . in order to address some of the limitations arising out of using literature - based parameters , indian - based estimates were used when available . additionally , estimates varied widely in the probabilistic analysis and the noninvasive ventilation strategy was still preferred . while the best estimates for cost - effectiveness analysis would ideally come from a large randomized controlled trial from different hospitals in india , this analysis may provide the rationale for perusing that type of analysis . this analysis establishes preliminary evidence for the cost - effectiveness of ward - based niv treatment for copd - induced respiratory failure in hospitals without icus in india . in this analysis , we found that noninvasive ventilation treatment was cost - effective at 61 ( usd , 2012)/qalys , substantially less than 1 xgdp / qaly , which in india is 1489 ( usd , 2012 ) . as 40% of the hospitals in india have no icu , ward - based noninvasive ventilation may become a crucial new option to provide some life - saving respiratory support . future research needs to assess the cost - effectiveness of noninvasive treatment in other low- and middle - income countries , as the potential benefits may be substantial .

the major objectives of root canal therapy are removal of the pathologic pulp , shaping and cleaning of the root canal system , disinfection of the contaminated root canals and three dimensional obturation to prevent reinfection.1 the success of endodontic therapy depends on adequate access , thorough biomechanical preparation and proper obturation of the root canal system . endodontically treated teeth are widely considered to be more susceptible to fracture than vital teeth . the reasons most often reported have been the removal of tooth structure during endodontic therapy , dehydration of dentin after endodontic therapy and excessive pressure during obturation.2,3 the strength of the endodontically treated teeth is directly related to the method of canal preparation and the amount of remaining sound tooth structure . it is observed that the greatest incidence of vertical root fracture occurs in teeth that have undergone endodontic therapy.4 vertical root fracture is a longitudinal fracture of the root , extending throughout the entire thickness of dentin from the root canal to the periodontium . it is of serious clinical concern and has an unfavorable prognosis resulting in extraction of the tooth or resection of the affected root.5,6 although obturation may not necessarily be the most critical stage in root canal therapy , it should still be performed according to the highest clinical standards . gutta - percha has widely been accepted for years as the gold standard filling material to obturate root canals . furthermore , the adhesive strength between the root canal walls , endodontic sealers , and gutta - percha was found to be very weak.7 although very few materials have seriously challenged gutta - percha and sealer in majority of filling situations , research continues to find alternatives that may seal better and also mechanically reinforce compromised roots.7 the bonding concept of the root filling material is hampered by the lack of a chemical union between the polyisoprene component of gutta - percha and methacrylate - based resin sealers.8 recently , two challenging strategies have been employed to effectively reinforce the endodontically treated tooth structure . a new material resilon ( pentron clinical technologies , wallington , ct ) was introduced as a better alternative to gutta - percha . this synthetic polymer claimed not only to provide a better seal but also reinforces the tooth structure through a combination of primer , dual cure sealer and resin obturating material . the polyester chemistry containing bioactive and radiopaque fillers made it to possess better handling characteristics and look alike gutta - percha . in addition , when used in conjunction with a resin - based sealant or bonding agent it forms a monoblock within the canals that bonds to the dentinal walls , it appears logical that they have the potential to strengthen the walls against fracture.1,8 a second strategy has been employed in which an unusual resin is created by first reacting one of the isocyanato groups of a diisocyanate with the hydroxyl group of a hydroxyl terminated polybutadiene as the latter is bondable to hydrophobic polyisoprene . this is followed by grafting of a hydrophilic methacrylate functional group to the other isocyanato group of the diisocyanate producing a gutta - percha resin coating that is bondable to a methacrylate - based resin sealer . the resin coated gutta - percha is recommended to be used with a recently modified patented version of a hydrophilic methacrylate - based dual - cured resin sealer ( endorez).9 in the light of incidence of vertical root fractures associated with gutta - percha filling techniques , this study was undertaken to compare the fracture resistance of roots endodontically treated with different resin - based adhesive sealers with conventional lateral condensation technique . a total of 60 single canalled extracted maxillary anterior teeth , caries free and periodontally compromised , which was confirmed by the department of periodontology , km shah dental college , were collected and cleaned of soft tissue debris and calculus and stored in phosphate buffered saline ( pbs , ph 7.2 ) containing 0.1% sodium azide to inhibit bacterial growth for a maximum of 5 days . caries free teeth , with a single root and a single , straight canal confirmed radiographically , completely formed apices with root lengths ranging from 14 to 16 mm and bucco lingual diameter ranging from 6 to 8 mm were included for the present study . all the teeth were carefully examined under 16 magnifications with the help of digital operating microscope ( seiler dental microscope , seiler instruments inc . , st . louis , usa ) to rule out preexisting fractures and all unacceptable ones were discarded . the selected teeth were sectioned at the cement - enamel junction with a diamond disk operated on a slow speed micromotor hand piece under a constant water coolant flow . the first group served as a negative control group containing the roots neither instrumented nor obturated . in the remaining four groups , the working length was determined by introducing a size 10 k file into the canal until it exited from the apex and the final working length was set 1 mm short of that length . after the introduction of hand files and establishment of a glide path , an automated torque control endomotor with an attached reduction gear hand piece was used with profile rotary series to clean and shape the root canals using glyde as a lubricant and a chelator ( figure 1 ) . instrumentation of the root canals with profile rotary series instruments and torque control endomotor with a mounted reduction gear hand piece . profile orifice shapers ( o.s ) , profile 0.06 and profile 0.04 tapers , were the three types of instruments used in the shaping of root canals . instrumentation was initiated by the introduction of profile o.s 4 ( # 0.07/50 ) and profile o.s 3 ( # 0.06/40 ) files until the point of resistance without exerting any pressure . roots were then submitted to chemomechanical preparation with the following sequence : # 30/.06 , # 25/.06 , # 20/.06 , # 30/.04 , # 25/.04 , # 20/.04 , and finally # 30/.06 . in between each preparation 2 ml after completion of instrumentation , all specimens received a final flush of 5 ml 17% of edta irrigating solution following manufacturer s instructions followed by 5 ml of 2.5% naocl solution in order to remove the smear layer . then the specimens were randomly assigned into five experimental groups ( n = 12 ) per group as follows . neither instrumentation nor obturation was done in the samples in this group ; the root canal openings were sealed with cavit . these teeth received a root filling by cold lateral condensation technique with gutta - percha cones and endoflas fs ( zoe - based sealer ) ( sanlor dental products , usa ) . the third group received a root filling by cold lateral condensation technique with gutta - percha and ah plus ( dentsply , detrey , germeny ) an epoxy resin - based root canal sealer . the fourth group received a root filling by lateral condensation using realseal resilon points and realseal dual cure resin sealer ( pentron clinical technologies , wallingford , ct ) ( figure 2 ) . after checking the snug fit of the master resilon cone , the realseal primer was dispensed onto a microbrush available in the kit ( figure 3 ) , the excess removed by using paper points . the apical third of the master cone was coated with the sealer and placed into canal and then a size 20 finger spreader was inserted , rotated and withdrawn . an accessory resilon cone coated with a thin layer of sealer was placed into the space created by the spreader and process repeated until the canal was completely obturated . the coronal portion of the sealer was subsequently light cured for 40 s , to stabilize the material , enabling excess resilon to be removed with a hot instrument . realseal resilon points and realseal dual cure resin sealer ( pentron clinical technologies , wallingford , ct ) . the realseal primer dispensed on to a microbrush , the primer applied on the root canal walls with the help of the microbrush , excess primer was removed by using paper points . the fifth group was root canals filled with cold lateral condensation with endorez resin coated gutta - percha points and endorez resin - based sealer ( ultradent , south jordan , usa ) ( figure 4 ) . after checking the snug fit of the master endorez cone , the mixed sealer was injected into the root canal via 30-gauge navitip ( ultradent ) attached to skini syringe ( ultradent ) ( figure 5 ) . the former was inserted to 2 - 3 mm short of working length and slowly withdrawn to fill the entire canal with sealer avoiding entrapment of air . the prefit master cone ( 0.06 taper resin coated , iso no . 30 ) was then inserted into the canal to the working length , followed by the passive placement of multiple , accessory 0.02 taper , no . # 20 resin coated gutta - percha cones , to reduce the sealer volume and to avoid scraping off of the resin coating with the use of spreader / plugger . the coronal portion of the sealer was subsequently light cured for 40 s , to stabilize the material , enabling excess gutta - percha to be removed with a hot instrument . endorez resin coated gutta - percha points and endorez resin - based sealer ( ultradent , south jordan , usa ) . all roots were kept at 37c with 100% humidity for 2 weeks to allow the sealers to set completely after which the samples were prepared to be subjected to mechanical strength testing . an acrylic block with a simulated periodontal ligament created with the help of vinyl polysiloxane light body rubber base impression material was fabricated to allow for adequate stabilization of the specimens during testing procedures . after the resin had set , the blocks with embedded roots were mounted on to the loading frame of material testing system ( minneapolis , minnesota , usa ) ( figure 6 ) . a stainless steel pin simulating a size 40 spreader was manufactured and fixed into the cross head and placed directly above the root canal orifice . extreme care was taken to ensure that all the roots were embedded in an exact line parallel to the long axis of the pin in order to prevent any unusual oblique stresses on the root canal walls . the testing machine was calibrated with these components to vertically drive the pin into the canal at a cross head speed of 5 mm / min during which it penetrated the root canal and force was applied to the root until it fractured . the block with embedded root were mounted on to the loading frame of material testing system . fracture was defined as the point at which a sharp and an instantaneous drop>25% of the applied force was observed for each root and so the machine was adjusted to terminate the test when a 25% reduction of force was observed . the force applied to the root canal was recorded in the form of a graphical representation in addition to the continuous digital display of different test parameters ( graph 1 ) . throughout the test , the loads at which different root specimens fractured were recorded in newtons and the data were subjected to statistical analysis . a complete graphical representation of the entire testing procedure on a particular specimen ( axial force vs. displacement graph ) . neither instrumentation nor obturation was done in the samples in this group ; the root canal openings were sealed with cavit . these teeth received a root filling by cold lateral condensation technique with gutta - percha cones and endoflas fs ( zoe - based sealer ) ( sanlor dental products , usa ) . the third group received a root filling by cold lateral condensation technique with gutta - percha and ah plus ( dentsply , detrey , germeny ) an epoxy resin - based root canal sealer . the fourth group received a root filling by lateral condensation using realseal resilon points and realseal dual cure resin sealer ( pentron clinical technologies , wallingford , ct ) ( figure 2 ) . after checking the snug fit of the master resilon cone , the realseal primer was dispensed onto a microbrush available in the kit ( figure 3 ) , the excess removed by using paper points . the apical third of the master cone was coated with the sealer and placed into canal and then a size 20 finger spreader was inserted , rotated and withdrawn . an accessory resilon cone coated with a thin layer of sealer was placed into the space created by the spreader and process repeated until the canal was completely obturated . the coronal portion of the sealer was subsequently light cured for 40 s , to stabilize the material , enabling excess resilon to be removed with a hot instrument . realseal resilon points and realseal dual cure resin sealer ( pentron clinical technologies , wallingford , ct ) . the realseal primer dispensed on to a microbrush , the primer applied on the root canal walls with the help of the microbrush , excess primer was removed by using paper points . the fifth group was root canals filled with cold lateral condensation with endorez resin coated gutta - percha points and endorez resin - based sealer ( ultradent , south jordan , usa ) ( figure 4 ) . after checking the snug fit of the master endorez cone , the mixed sealer was injected into the root canal via 30-gauge navitip ( ultradent ) attached to skini syringe ( ultradent ) ( figure 5 ) . the former was inserted to 2 - 3 mm short of working length and slowly withdrawn to fill the entire canal with sealer avoiding entrapment of air . the prefit master cone ( 0.06 taper resin coated , iso no . 30 ) was then inserted into the canal to the working length , followed by the passive placement of multiple , accessory 0.02 taper , no . # 20 resin coated gutta - percha cones , to reduce the sealer volume and to avoid scraping off of the resin coating with the use of spreader / plugger . the coronal portion of the sealer was subsequently light cured for 40 s , to stabilize the material , enabling excess gutta - percha to be removed with a hot instrument . endorez resin coated gutta - percha points and endorez resin - based sealer ( ultradent , south jordan , usa ) . all roots were kept at 37c with 100% humidity for 2 weeks to allow the sealers to set completely after which the samples were prepared to be subjected to mechanical strength testing . an acrylic block with a simulated periodontal ligament created with the help of vinyl polysiloxane light body rubber base impression material was fabricated to allow for adequate stabilization of the specimens during testing procedures . after the resin had set , the blocks with embedded roots were mounted on to the loading frame of material testing system ( minneapolis , minnesota , usa ) ( figure 6 ) . a stainless steel pin simulating a size 40 spreader was manufactured and fixed into the cross head and placed directly above the root canal orifice . extreme care was taken to ensure that all the roots were embedded in an exact line parallel to the long axis of the pin in order to prevent any unusual oblique stresses on the root canal walls . the testing machine was calibrated with these components to vertically drive the pin into the canal at a cross head speed of 5 mm / min during which it penetrated the root canal and force was applied to the root until it fractured . the block with embedded root fracture was defined as the point at which a sharp and an instantaneous drop>25% of the applied force was observed for each root and so the machine was adjusted to terminate the test when a 25% reduction of force was observed . the force applied to the root canal was recorded in the form of a graphical representation in addition to the continuous digital display of different test parameters ( graph 1 ) . throughout the test , the loads at which different root specimens fractured were recorded in newtons and the data were subjected to statistical analysis . a complete graphical representation of the entire testing procedure on a particular specimen ( axial force vs. displacement graph ) . firstly one - way anova test followed by post - hoc scheffe multiple comparison tests on both the variables of length and faciolingual diameters of different samples were performed and it was concluded that all the samples of the experimental groups were completely well - balanced concerning the defined constraints . the results of fracture resistance obtained were measured in newtons for each specimen has been tabulated in ( table 1 ) . the descriptive statistics of loads of various specimens of different groups are mentioned in ( table 2 ) . descriptive statistics of the loads to fracture different samples . a graphical representation of the mean load values to fracture roots obturated with different sealers is shown below ( graph 2 ) . a graphical representation of the mean load values to fracture roots obturated with different sealers . considering this statistical situation a more detailed analysis one - way anova test was called for ( table 3 ) . further this was substantiated with multiple comparisons through post - hoc tukey hsd multiple comparison test ( table 4 ) . one - way anova revealed highly significant difference between groups ( p < 0.0001 ) . statistically significant difference is noted in fracture resistance values of control group and certain test groups i.e. , the control group displayed significantly higher values than both the gutta - percha groups ( group ii and iii ) ( p < 0.05 ) . while groups iv and v displayed values lower than the control group but were statistically insignificant ( p > 0.05 ) . among the test groups , resilon group ( group iv ) and endorez group ( group v ) displayed significantly higher values than the gutta - percha groups ( group ii - endoflas and group iii - ah plus ) ( p < 0.05 ) . furthermore , there was no statistically significant difference between the values of group iv and group v ( p > 0.05 ) . similarly , there was no statistically significant difference between the values of group ii and group iii ( p > 0.05 ) . the primary goal of endodontics is not only to restore the tooth structure but also to increase the inherent strength of the remaining tooth structure . although the use of gutta - percha with an insoluble root canal sealer can be seen as a gold standard of root canal fillings , the ability of these materials to reinforce an endodontically treated root is discussed with controversy . certain resin - based materials have been proposed since long to reinforce the endodontically treated teeth like diaket,10 hydron,11 ketac endo aplicap12 ah 26 , ah plus13,14 endoresin.15,16 the development of bonded obturating materials is in congruence with the efforts to provide a more effective seal apically as well as coronally . the adhesion between dental structures and resin - based sealers is the result of a physicochemical interaction across the interface , allowing the union between filling material and root canal wall.17,18 this process is important in static and dynamic situations . in static circumstances , the adhesion eliminates spaces that allow the infiltration of fluids into the sealer / dentine interface.19 in dynamic situations , the adhesion is necessary to avoid the sealer dislodgment during operative procedures . therefore , the endodontic filling materials may enhance the ability of root filled teeth to resist fracture.20,21 because the resin core , sealant , and the dentinal wall all are attached , it appears logical that they have the potential to strengthen the walls against fracture . however , very few studies have been conducted on this aspect to support the hypothesis regarding the root reinforcement ability of the newer resin - based systems . all the root canals were enlarged by a single operator to minimize operator variation.13 canal shape after preparation with hand files can be quite irregular.22 from a fracture mechanics point of view , the presence of structural defects , cracks or canal irregularities is likely to play a major role in determining fracture strength.23 rotary niti canal preparation using profile rotary instruments did not reduce fracture susceptibility of the root and also the roots were significantly weakened by the preparation with greater taper instruments.24 during the root canal preparation freshly prepared 1% sodium hypochlorite was used as an irrigant as it is the most commonly used irrigant , and a low concentration was used to minimize the adverse effect on dentin mechanical properties.1 final rinse was done with edta followed by naocl to enhance the bonding of the materials tested to the dentinal surface of the root . this was done in accordance with the protocol recommended by weiger et al . to use edta followed by naocl , which seemed to optimize adhesion of sealers to the root canal walls.25 root canal obturation has been implicated as the major cause of vertical root fracture . in lateral condensation , the strain is generated by the wedging effect of the spreader because it laterally compacts the gutta - percha and adapts it to canal wall while vertical condensation creates strains as the mass of gutta - percha is compacted apically with pluggers under consistent vertical load.5,26 however , lateral condensation technique was used in this study because it is a more widely recommended and a proven classic technique,7 which facilitates comparison with previous studies.1,27 furthermore , the principal reason is that endorez system is only provided in the form of cones and not yet available in any other form for vertical or thermal condensation.3 strength testing is the methodology that has been used to study the influence of filling materials on the fracture resistance of teeth submitted to root canal treatment9,12,28 as performed in this study . in this study , the force was applied along the long axis of the root with a stainless steel pin , which produced root fracture when contact was made between the pin and the walls of the canal opening . the method adapted in this study to fracture the root specimens was chosen because it provided force distribution from inside the root canal and fractures occurring as a result of forces transmitted via the obturating material.5 this resembled root fracture of endodontic origin or from a post.27 stresses generated from inside the root canal were transmitted through the root to the surface where the interdentin bonding failed.29 the test was terminated after a 25% drop in the maximum force recorded similar to that used in the previous studies.1,8,13 the method presented in this study for measuring the fracture strength of obturating material has proven to be effective and reproducible . it is simple and easy to duplicate and reliable in accordance with the results of previously published findings . the following conclusions were drawn from this study : intact roots have greater fracture resistance than that of the instrumented roots.intact roots have significantly greater fracture resistance than the gutta - percha filled roots.the roots obturated with both the newer resin - based adhesive systems i.e. realseal and endorez have significantly greater fracture resistance than the gutta - percha filled roots.there is no statistically significant difference in fracture resistance of the roots obturated with gutta - percha / endoflas fs and gutta - percha / ah plus sealer.there is no statistically significant difference in fracture resistance of roots obturated with resilon / realseal sealer and the intact roots.there is no statistically significant difference in fracture resistance of roots obturated with resin coated gutta - percha / endorez sealer and the intact roots.there is no statistically significant difference in fracture resistance of roots obturated with resilon / realseal sealer , and resin coated gutta - percha / endorez sealer . intact roots have significantly greater fracture resistance than the gutta - percha filled roots . the roots obturated with both the newer resin - based adhesive systems i.e. realseal and endorez have significantly greater fracture resistance than the gutta - percha filled roots . there is no statistically significant difference in fracture resistance of the roots obturated with gutta - percha / endoflas fs and gutta - percha / ah plus sealer . there is no statistically significant difference in fracture resistance of roots obturated with resilon / realseal sealer and the intact roots . there is no statistically significant difference in fracture resistance of roots obturated with resin coated gutta - percha / endorez sealer and the intact roots . there is no statistically significant difference in fracture resistance of roots obturated with resilon / realseal sealer , and resin coated gutta - percha / endorez sealer . although the present results concerning the adhesive root canal filling materials realseal and endorez to reinforce the endodontically treated roots are very promising some care should be taken in the transfer of these findings to the long term clinical situation . further in vivo and biocompatibility tests involving newer resin - based systems will be necessary to determine whether the results in vitro will be validated . clinical long - term studies are necessary to collect evidence - based data to support the confident use of these materials .

background : to compare fracture resistance of endodontically treated teeth obturated with different resin - based adhesive sealers with a conventional obturation technique.materials and methods : a total of 60 single canaled teeth were divided into five groups . the first group was taken as a negative control . the rest of the groups were shaped using profile rotary files ( dentsply maillefer , ballaigues , switzerland ) . the second group was obturated with gutta - percha and a zoe - based sealer endoflas fs ( sanlor dental products , usa ) . the third group was obturated with gutta - percha and an epoxy - based sealer ah plus ( dentsply , detrey , germany ) . the fourth group was obturated with resilon ( pentron clinical technologies , wallingford , ct ) and realseal sealer ( pentron clinical technologies ) . the fifth group was obturated with endorez points and endorez sealer ( both from ultradent , south jordan , ut ) . roots were then embedded into acrylic blocks and were then fixed into a material testing system and loaded with a stainless steel pin with a crosshead speed of 5 mm / min until fracture . the load at which the specimen fractured was recorded in newtons.results:it was found that forces at fracture were statistically significant for the newer resin systems , resilon , and endorez.conclusion:it was concluded that roots obturated with newer resin systems ( resilon and endorez ) enhanced the root strength almost up to the level of the intact roots .

Introduction Materials and Methods Group I: Control group Group II: Endoflas FS group Group III: AH Plus group Group IV: RealSeal group Group V: EndoREZ group Observations and Results Discussion Conclusions

the major objectives of root canal therapy are removal of the pathologic pulp , shaping and cleaning of the root canal system , disinfection of the contaminated root canals and three dimensional obturation to prevent reinfection.1 the success of endodontic therapy depends on adequate access , thorough biomechanical preparation and proper obturation of the root canal system . endodontically treated teeth are widely considered to be more susceptible to fracture than vital teeth . the reasons most often reported have been the removal of tooth structure during endodontic therapy , dehydration of dentin after endodontic therapy and excessive pressure during obturation.2,3 the strength of the endodontically treated teeth is directly related to the method of canal preparation and the amount of remaining sound tooth structure . it is observed that the greatest incidence of vertical root fracture occurs in teeth that have undergone endodontic therapy.4 vertical root fracture is a longitudinal fracture of the root , extending throughout the entire thickness of dentin from the root canal to the periodontium . it is of serious clinical concern and has an unfavorable prognosis resulting in extraction of the tooth or resection of the affected root.5,6 although obturation may not necessarily be the most critical stage in root canal therapy , it should still be performed according to the highest clinical standards . gutta - percha has widely been accepted for years as the gold standard filling material to obturate root canals . furthermore , the adhesive strength between the root canal walls , endodontic sealers , and gutta - percha was found to be very weak.7 although very few materials have seriously challenged gutta - percha and sealer in majority of filling situations , research continues to find alternatives that may seal better and also mechanically reinforce compromised roots.7 the bonding concept of the root filling material is hampered by the lack of a chemical union between the polyisoprene component of gutta - percha and methacrylate - based resin sealers.8 recently , two challenging strategies have been employed to effectively reinforce the endodontically treated tooth structure . a new material resilon ( pentron clinical technologies , wallington , ct ) was introduced as a better alternative to gutta - percha . the polyester chemistry containing bioactive and radiopaque fillers made it to possess better handling characteristics and look alike gutta - percha . in addition , when used in conjunction with a resin - based sealant or bonding agent it forms a monoblock within the canals that bonds to the dentinal walls , it appears logical that they have the potential to strengthen the walls against fracture.1,8 a second strategy has been employed in which an unusual resin is created by first reacting one of the isocyanato groups of a diisocyanate with the hydroxyl group of a hydroxyl terminated polybutadiene as the latter is bondable to hydrophobic polyisoprene . this is followed by grafting of a hydrophilic methacrylate functional group to the other isocyanato group of the diisocyanate producing a gutta - percha resin coating that is bondable to a methacrylate - based resin sealer . the resin coated gutta - percha is recommended to be used with a recently modified patented version of a hydrophilic methacrylate - based dual - cured resin sealer ( endorez).9 in the light of incidence of vertical root fractures associated with gutta - percha filling techniques , this study was undertaken to compare the fracture resistance of roots endodontically treated with different resin - based adhesive sealers with conventional lateral condensation technique . a total of 60 single canalled extracted maxillary anterior teeth , caries free and periodontally compromised , which was confirmed by the department of periodontology , km shah dental college , were collected and cleaned of soft tissue debris and calculus and stored in phosphate buffered saline ( pbs , ph 7.2 ) containing 0.1% sodium azide to inhibit bacterial growth for a maximum of 5 days . caries free teeth , with a single root and a single , straight canal confirmed radiographically , completely formed apices with root lengths ranging from 14 to 16 mm and bucco lingual diameter ranging from 6 to 8 mm were included for the present study . louis , usa ) to rule out preexisting fractures and all unacceptable ones were discarded . the selected teeth were sectioned at the cement - enamel junction with a diamond disk operated on a slow speed micromotor hand piece under a constant water coolant flow . the first group served as a negative control group containing the roots neither instrumented nor obturated . after the introduction of hand files and establishment of a glide path , an automated torque control endomotor with an attached reduction gear hand piece was used with profile rotary series to clean and shape the root canals using glyde as a lubricant and a chelator ( figure 1 ) . instrumentation of the root canals with profile rotary series instruments and torque control endomotor with a mounted reduction gear hand piece . roots were then submitted to chemomechanical preparation with the following sequence : # 30/.06 , # 25/.06 , # 20/.06 , # 30/.04 , # 25/.04 , # 20/.04 , and finally # 30/.06 . these teeth received a root filling by cold lateral condensation technique with gutta - percha cones and endoflas fs ( zoe - based sealer ) ( sanlor dental products , usa ) . the third group received a root filling by cold lateral condensation technique with gutta - percha and ah plus ( dentsply , detrey , germeny ) an epoxy resin - based root canal sealer . the fourth group received a root filling by lateral condensation using realseal resilon points and realseal dual cure resin sealer ( pentron clinical technologies , wallingford , ct ) ( figure 2 ) . the coronal portion of the sealer was subsequently light cured for 40 s , to stabilize the material , enabling excess resilon to be removed with a hot instrument . realseal resilon points and realseal dual cure resin sealer ( pentron clinical technologies , wallingford , ct ) . the realseal primer dispensed on to a microbrush , the primer applied on the root canal walls with the help of the microbrush , excess primer was removed by using paper points . the fifth group was root canals filled with cold lateral condensation with endorez resin coated gutta - percha points and endorez resin - based sealer ( ultradent , south jordan , usa ) ( figure 4 ) . after checking the snug fit of the master endorez cone , the mixed sealer was injected into the root canal via 30-gauge navitip ( ultradent ) attached to skini syringe ( ultradent ) ( figure 5 ) . # 20 resin coated gutta - percha cones , to reduce the sealer volume and to avoid scraping off of the resin coating with the use of spreader / plugger . the coronal portion of the sealer was subsequently light cured for 40 s , to stabilize the material , enabling excess gutta - percha to be removed with a hot instrument . endorez resin coated gutta - percha points and endorez resin - based sealer ( ultradent , south jordan , usa ) . all roots were kept at 37c with 100% humidity for 2 weeks to allow the sealers to set completely after which the samples were prepared to be subjected to mechanical strength testing . an acrylic block with a simulated periodontal ligament created with the help of vinyl polysiloxane light body rubber base impression material was fabricated to allow for adequate stabilization of the specimens during testing procedures . after the resin had set , the blocks with embedded roots were mounted on to the loading frame of material testing system ( minneapolis , minnesota , usa ) ( figure 6 ) . a stainless steel pin simulating a size 40 spreader was manufactured and fixed into the cross head and placed directly above the root canal orifice . extreme care was taken to ensure that all the roots were embedded in an exact line parallel to the long axis of the pin in order to prevent any unusual oblique stresses on the root canal walls . the testing machine was calibrated with these components to vertically drive the pin into the canal at a cross head speed of 5 mm / min during which it penetrated the root canal and force was applied to the root until it fractured . the block with embedded root were mounted on to the loading frame of material testing system . fracture was defined as the point at which a sharp and an instantaneous drop>25% of the applied force was observed for each root and so the machine was adjusted to terminate the test when a 25% reduction of force was observed . the force applied to the root canal was recorded in the form of a graphical representation in addition to the continuous digital display of different test parameters ( graph 1 ) . throughout the test , the loads at which different root specimens fractured were recorded in newtons and the data were subjected to statistical analysis . these teeth received a root filling by cold lateral condensation technique with gutta - percha cones and endoflas fs ( zoe - based sealer ) ( sanlor dental products , usa ) . the third group received a root filling by cold lateral condensation technique with gutta - percha and ah plus ( dentsply , detrey , germeny ) an epoxy resin - based root canal sealer . the fourth group received a root filling by lateral condensation using realseal resilon points and realseal dual cure resin sealer ( pentron clinical technologies , wallingford , ct ) ( figure 2 ) . the coronal portion of the sealer was subsequently light cured for 40 s , to stabilize the material , enabling excess resilon to be removed with a hot instrument . realseal resilon points and realseal dual cure resin sealer ( pentron clinical technologies , wallingford , ct ) . the realseal primer dispensed on to a microbrush , the primer applied on the root canal walls with the help of the microbrush , excess primer was removed by using paper points . the fifth group was root canals filled with cold lateral condensation with endorez resin coated gutta - percha points and endorez resin - based sealer ( ultradent , south jordan , usa ) ( figure 4 ) . after checking the snug fit of the master endorez cone , the mixed sealer was injected into the root canal via 30-gauge navitip ( ultradent ) attached to skini syringe ( ultradent ) ( figure 5 ) . # 20 resin coated gutta - percha cones , to reduce the sealer volume and to avoid scraping off of the resin coating with the use of spreader / plugger . the coronal portion of the sealer was subsequently light cured for 40 s , to stabilize the material , enabling excess gutta - percha to be removed with a hot instrument . endorez resin coated gutta - percha points and endorez resin - based sealer ( ultradent , south jordan , usa ) . all roots were kept at 37c with 100% humidity for 2 weeks to allow the sealers to set completely after which the samples were prepared to be subjected to mechanical strength testing . an acrylic block with a simulated periodontal ligament created with the help of vinyl polysiloxane light body rubber base impression material was fabricated to allow for adequate stabilization of the specimens during testing procedures . after the resin had set , the blocks with embedded roots were mounted on to the loading frame of material testing system ( minneapolis , minnesota , usa ) ( figure 6 ) . a stainless steel pin simulating a size 40 spreader was manufactured and fixed into the cross head and placed directly above the root canal orifice . extreme care was taken to ensure that all the roots were embedded in an exact line parallel to the long axis of the pin in order to prevent any unusual oblique stresses on the root canal walls . the testing machine was calibrated with these components to vertically drive the pin into the canal at a cross head speed of 5 mm / min during which it penetrated the root canal and force was applied to the root until it fractured . the block with embedded root fracture was defined as the point at which a sharp and an instantaneous drop>25% of the applied force was observed for each root and so the machine was adjusted to terminate the test when a 25% reduction of force was observed . the force applied to the root canal was recorded in the form of a graphical representation in addition to the continuous digital display of different test parameters ( graph 1 ) . throughout the test , the loads at which different root specimens fractured were recorded in newtons and the data were subjected to statistical analysis . firstly one - way anova test followed by post - hoc scheffe multiple comparison tests on both the variables of length and faciolingual diameters of different samples were performed and it was concluded that all the samples of the experimental groups were completely well - balanced concerning the defined constraints . a graphical representation of the mean load values to fracture roots obturated with different sealers is shown below ( graph 2 ) . a graphical representation of the mean load values to fracture roots obturated with different sealers . statistically significant difference is noted in fracture resistance values of control group and certain test groups i.e. , the control group displayed significantly higher values than both the gutta - percha groups ( group ii and iii ) ( p < 0.05 ) . among the test groups , resilon group ( group iv ) and endorez group ( group v ) displayed significantly higher values than the gutta - percha groups ( group ii - endoflas and group iii - ah plus ) ( p < 0.05 ) . although the use of gutta - percha with an insoluble root canal sealer can be seen as a gold standard of root canal fillings , the ability of these materials to reinforce an endodontically treated root is discussed with controversy . certain resin - based materials have been proposed since long to reinforce the endodontically treated teeth like diaket,10 hydron,11 ketac endo aplicap12 ah 26 , ah plus13,14 endoresin.15,16 the development of bonded obturating materials is in congruence with the efforts to provide a more effective seal apically as well as coronally . the adhesion between dental structures and resin - based sealers is the result of a physicochemical interaction across the interface , allowing the union between filling material and root canal wall.17,18 this process is important in static and dynamic situations . however , very few studies have been conducted on this aspect to support the hypothesis regarding the root reinforcement ability of the newer resin - based systems . all the root canals were enlarged by a single operator to minimize operator variation.13 canal shape after preparation with hand files can be quite irregular.22 from a fracture mechanics point of view , the presence of structural defects , cracks or canal irregularities is likely to play a major role in determining fracture strength.23 rotary niti canal preparation using profile rotary instruments did not reduce fracture susceptibility of the root and also the roots were significantly weakened by the preparation with greater taper instruments.24 during the root canal preparation freshly prepared 1% sodium hypochlorite was used as an irrigant as it is the most commonly used irrigant , and a low concentration was used to minimize the adverse effect on dentin mechanical properties.1 final rinse was done with edta followed by naocl to enhance the bonding of the materials tested to the dentinal surface of the root . in lateral condensation , the strain is generated by the wedging effect of the spreader because it laterally compacts the gutta - percha and adapts it to canal wall while vertical condensation creates strains as the mass of gutta - percha is compacted apically with pluggers under consistent vertical load.5,26 however , lateral condensation technique was used in this study because it is a more widely recommended and a proven classic technique,7 which facilitates comparison with previous studies.1,27 furthermore , the principal reason is that endorez system is only provided in the form of cones and not yet available in any other form for vertical or thermal condensation.3 strength testing is the methodology that has been used to study the influence of filling materials on the fracture resistance of teeth submitted to root canal treatment9,12,28 as performed in this study . in this study , the force was applied along the long axis of the root with a stainless steel pin , which produced root fracture when contact was made between the pin and the walls of the canal opening . the method adapted in this study to fracture the root specimens was chosen because it provided force distribution from inside the root canal and fractures occurring as a result of forces transmitted via the obturating material.5 this resembled root fracture of endodontic origin or from a post.27 stresses generated from inside the root canal were transmitted through the root to the surface where the interdentin bonding failed.29 the test was terminated after a 25% drop in the maximum force recorded similar to that used in the previous studies.1,8,13 the method presented in this study for measuring the fracture strength of obturating material has proven to be effective and reproducible . the following conclusions were drawn from this study : intact roots have greater fracture resistance than that of the instrumented roots.intact roots have significantly greater fracture resistance than the gutta - percha filled roots.the roots obturated with both the newer resin - based adhesive systems i.e. realseal and endorez have significantly greater fracture resistance than the gutta - percha filled roots.there is no statistically significant difference in fracture resistance of the roots obturated with gutta - percha / endoflas fs and gutta - percha / ah plus sealer.there is no statistically significant difference in fracture resistance of roots obturated with resilon / realseal sealer and the intact roots.there is no statistically significant difference in fracture resistance of roots obturated with resin coated gutta - percha / endorez sealer and the intact roots.there is no statistically significant difference in fracture resistance of roots obturated with resilon / realseal sealer , and resin coated gutta - percha / endorez sealer . intact roots have significantly greater fracture resistance than the gutta - percha filled roots . the roots obturated with both the newer resin - based adhesive systems i.e. realseal and endorez have significantly greater fracture resistance than the gutta - percha filled roots . there is no statistically significant difference in fracture resistance of the roots obturated with gutta - percha / endoflas fs and gutta - percha / ah plus sealer . there is no statistically significant difference in fracture resistance of roots obturated with resilon / realseal sealer and the intact roots . there is no statistically significant difference in fracture resistance of roots obturated with resin coated gutta - percha / endorez sealer and the intact roots . there is no statistically significant difference in fracture resistance of roots obturated with resilon / realseal sealer , and resin coated gutta - percha / endorez sealer . although the present results concerning the adhesive root canal filling materials realseal and endorez to reinforce the endodontically treated roots are very promising some care should be taken in the transfer of these findings to the long term clinical situation . further in vivo and biocompatibility tests involving newer resin - based systems will be necessary to determine whether the results in vitro will be validated .

the bogalusa heart study is being conducted in the semirural , biracial ( 65% white and 35% black ) community of bogalusa , louisiana . between 1976 and 1994 , in addition , eight cross - sectional surveys were conducted between 1978 and 2002 using young adults who had been examined previously as children . a detailed description of the study design , participation , and protocols was published elsewhere ( 12 ) . this panel design , based on repeated cross - sectional examinations conducted approximately every 34 years , resulted in serial observations from childhood to young adulthood , allowing longitudinal analyses . subjects from six cross - sectional studies of children who participated in at least one of the 14 cross - sectional surveys of children and adults were eligible for this retrospective cohort study . of these , a total of 1,988 fasting subjects ( 68% white and 43% male ) were selected from the last three surveys of adults from 19952002 . at the baseline examination , the children with a history of treatment of diabetes or those who had a fasting glucose level 7 mmol / l were excluded . at the initial screening , the mean sd age was 10.9 4.0 years ( range 418 ) . at the most recent screening , the number of screenings and observations between childhood and adulthood ranged from 2 to 9 . in all , 91% of subjects were screened 3 times and 63% were screened 46 times , with a total of 9,232 sets of observations . on the basis of the data at the last survey , adult subjects were classified as normoglycemic , pre - diabetic , or diabetic according to the american diabetes association criteria ( 7 ) . individuals were considered normoglycemic ( n = 1,838 ) if they had a fasting glucose level < 5.6 mmol / l , pre - diabetic ( n = 90 ) if their fasting glucose level was between 5.6 and 6.9 mmol / l , or diabetic ( n = 60 ) if 1 ) their fasting glucose level was 7 the institutional review board approved consent forms used for these surveys , and informed consent was obtained from adult study participants and from parents or guardians of children . identical protocols were used by trained examiners , nurses , and technicians across all surveys ; procedures for general examination were reported previously ( 13 ) . in brief , subjects were instructed to fast for 12 h before the screening , with compliance ascertained by an interview on the day of examination . serum samples were obtained from antecubital venous blood and kept at 4c until analysis the following day . each screening day , on a random 10% subsample , a second blood sample was collected , labeled , and analyzed in a blind fashion to estimate the measurement error . all measurements were made in replicate , and mean values were used . in the longitudinal analysis , bmi ( weight in kilograms divided by the square of height in meters ) was used as a measure of overall adiposity and subscapular skinfold measured with lange skinfold calipers was used for truncal fatness . blood pressure measurements were obtained on the right arm with the subjects in a relaxed , sitting position . systolic and diastolic blood pressures were recorded at the first and fifth korotkoff phases using a mercury sphygmomanometer . blood pressure levels were reported as the mean of six replicate readings , with three taken by each of two randomly assigned and trained observers . mean arterial pressure ( map ) , calculated as diastolic blood pressure plus one - third pulse pressure , was used in the analysis . from 1973 to 1986 , cholesterol and triglycerides levels were measured using chemical procedures on a autoanalyzer ii ( technicon ) according to the laboratory manual of the lipid research clinics program . these variables were determined by enzymatic procedures on a vp instrument ( abbott ) between 1987 and 1996 and on the hitachi 902 automatic analyzer ( roche ) afterward . both chemical and enzymatic procedures met the performance requirements of the lipid standardization program of the centers for disease control and prevention , which routinely monitored the precision and accuracy of cholesterol , triglyceride , and hdl cholesterol measurements since the beginning of this study . measurements on the quality - control samples assigned by the agency showed no consistent bias over time within or between surveys . serum lipoprotein cholesterol levels were analyzed by using a combination of heparin - calcium precipitation and agar - agarose gel electrophoresis procedures ( 14 ) . the intraclass correlation coefficients between the blind duplicate ( 10% random sample ) values ranged from 0.86 to 0.98 for hdl cholesterol , from 0.86 to 0.98 for ldl cholesterol , and from 0.88 to 0.99 for triglycerides . on the basis of the national cholesterol education program adult treatment panel iii guidelines ( 15 ) , mmol / l ) , high triglycerides ( 2.26 mmol / l ) , or low hdl cholesterol ( < 1.03 mmol / l ) or if they were taking medication for dyslipidemia and as having metabolic syndrome if they had at least three risk factors consistent with that diagnosis . from 1976 to 1991 , plasma glucose was measured by a glucose oxidase method using a glucose analyzer ( beckman ) . since then , it has been measured enzymatically as part of a multichemistry ( sma20 ) profile . plasma immunoreactive insulin levels were measured by a commercial radioimmunoassay kit ( phadebas ; pharmacia ) . the intraclass correlation coefficients between the blind duplicate values ranged from 0.94 to 0.98 for insulin and from 0.86 to 0.98 for glucose . > 108 pmol / l , a value considered indicative of insulin resistance in normoglycemic subjects ( 16 ) . in addition , an index of insulin resistance was calculated according to the homeostasis model assessment for insulin resistance formula : homa - ir = [ insulin ( microunits per milliliter ) glucose ( millimoles per liter)/22.5 ] . all of the statistical analyses were performed with sas ( version 9.1 ; sas institute , cary , nc ) . in the analyses , the race - sex groups were combined to increase statistical power and to simplify the presentation . values for triglycerides , glucose , and insulin and homa - ir variables used in the analyses were log transformed to improve normality as necessary . mean levels of risk variables in 4- to 11- , 12- to 18- , and 19-year - old age - groups corresponding with the preadolescence , adolescence , and adulthood period were compared by their diabetes status in adulthood ( normoglycemic versus pre - diabetic , normoglycemic versus diabetic , and pre - diabetic versus diabetic ) . a single measurement per subject was used to calculate mean levels of risk variable within age - groups . general linear models were used to examine impaired glucose regulation differences in risk factor variables , adjusted for age , race , and sex . by using data from the last survey in adulthood , significant differences in the prevalence of pre - diabetes and diabetes by race and sex and metabolic syndrome and its related cardiovascular risk factors by diabetes status were tested by pearson 's test . multivariate analyses ( generalized estimation equation method ) were used to determine which longitudinal changes in risk variables since childhood predicted adult diabetes status . the model included bmi , map , hdl cholesterol , ldl cholesterol , triglycerides , glucose , and insulin , adjusted for age , age , race , sex , and sex- by - race interaction , as applicable . nonsignificant terms ( p > 0.05 ) were removed from the model by backward stepwise procedure . identical protocols were used by trained examiners , nurses , and technicians across all surveys ; procedures for general examination were reported previously ( 13 ) . in brief , subjects were instructed to fast for 12 h before the screening , with compliance ascertained by an interview on the day of examination . serum samples were obtained from antecubital venous blood and kept at 4c until analysis the following day . each screening day , on a random 10% subsample , a second blood sample was collected , labeled , and analyzed in a blind fashion to estimate the measurement error . all measurements were made in replicate , and mean values were used . in the longitudinal analysis , bmi ( weight in kilograms divided by the square of height in meters ) was used as a measure of overall adiposity and subscapular skinfold measured with lange skinfold calipers was used for truncal fatness . blood pressure measurements were obtained on the right arm with the subjects in a relaxed , sitting position . systolic and diastolic blood pressures were recorded at the first and fifth korotkoff phases using a mercury sphygmomanometer . blood pressure levels were reported as the mean of six replicate readings , with three taken by each of two randomly assigned and trained observers . mean arterial pressure ( map ) , calculated as diastolic blood pressure plus one - third pulse pressure , was used in the analysis . from 1973 to 1986 , cholesterol and triglycerides levels were measured using chemical procedures on a autoanalyzer ii ( technicon ) according to the laboratory manual of the lipid research clinics program . these variables were determined by enzymatic procedures on a vp instrument ( abbott ) between 1987 and 1996 and on the hitachi 902 automatic analyzer ( roche ) afterward . both chemical and enzymatic procedures met the performance requirements of the lipid standardization program of the centers for disease control and prevention , which routinely monitored the precision and accuracy of cholesterol , triglyceride , and hdl cholesterol measurements since the beginning of this study . measurements on the quality - control samples assigned by the agency showed no consistent bias over time within or between surveys . serum lipoprotein cholesterol levels were analyzed by using a combination of heparin - calcium precipitation and agar - agarose gel electrophoresis procedures ( 14 ) . the intraclass correlation coefficients between the blind duplicate ( 10% random sample ) values ranged from 0.86 to 0.98 for hdl cholesterol , from 0.86 to 0.98 for ldl cholesterol , and from 0.88 to 0.99 for triglycerides . on the basis of the national cholesterol education program adult treatment panel iii guidelines ( 15 ) , mmol / l ) , or low hdl cholesterol ( < 1.03 mmol / l ) or if they were taking medication for dyslipidemia and as having metabolic syndrome if they had at least three risk factors consistent with that diagnosis . from 1976 to 1991 , plasma glucose was measured by a glucose oxidase method using a glucose analyzer ( beckman ) . since then , it has been measured enzymatically as part of a multichemistry ( sma20 ) profile . plasma immunoreactive insulin levels were measured by a commercial radioimmunoassay kit ( phadebas ; pharmacia ) . the intraclass correlation coefficients between the blind duplicate values ranged from 0.94 to 0.98 for insulin and from 0.86 to 0.98 for glucose . hyperinsulinemia was arbitrarily defined as a fasting value > 108 pmol / l , a value considered indicative of insulin resistance in normoglycemic subjects ( 16 ) . in addition , an index of insulin resistance was calculated according to the homeostasis model assessment for insulin resistance formula : homa - ir = [ insulin ( microunits per milliliter ) glucose ( millimoles per liter)/22.5 ] . all of the statistical analyses were performed with sas ( version 9.1 ; sas institute , cary , nc ) . in the analyses , the race - sex groups were combined to increase statistical power and to simplify the presentation . values for triglycerides , glucose , and insulin and homa - ir variables used in the analyses were log transformed to improve normality as necessary . mean levels of risk variables in 4- to 11- , 12- to 18- , and 19-year - old age - groups corresponding with the preadolescence , adolescence , and adulthood period were compared by their diabetes status in adulthood ( normoglycemic versus pre - diabetic , normoglycemic versus diabetic , and pre - diabetic versus diabetic ) . a single measurement per subject was used to calculate mean levels of risk variable within age - groups . general linear models were used to examine impaired glucose regulation differences in risk factor variables , adjusted for age , race , and sex . by using data from the last survey in adulthood , significant differences in the prevalence of pre - diabetes and diabetes by race and sex and metabolic syndrome and its related cardiovascular risk factors by diabetes status were tested by pearson 's test . multivariate analyses ( generalized estimation equation method ) were used to determine which longitudinal changes in risk variables since childhood predicted adult diabetes status . the model included bmi , map , hdl cholesterol , ldl cholesterol , triglycerides , glucose , and insulin , adjusted for age , age , race , sex , and sex- by - race interaction , as applicable . nonsignificant terms ( p > 0.05 ) were removed from the model by backward stepwise procedure . mean levels of anthropometric , hemodynamic , and metabolic variables at baseline are presented in table 1 by diabetes status . the pre - diabetic group had significantly higher age , ldl cholesterol , and glucose than the normoglycemic group . with the exceptions of diastolic blood pressure and ldl cholesterol , the diabetic group versus normoglycemic group showed significantly higher age , subscapular skinfold , bmi , systolic blood pressure , map , triglycerides , glucose , insulin , and homa - ir and lower hdl cholesterol . compared with the pre - diabetic group , diabetic subjects had significantly higher subscapular skinfold , bmi , and triglycerides and lower hdl cholesterol . the prevalence of metabolic syndrome and its related cardiovascular risk factors in adulthood at the last survey is shown in table 1 by diabetes status . compared with the normoglycemic group , obesity in terms of excess generalized adiposity ( bmi ) , hypertension , dyslipidemia , and hyperinsulinemia ( indicative of insulin resistance ) , and metabolic syndrome were significantly more prevalent among the pre - diabetic ( except hdl cholesterol ) and diabetic ( except ldl cholesterol ) groups . compared with the pre - diabetic group , high risk levels of hdl cholesterol were more prevalent among the diabetic group . the overall prevalence of the metabolic syndrome was 18.4% in the total sample ( data not shown ) . mean levels of variables of metabolic syndrome in childhood ( 411 years ) , adolescence ( 1218 years ) , and adulthood ( 1944 years ) are shown in fig . 1 by diabetes status in adulthood . the pre - diabetic group versus normoglycemic individuals showed higher levels of glucose and subscapular skinfold from childhood through adulthood ; higher levels of ldl cholesterol , insulin , and homa - ir in adolescence and adulthood ; and higher levels of bmi , map , and triglycerides in adulthood . on the other hand , the diabetic group versus the normoglycemic group had higher levels of subscapular skinfold , bmi , triglycerides , glucose , insulin , and homa - ir and lower levels of hdl cholesterol from childhood through adulthood and higher levels of map in adolescence and adulthood . in addition , the diabetic group versus the pre - diabetic group had higher levels of subscapular skinfold in adolescence and adulthood , higher bmi in adolescence , and lower levels of hdl cholesterol and ldl cholesterol and higher levels of glucose and homa - ir in adulthood . as shown in supplementary fig . 1 of the online appendix ( available at http://dx.doi.org/10.2337/dc08-0898 ) , white male subjects had a significantly higher prevalence of pre - diabetes than white female subjects . with respect to diabetes , white male versus white female subjects and black female versus white female subjects had a significantly greater prevalence . male versus female subjects with pre - diabetes and black versus white subjects with diabetes had a significantly higher prevalence ; overall prevalences of pre - diabetes and diabetes were 4.5 and 3.0% in the total sample , respectively ( data not shown ) . longitudinal rates of change in risk variables of metabolic syndrome by diabetes status were observed ( supplementary table 1 of the online appendix ) . compared with the normoglycemic group , the rates of increase in subscapular skinfold , bmi , triglycerides , glucose , insulin , and homa - ir were significantly higher among the pre - diabetic and diabetic groups and the rates of increase in diastolic blood pressure and map were significantly higher among the diabetic group . independent relationships between adverse longitudinal changes in risk variables since childhood and adult pre - diabetes and diabetes conditions were determined in the multivariate models , presented in table 2 . adverse changes in ldl cholesterol and glucose were independently associated with pre - diabetes status and adverse changes in bmi , hdl cholesterol , and glucose with diabetes status . when homa - ir index , instead of glucose and insulin , was included in the models , homa - ir index , ldl cholesterol , and glucose were significant independent predictors in the pre - diabetic group and homa - ir , bmi , and hdl cholesterol were predictors in the diabetic group ; further , alternate multivariate analyses using subscapular skinfold , instead of bmi , gave essentially identical results ( data not shown ) . these observations explore the natural history of impaired glucose regulation and diabetes status in a community - based population of children free from a selection bias monitored longitudinally over a period of 21 years . data linked the conditions of pre - diabetes and type 2 diabetes in young adults with concurrent longitudinal changes in some metabolic syndrome risk variables from childhood to young adulthood . the present population study shows that among the metabolic syndrome risk variables , in a comparison with normoglycemic subjects , glucose was consistently higher from childhood through adulthood in both pre - diabetic and diabetic subjects ; ldl cholesterol , insulin , and homa - ir were higher in pre - diabetic subjects since adolescence ; and obesity , triglycerides , insulin , and homa - ir were higher and hdl cholesterol was lower in diabetic subjects beginning in childhood . in terms of adverse longitudinal changes from childhood to adulthood , ldl cholesterol and glucose were independently related with pre - diabetic status , whereas obesity , hdl cholesterol , and glucose were related to diabetic status . in the current study , the prevalence of both pre - diabetes , which may represent an impaired fasting glucose state , and diabetes was lower than that found previously ( 1,8,10 ) the observed differences of sex ( male > female ) in the pre - diabetic group ( 1,8,10 ) and race ( black > white ) in the diabetic group ( 1 ) in the study cohort are in agreement with the earlier reports . further , compared with other race - sex groups , pre - diabetes ( 10 ) and diabetes ( 1 ) were less prevalent among white females . it is of interest that levels of obesity were higher beginning in childhood , changed adversely through adulthood , and related independently with diabetes in adulthood . this observation is consistent with the known tracking of risk factor variables over time and especially of childhood obesity in predicting adulthood obesity ( 17 ) . the persistent elevation of obesity has influenced the onset of type 2 diabetes occurring at a younger age ( 17,18 ) . with respect to the longitudinal changes in this present study cohort , obesity was the most consistent predictor of adverse changes leading to diabetes , regardless of age , race , or sex . a number of studies have shown baseline obesity as an independent and modifiable risk factor for type 2 diabetes ( 18,19 ) . although of different populations , studies showed obesity in young children and adolescents as a strong predictor of subsequent diabetes ( 17,18 ) . such observations suggest the molecular mechanisms by which obesity plays a part in glucose intolerance are complex and include a combination of genetic factors and mechanisms in which skeletal myocytes and central adipocytes play a role ( 19 ) . this study demonstrates that both pre - diabetic and diabetic groups displayed excess basal glucose , insulin , and homa - ir index values at least by adolescence . of note , further , glucose , but not insulin , along with lipid and obesity ( in the diabetic group ) variables were the independent predictors of adverse longitudinal changes of impaired glucose regulation . of interest , blood pressure was not independently associated in the models , which is consistent with an earlier study in childhood and adolescence ( 18 ) . although blood pressure and insulin were individually predictors of diabetes , they were not independently correlated once obesity , glucose , and lipid variables were introduced in the multivariate analyses ( 18 ) . the deterioration in glucose levels to pre - diabetes or diabetes that , after a relatively stable period , occurs as a rapid , incremental increase accompanied by a decline in insulin sensitivity has been noted ( 20 ) . in the current cohort study , both showed progressively increased glucose levels beginning in early life , before the onset of impaired glucose regulation status , suggesting that even small changes in glucose levels may be a marker of altered carbohydrate - insulin imbalance . in the present study , adverse changes in ldl cholesterol and hdl cholesterol were independently correlated among the pre - diabetic and diabetic groups . of relevance to this present finding , hdl cholesterol was demonstrated as an independent modifiable predictor of diabetes in childhood and adolescence in pima indians ( 18 ) . individuals with diabetes usually have increased triglycerides and decreased hdl cholesterol resulting from the release of fatty acids from adipose tissue , the elevation of delivery of free fatty acids to the liver , and the hepatic synthesis of vldls ( 21 ) . this abnormal lipid profile is characterized by modestly elevated ldl cholesterol and high triglycerides levels with a markedly increased cardiovascular risk among diabetic patients ( 22 ) . perhaps most important is the observation that young adults with pre - diabetes and diabetes showed an increased prevalence of metabolic syndrome and its multiple risk factors . the observed overall prevalence of metabolic syndrome is in agreement with an earlier report ( 23 ) . also , metabolic syndrome status was more prevalent in the pre - diabetic and diabetic groups , as might be expected and is consistent with previous findings ( 11 ) . a pre - diabetic status reflects an atherogenic profile of metabolic syndrome risk variables long before an overt clinical macrovascular event ( 24 ) . further , these multiple risk factors related to autopsy findings of coronary atherosclerosis that we studied in our young individuals , evidence of silent , subclinical disease that evolves from childhood . as limitations , an established simple surrogate measure homa - ir index applicable to population studies was used . also , because of the lack of dietary intake data , this study did not address the role of diet in the regulation of glucose homeostasis and related risk of obesity , cardiovascular disease , and type 2 diabetes ( 25 ) . the fasting status was based on self - report . in summary , these findings indicate that adverse levels of risk variables of metabolic syndrome , adiposity , and measures of glucose homeostasis in particular , and their accelerated rates of change since childhood , characterizes the early natural history of carbohydrate - insulin imbalance . the current findings reinforce a primary role for early prevention of and intervention for these risk factors , especially obesity , beginning in childhood .

objective that type 2 diabetes is associated with the metabolic syndrome is known . however , information is lacking regarding the long - term and adverse changes of metabolic syndrome variables in the development of type 2 diabetes from childhood to adulthood.research design and methods observations were examined , retrospectively , in a community - based cohort of normoglycemic ( n = 1,838 ) , pre - diabetic ( n = 90 ) , and type 2 diabetic ( n = 60 ) subjects followed serially for cardiovascular risk factors during childhood ( 411 years ) , adolescence ( 1218 years ) , and adulthood ( 1944 years).results diabetic subjects versus normoglycemic subjects had significantly higher levels of subscapular skinfold , bmi , triglycerides , glucose , insulin , and homeostasis model assessment of insulin resistance and lower levels of hdl cholesterol beginning in childhood and higher levels of mean arterial pressure ( map ) in adolescence and adulthood . in a multivariate model including bmi , map , hdl cholesterol , ldl cholesterol , triglycerides , glucose , and insulin , adjusted for age , age2 , race , sex , and race sex interaction , adverse changes in glucose and ldl cholesterol were independently associated with pre - diabetic subjects , whereas adverse changes in bmi , glucose , and hdl cholesterol were associated with diabetic subjects . as young adults , pre - diabetic and diabetic groups displayed a significantly higher prevalence of obesity , hypertension , dyslipidemia , hyperinsulinemia , and metabolic syndrome.conclusionsthese findings indicate that adverse levels of risk variables of metabolic syndrome , adiposity , and measures of glucose homeostasis accelerating since childhood characterize the early natural history of type 2 diabetes and underscore the importance of early prevention and intervention on risk factors beginning in childhood .

RESEARCH DESIGN AND METHODS General examination Laboratory analyses Statistical analysis RESULTS CONCLUSIONS Supplementary Material

the bogalusa heart study is being conducted in the semirural , biracial ( 65% white and 35% black ) community of bogalusa , louisiana . between 1976 and 1994 , in addition , eight cross - sectional surveys were conducted between 1978 and 2002 using young adults who had been examined previously as children . a detailed description of the study design , participation , and protocols was published elsewhere ( 12 ) . this panel design , based on repeated cross - sectional examinations conducted approximately every 34 years , resulted in serial observations from childhood to young adulthood , allowing longitudinal analyses . subjects from six cross - sectional studies of children who participated in at least one of the 14 cross - sectional surveys of children and adults were eligible for this retrospective cohort study . at the baseline examination , the children with a history of treatment of diabetes or those who had a fasting glucose level 7 mmol / l were excluded . at the most recent screening , the number of screenings and observations between childhood and adulthood ranged from 2 to 9 . in all , 91% of subjects were screened 3 times and 63% were screened 46 times , with a total of 9,232 sets of observations . on the basis of the data at the last survey , adult subjects were classified as normoglycemic , pre - diabetic , or diabetic according to the american diabetes association criteria ( 7 ) . individuals were considered normoglycemic ( n = 1,838 ) if they had a fasting glucose level < 5.6 mmol / l , pre - diabetic ( n = 90 ) if their fasting glucose level was between 5.6 and 6.9 mmol / l , or diabetic ( n = 60 ) if 1 ) their fasting glucose level was 7 the institutional review board approved consent forms used for these surveys , and informed consent was obtained from adult study participants and from parents or guardians of children . identical protocols were used by trained examiners , nurses , and technicians across all surveys ; procedures for general examination were reported previously ( 13 ) . each screening day , on a random 10% subsample , a second blood sample was collected , labeled , and analyzed in a blind fashion to estimate the measurement error . all measurements were made in replicate , and mean values were used . in the longitudinal analysis , bmi ( weight in kilograms divided by the square of height in meters ) was used as a measure of overall adiposity and subscapular skinfold measured with lange skinfold calipers was used for truncal fatness . blood pressure measurements were obtained on the right arm with the subjects in a relaxed , sitting position . systolic and diastolic blood pressures were recorded at the first and fifth korotkoff phases using a mercury sphygmomanometer . blood pressure levels were reported as the mean of six replicate readings , with three taken by each of two randomly assigned and trained observers . mean arterial pressure ( map ) , calculated as diastolic blood pressure plus one - third pulse pressure , was used in the analysis . both chemical and enzymatic procedures met the performance requirements of the lipid standardization program of the centers for disease control and prevention , which routinely monitored the precision and accuracy of cholesterol , triglyceride , and hdl cholesterol measurements since the beginning of this study . the intraclass correlation coefficients between the blind duplicate ( 10% random sample ) values ranged from 0.86 to 0.98 for hdl cholesterol , from 0.86 to 0.98 for ldl cholesterol , and from 0.88 to 0.99 for triglycerides . on the basis of the national cholesterol education program adult treatment panel iii guidelines ( 15 ) , mmol / l ) , high triglycerides ( 2.26 mmol / l ) , or low hdl cholesterol ( < 1.03 mmol / l ) or if they were taking medication for dyslipidemia and as having metabolic syndrome if they had at least three risk factors consistent with that diagnosis . the intraclass correlation coefficients between the blind duplicate values ranged from 0.94 to 0.98 for insulin and from 0.86 to 0.98 for glucose . > 108 pmol / l , a value considered indicative of insulin resistance in normoglycemic subjects ( 16 ) . in addition , an index of insulin resistance was calculated according to the homeostasis model assessment for insulin resistance formula : homa - ir = [ insulin ( microunits per milliliter ) glucose ( millimoles per liter)/22.5 ] . in the analyses , the race - sex groups were combined to increase statistical power and to simplify the presentation . values for triglycerides , glucose , and insulin and homa - ir variables used in the analyses were log transformed to improve normality as necessary . mean levels of risk variables in 4- to 11- , 12- to 18- , and 19-year - old age - groups corresponding with the preadolescence , adolescence , and adulthood period were compared by their diabetes status in adulthood ( normoglycemic versus pre - diabetic , normoglycemic versus diabetic , and pre - diabetic versus diabetic ) . a single measurement per subject was used to calculate mean levels of risk variable within age - groups . general linear models were used to examine impaired glucose regulation differences in risk factor variables , adjusted for age , race , and sex . by using data from the last survey in adulthood , significant differences in the prevalence of pre - diabetes and diabetes by race and sex and metabolic syndrome and its related cardiovascular risk factors by diabetes status were tested by pearson 's test . multivariate analyses ( generalized estimation equation method ) were used to determine which longitudinal changes in risk variables since childhood predicted adult diabetes status . the model included bmi , map , hdl cholesterol , ldl cholesterol , triglycerides , glucose , and insulin , adjusted for age , age , race , sex , and sex- by - race interaction , as applicable . nonsignificant terms ( p > 0.05 ) were removed from the model by backward stepwise procedure . identical protocols were used by trained examiners , nurses , and technicians across all surveys ; procedures for general examination were reported previously ( 13 ) . serum samples were obtained from antecubital venous blood and kept at 4c until analysis the following day . each screening day , on a random 10% subsample , a second blood sample was collected , labeled , and analyzed in a blind fashion to estimate the measurement error . all measurements were made in replicate , and mean values were used . in the longitudinal analysis , bmi ( weight in kilograms divided by the square of height in meters ) was used as a measure of overall adiposity and subscapular skinfold measured with lange skinfold calipers was used for truncal fatness . blood pressure measurements were obtained on the right arm with the subjects in a relaxed , sitting position . mean arterial pressure ( map ) , calculated as diastolic blood pressure plus one - third pulse pressure , was used in the analysis . these variables were determined by enzymatic procedures on a vp instrument ( abbott ) between 1987 and 1996 and on the hitachi 902 automatic analyzer ( roche ) afterward . both chemical and enzymatic procedures met the performance requirements of the lipid standardization program of the centers for disease control and prevention , which routinely monitored the precision and accuracy of cholesterol , triglyceride , and hdl cholesterol measurements since the beginning of this study . serum lipoprotein cholesterol levels were analyzed by using a combination of heparin - calcium precipitation and agar - agarose gel electrophoresis procedures ( 14 ) . the intraclass correlation coefficients between the blind duplicate ( 10% random sample ) values ranged from 0.86 to 0.98 for hdl cholesterol , from 0.86 to 0.98 for ldl cholesterol , and from 0.88 to 0.99 for triglycerides . on the basis of the national cholesterol education program adult treatment panel iii guidelines ( 15 ) , mmol / l ) , or low hdl cholesterol ( < 1.03 mmol / l ) or if they were taking medication for dyslipidemia and as having metabolic syndrome if they had at least three risk factors consistent with that diagnosis . from 1976 to 1991 , plasma glucose was measured by a glucose oxidase method using a glucose analyzer ( beckman ) . since then , it has been measured enzymatically as part of a multichemistry ( sma20 ) profile . hyperinsulinemia was arbitrarily defined as a fasting value > 108 pmol / l , a value considered indicative of insulin resistance in normoglycemic subjects ( 16 ) . in addition , an index of insulin resistance was calculated according to the homeostasis model assessment for insulin resistance formula : homa - ir = [ insulin ( microunits per milliliter ) glucose ( millimoles per liter)/22.5 ] . all of the statistical analyses were performed with sas ( version 9.1 ; sas institute , cary , nc ) . in the analyses , the race - sex groups were combined to increase statistical power and to simplify the presentation . values for triglycerides , glucose , and insulin and homa - ir variables used in the analyses were log transformed to improve normality as necessary . mean levels of risk variables in 4- to 11- , 12- to 18- , and 19-year - old age - groups corresponding with the preadolescence , adolescence , and adulthood period were compared by their diabetes status in adulthood ( normoglycemic versus pre - diabetic , normoglycemic versus diabetic , and pre - diabetic versus diabetic ) . a single measurement per subject was used to calculate mean levels of risk variable within age - groups . general linear models were used to examine impaired glucose regulation differences in risk factor variables , adjusted for age , race , and sex . by using data from the last survey in adulthood , significant differences in the prevalence of pre - diabetes and diabetes by race and sex and metabolic syndrome and its related cardiovascular risk factors by diabetes status were tested by pearson 's test . multivariate analyses ( generalized estimation equation method ) were used to determine which longitudinal changes in risk variables since childhood predicted adult diabetes status . the model included bmi , map , hdl cholesterol , ldl cholesterol , triglycerides , glucose , and insulin , adjusted for age , age , race , sex , and sex- by - race interaction , as applicable . mean levels of anthropometric , hemodynamic , and metabolic variables at baseline are presented in table 1 by diabetes status . the pre - diabetic group had significantly higher age , ldl cholesterol , and glucose than the normoglycemic group . with the exceptions of diastolic blood pressure and ldl cholesterol , the diabetic group versus normoglycemic group showed significantly higher age , subscapular skinfold , bmi , systolic blood pressure , map , triglycerides , glucose , insulin , and homa - ir and lower hdl cholesterol . compared with the pre - diabetic group , diabetic subjects had significantly higher subscapular skinfold , bmi , and triglycerides and lower hdl cholesterol . the prevalence of metabolic syndrome and its related cardiovascular risk factors in adulthood at the last survey is shown in table 1 by diabetes status . compared with the normoglycemic group , obesity in terms of excess generalized adiposity ( bmi ) , hypertension , dyslipidemia , and hyperinsulinemia ( indicative of insulin resistance ) , and metabolic syndrome were significantly more prevalent among the pre - diabetic ( except hdl cholesterol ) and diabetic ( except ldl cholesterol ) groups . compared with the pre - diabetic group , high risk levels of hdl cholesterol were more prevalent among the diabetic group . the overall prevalence of the metabolic syndrome was 18.4% in the total sample ( data not shown ) . mean levels of variables of metabolic syndrome in childhood ( 411 years ) , adolescence ( 1218 years ) , and adulthood ( 1944 years ) are shown in fig . the pre - diabetic group versus normoglycemic individuals showed higher levels of glucose and subscapular skinfold from childhood through adulthood ; higher levels of ldl cholesterol , insulin , and homa - ir in adolescence and adulthood ; and higher levels of bmi , map , and triglycerides in adulthood . on the other hand , the diabetic group versus the normoglycemic group had higher levels of subscapular skinfold , bmi , triglycerides , glucose , insulin , and homa - ir and lower levels of hdl cholesterol from childhood through adulthood and higher levels of map in adolescence and adulthood . in addition , the diabetic group versus the pre - diabetic group had higher levels of subscapular skinfold in adolescence and adulthood , higher bmi in adolescence , and lower levels of hdl cholesterol and ldl cholesterol and higher levels of glucose and homa - ir in adulthood . as shown in supplementary fig . 1 of the online appendix ( available at http://dx.doi.org/10.2337/dc08-0898 ) , white male subjects had a significantly higher prevalence of pre - diabetes than white female subjects . with respect to diabetes , white male versus white female subjects and black female versus white female subjects had a significantly greater prevalence . male versus female subjects with pre - diabetes and black versus white subjects with diabetes had a significantly higher prevalence ; overall prevalences of pre - diabetes and diabetes were 4.5 and 3.0% in the total sample , respectively ( data not shown ) . longitudinal rates of change in risk variables of metabolic syndrome by diabetes status were observed ( supplementary table 1 of the online appendix ) . compared with the normoglycemic group , the rates of increase in subscapular skinfold , bmi , triglycerides , glucose , insulin , and homa - ir were significantly higher among the pre - diabetic and diabetic groups and the rates of increase in diastolic blood pressure and map were significantly higher among the diabetic group . independent relationships between adverse longitudinal changes in risk variables since childhood and adult pre - diabetes and diabetes conditions were determined in the multivariate models , presented in table 2 . adverse changes in ldl cholesterol and glucose were independently associated with pre - diabetes status and adverse changes in bmi , hdl cholesterol , and glucose with diabetes status . when homa - ir index , instead of glucose and insulin , was included in the models , homa - ir index , ldl cholesterol , and glucose were significant independent predictors in the pre - diabetic group and homa - ir , bmi , and hdl cholesterol were predictors in the diabetic group ; further , alternate multivariate analyses using subscapular skinfold , instead of bmi , gave essentially identical results ( data not shown ) . these observations explore the natural history of impaired glucose regulation and diabetes status in a community - based population of children free from a selection bias monitored longitudinally over a period of 21 years . data linked the conditions of pre - diabetes and type 2 diabetes in young adults with concurrent longitudinal changes in some metabolic syndrome risk variables from childhood to young adulthood . the present population study shows that among the metabolic syndrome risk variables , in a comparison with normoglycemic subjects , glucose was consistently higher from childhood through adulthood in both pre - diabetic and diabetic subjects ; ldl cholesterol , insulin , and homa - ir were higher in pre - diabetic subjects since adolescence ; and obesity , triglycerides , insulin , and homa - ir were higher and hdl cholesterol was lower in diabetic subjects beginning in childhood . in terms of adverse longitudinal changes from childhood to adulthood , ldl cholesterol and glucose were independently related with pre - diabetic status , whereas obesity , hdl cholesterol , and glucose were related to diabetic status . in the current study , the prevalence of both pre - diabetes , which may represent an impaired fasting glucose state , and diabetes was lower than that found previously ( 1,8,10 ) the observed differences of sex ( male > female ) in the pre - diabetic group ( 1,8,10 ) and race ( black > white ) in the diabetic group ( 1 ) in the study cohort are in agreement with the earlier reports . further , compared with other race - sex groups , pre - diabetes ( 10 ) and diabetes ( 1 ) were less prevalent among white females . it is of interest that levels of obesity were higher beginning in childhood , changed adversely through adulthood , and related independently with diabetes in adulthood . this observation is consistent with the known tracking of risk factor variables over time and especially of childhood obesity in predicting adulthood obesity ( 17 ) . the persistent elevation of obesity has influenced the onset of type 2 diabetes occurring at a younger age ( 17,18 ) . with respect to the longitudinal changes in this present study cohort , obesity was the most consistent predictor of adverse changes leading to diabetes , regardless of age , race , or sex . a number of studies have shown baseline obesity as an independent and modifiable risk factor for type 2 diabetes ( 18,19 ) . such observations suggest the molecular mechanisms by which obesity plays a part in glucose intolerance are complex and include a combination of genetic factors and mechanisms in which skeletal myocytes and central adipocytes play a role ( 19 ) . this study demonstrates that both pre - diabetic and diabetic groups displayed excess basal glucose , insulin , and homa - ir index values at least by adolescence . of note , further , glucose , but not insulin , along with lipid and obesity ( in the diabetic group ) variables were the independent predictors of adverse longitudinal changes of impaired glucose regulation . of interest , blood pressure was not independently associated in the models , which is consistent with an earlier study in childhood and adolescence ( 18 ) . although blood pressure and insulin were individually predictors of diabetes , they were not independently correlated once obesity , glucose , and lipid variables were introduced in the multivariate analyses ( 18 ) . the deterioration in glucose levels to pre - diabetes or diabetes that , after a relatively stable period , occurs as a rapid , incremental increase accompanied by a decline in insulin sensitivity has been noted ( 20 ) . in the current cohort study , both showed progressively increased glucose levels beginning in early life , before the onset of impaired glucose regulation status , suggesting that even small changes in glucose levels may be a marker of altered carbohydrate - insulin imbalance . in the present study , adverse changes in ldl cholesterol and hdl cholesterol were independently correlated among the pre - diabetic and diabetic groups . of relevance to this present finding , hdl cholesterol was demonstrated as an independent modifiable predictor of diabetes in childhood and adolescence in pima indians ( 18 ) . individuals with diabetes usually have increased triglycerides and decreased hdl cholesterol resulting from the release of fatty acids from adipose tissue , the elevation of delivery of free fatty acids to the liver , and the hepatic synthesis of vldls ( 21 ) . this abnormal lipid profile is characterized by modestly elevated ldl cholesterol and high triglycerides levels with a markedly increased cardiovascular risk among diabetic patients ( 22 ) . perhaps most important is the observation that young adults with pre - diabetes and diabetes showed an increased prevalence of metabolic syndrome and its multiple risk factors . the observed overall prevalence of metabolic syndrome is in agreement with an earlier report ( 23 ) . also , metabolic syndrome status was more prevalent in the pre - diabetic and diabetic groups , as might be expected and is consistent with previous findings ( 11 ) . a pre - diabetic status reflects an atherogenic profile of metabolic syndrome risk variables long before an overt clinical macrovascular event ( 24 ) . further , these multiple risk factors related to autopsy findings of coronary atherosclerosis that we studied in our young individuals , evidence of silent , subclinical disease that evolves from childhood . also , because of the lack of dietary intake data , this study did not address the role of diet in the regulation of glucose homeostasis and related risk of obesity , cardiovascular disease , and type 2 diabetes ( 25 ) . in summary , these findings indicate that adverse levels of risk variables of metabolic syndrome , adiposity , and measures of glucose homeostasis in particular , and their accelerated rates of change since childhood , characterizes the early natural history of carbohydrate - insulin imbalance . the current findings reinforce a primary role for early prevention of and intervention for these risk factors , especially obesity , beginning in childhood .

menarche is a vital maturational event of puberty in female adolescents . unlike other pubertal changes that are gradual and continuous , it is highly correlated with other pubertal characteristics and is , therefore , preferred as a benchmark for sexual maturation . the timing of menarche is an important determinant of population size , reproductive performance , and other chronic outcomes , such as cancers of the reproductive organs ( 1 ) . early onset of menarche has been associated with premature marriage and first childbirth , especially in developing countries , and is a risk factor for breast - cancer , ovarian cancer , and other diseases ( 1,2 ) . during the past century , there has been a secular ( time - related ) trend towards an earlier onset of menarche in most developed countries , with a decline of 23 months per decade in europe and the united states ( 3 ) . general improvements in nutrition and health have been suggested to explain the downward trend ( 3,4 ) . the earlier onset of menarche has also been observed in some developing countries , such as bangladesh . the most recent study of unmarried female adolescents conducted in 1996 in a rural area of bangladesh reported an average age at menarche of 13.0 years ( 4 ) , which was substantially lower than the previous estimate of 15.8 years in 1976 ( 5 ) . the relatively high age at menarche in 1976 was linked to severe malnutrition caused by inflation , famine , and flooding following bangladesh 's war for independence in 1971 ( 5 ) . the association between the nutritional status and the onset of menarche has been well - documented ( 46 ) . in general , adolescents who are taller and heavier with a greater body - fat mass tend to reach menarche at younger ages ( 46 ) . female adolescents in bangladesh , particularly those in rural areas , are largely affected by undernutrition , with 48% and 59% being stunted ( height - for - age <3 percentile ) and thin ( body mass index [ bmi]-for - age < 5 percentile ) respectively , applying the national center for health statistics reference ( 7 ) . considering the high prevalence of undernutrition and a lack of significant improvement in nutritional status during recent years ( 7 ) , it is not clear whether the age at menarche has continued to decrease in rural bangladesh . a marked seasonal pattern of menarche has previously been reported such that the monthly occurrence of menarche is not uniformly distributed throughout the year but peaks show up in summer and winter ( 8,9 ) . seasonal factors , such as length of daylight , ambient temperatures , and the psychological effects of reduced stress and increased relaxation during school vacation , are some suggested explanations for this seasonal variation ( 811 ) . the present analysis was conducted to determine the age at menarche using self - reported date of onset in a group of female adolescents aged 1219 years in a rural area of bangladesh and to examine its association with marital and nutritional status and season . we believe that the study of onset of menarche is important not only from an anthropological point of view but also because the mean menarcheal age has been accepted as a particularly sensitive indicator of the biosocial status of the population ( 12 ) . the mean age at menarche in rural bangladesh was last estimated more than a decade ago , and a number of biological , social and ecological factors have likely changed since then . we believe that it is opportune to re - assess the onset of menarche and its association with various factors , particularly marital status , considering the numerous negative consequences of marriage and pregnancy in adolescence , which may be triggered by early onset of menarche . data were collected as part of a cohort study of the consequences of pregnancy and lactation on growth of adolescents in gaibandha district in rural northwest bangladesh ( 13 ) . this study was conducted in a smaller area ( 96 communities with 200 households in four unions of gaibandha district ) , which was part of the study area where a randomized community trial for assessing the impact of vitamin a and -carotene supplementation on maternal and infant survival called the jivita project was being conducted by our group . this rural and densely - populated ( 1,000 per sq km ) region is characterized by average to below - average socioeconomic status indicators , rates of health service - use , child health , and nutritional status compared to national statistics . at the outset , all the households in this area were visited by 96 local female workers from january to march 2005 to identify and list all married and unmarried adolescents aged less than 20 years . all enumerated adolescents were interviewed in the home to elicit dates of their birth , months and years of menarche , marital status , and pregnancy history using recall and to measure mid - upper arm circumference ( muac ) . home records of date of birth , when available , were requested and , in their absence , parental input was obtained to confirm the self - reported year and month of birth . various probes , such as age at school enrollment , differences in age between siblings , year of marriage , and tools , such as a local events calendar , were used for determining the year and month of birth . adolescents who were post - menarcheal at the time of enumeration were asked to recall the year and month of menarche . reference to age of school enrollment and the number of years spent in each class was used as recall probes as the majority of adolescents in the study area had received some schooling due to the government support for female education ( 14 ) . for example , the adolescents were asked in which grade they had started menstruating , along with the highest grade completed and its corresponding calendar year . also , the number of years for which a particular grade was repeated was recorded . family events , seasons , school examinations , and a local events calendar were used for probing the month of menarche . in addition , the mother or other female relatives living in the household were asked to help with the recall of the age at menarche . the year and month of marriage muac was assessed as a measure of nutritional status to the nearest millimetre following standard procedures ( 15 ) . of the 5,582 female adolescents enumerated , 100 ( 1.8% ) refused to participate in the study , and 1,365 ( 24.4% ) were excluded either due to not meeting the age criteria of 1219 years or for not being able to provide years of their birth . further , 29 ( 0.5% ) adolescents who were unable to report months of their menarche and 165 ( 3.0% ) who had previously been pregnant were excluded because the adolescent cohort study enrolled only never - pregnant adolescents ( 13 ) . the resulting sample included in the analysis comprised 3,923 ( 70.3% ) married nulliparous and unmarried female adolescents aged 1219 years . of the 3,923 female adolescents , 483 ( 12.3% ) were unable to report months of their birth . thus , the mean age at menarche was assessed by assigning a random month of birth to this group using microsoft excel 2000 ( microsoft corp , redmond , wa , usa ) . as some households had more than one female adolescent aged 1219 years and age at menarche of those living in the same households could be correlated , we estimated the mean age at menarche adjusting for the clustering of girls of the same households . the differences in age at menarche , age , and muac between married and unmarried adolescents and that in muac and age between pre- and post - menarcheal adolescents were examined using a t - test . a multiple linear regression analysis was conducted with age at menarche as the dependent variable and marital status , age , and muac as the independent variables . to examine the seasonality of menarche , frequencies of onset by calendar month and season were computed . seasons were defined as follows : winter = novemberfebruary ; summer = marchjune ; and monsoon = julyoctober . the uniformity of monthly and seasonal distribution of menarche was examined using a test ( 8,9 ) , accounting for the different number of days in each month . the observed frequencies of menarche for each month and season were compared against the expected uniform distribution [ 31/3653,452 ( total number of adolescents ) , 28/3653,452 , etc . ] . all analyses were performed using the stata software ( version 8.0 ) ( stata corp , college station , texas , usa ) . the bangladesh medical research council and the institutional review board at the bloomberg school of public health , johns hopkins university , baltimore , md , usa , approved the study . of the 3,923 female adolescents , 483 ( 12.3% ) were unable to report months of their birth . thus , the mean age at menarche was assessed by assigning a random month of birth to this group using microsoft excel 2000 ( microsoft corp , redmond , wa , usa ) . as some households had more than one female adolescent aged 1219 years and age at menarche of those living in the same households could be correlated , we estimated the mean age at menarche adjusting for the clustering of girls of the same households . the differences in age at menarche , age , and muac between married and unmarried adolescents and that in muac and age between pre- and post - menarcheal adolescents were examined using a t - test . a multiple linear regression analysis was conducted with age at menarche as the dependent variable and marital status , age , and muac as the independent variables . to examine the seasonality of menarche , frequencies of onset by calendar month and season were computed . seasons were defined as follows : winter = novemberfebruary ; summer = marchjune ; and monsoon = julyoctober . the uniformity of monthly and seasonal distribution of menarche was examined using a test ( 8,9 ) , accounting for the different number of days in each month . the observed frequencies of menarche for each month and season were compared against the expected uniform distribution [ 31/3653,452 ( total number of adolescents ) , 28/3653,452 , etc . ] . all analyses were performed using the stata software ( version 8.0 ) ( stata corp , college station , texas , usa ) . the bangladesh medical research council and the institutional review board at the bloomberg school of public health , johns hopkins university , baltimore , md , usa , approved the study . the mean ( sd ) age and muac of the adolescents included in the analysis ( n=3,923 ) were 15.1 ( 1.8 ) years and 22.4 ( 2.3 ) cm respectively . only 13% of the adolescents were married at the time of interview , although most ( 88% ) were already menstruating . of those who had already attained menarche , the mean age of onset of menarche was 12.8 ( 1.4 ) years . a higher proportion of married vs unmarried female adolescents was post - menarcheal ( 99% vs 86% , p<0.01 ) , and married adolescents had an earlier onset of menarche than unmarried girls ( 12.61.3 years vs 12.91.4 years , p<0.01 ) , a difference that remained significant after adjusting for age and muac ( =-0.44 , p<0.01 ) ( table ) . the mean muac was significantly higher among post- vs pre - menarcheal adolescents ( 22.82.0 vs 19.51.9 cm , p<0.01 ) , and there was a negative linear association between age at menarche and muac , after adjusting for age and marital status ( =-0.11 , p<0.001 ) ( table ) . factors associated with mean age at menarche among adolescents in rural bangladesh r=0.20 , f=250.31 , p=<0.01 ; muac = mid - upper arm circumference the monthly occurrence of menarche was not uniformly distributed throughout the year ( = 885.97 , df=11 , p<0.001 ) but had peaks in december and january and troughs in august and september ( fig . ) . more than half of the female adolescents reached menarche during the winter months whereas less than 20% had an onset in monsoon ( = 634.97 , df=2 , p<0.001 ) . frequency distribution of calendar month of onset of menarche among adolescent girls , aged 1219 years , in rural bangladesh ; expected frequencies for each month calculated as 31/3653,452 ( total number of adolescents , 28/3653,452 , 30/3653,452 , etc . ) the estimated mean age at menarche of 12.8 years reported from this study indicated a slightly earlier onset of menarche of the female adolescents in a rural area of bangladesh by 2 months compared to the mean of 13.0 ( 0.9 ) years that had been estimated in narayanganj district of bangladesh in 1996 ( 4 ) . while not directly tested , this apparent small difference in age at menarche compared to that found in a survey in 1996 may be attributable to differences in the timing of studies , location of study areas , and sample characteristics . while the present study included both married and unmarried female adolescents aged 1219 years , the previous study included only unmarried ones aged 1017 years ( 4 ) . on the other hand , one can conjecture that the mean age at menarche in rural bangladesh has lowered slightly over the past decade and that the downward trend in age of onset of menarche observed elsewhere ( 3 ) may also be occurring in rural bangladesh , albeit at a slower pace . in europe , a trend towards an earlier onset of menarche by 23 months per decade has been reported , with the smallest magnitude of decline of 1.1 months per decade observed in france ( 3 ) . analysis of the national health and nutrition examination survey ( nhanes ) in the usa indicated declines in age at menarche for all race / ethnic groups , with non - hispanic white females having a decline in the mean age at menarche from 13.3 years in the pre-1920 birth - cohort to 12.5 years in the 19801984 birth - cohort ( 16 ) . while the declining trend in age at menarche has been attributed to improvements in health and nutrition in developed countries , a large proportion of female adolescents in rural bangladesh is still affected by undernutrition as reflected by the high prevalence of stunting and thinness ( 7 ) . if real , factors associated with a potential declining trend in age at menarche in rural bangladesh remain to be elucidated . the accuracy of recall of the date depends on factors , such as interval of recall ( 17 ) . in the present study , the mean interval of recall was 2.5 years , and 63% of the adolescents had reached menarche within three years of the time of interview . previous studies that used a similar recall procedure reported a correlation coefficient of 0.83 and 0.67 between the actual and the recalled age at menarche when the mean interval of recall compared to occurrence of menarche was 4.8 years and 33 years respectively ( 18,19 ) . therefore , the recall errors are expected to be low in this study due to the relatively short interval of recall . the married adolescents reached menarche at an earlier age than the unmarried adolescents . in developing countries , such as bangladesh and pakistan , age at menarche is suggested to be positively associated with age at marriage and first childbirth ( 20 ) . a similar association has been found in some developed countries where the intervals between menarche and first marriage are much larger ( 20,21 ) . although the mechanisms of this positive association are poorly understood , in developing countries , menarche has been suggested to serve as a marker of maturity and readiness for marriage , signalling the parents to place their daughters in the marriage market ( 2,20 ) . the mean age at marriage in the jivita study area is 16.5 years despite the legal age of marriage being 18 years in bangladesh , and the first pregnancy ( conception ) occurs within an average of 13 months from marriage ( 13 ) . the inverse association between age at menarche and muac shown in this study corresponds with the previous findings that adolescents who are taller and heavier with a greater body - fat mass tend to attain menarche at younger ages ( 4,5 ) . the nutritional status of the adolescents was assessed by muac in this study whereas most previous studies measured weight , height , bmi , or skinfold thickness . in general , muac is a good indicator of nutritional status , serving as a proxy for wasting malnutrition and weight , particularly in adult women ( 22 ) . in a study of rural senegalese female adolescents , the mean muac was shown to be higher among post- vs pre - menarcheal girls ( 23 ) , coinciding with the present finding . the winter peak of menarche found in our study corresponds with the previous findings from rural bangladesh that about half of adolescents attained menarche in winter whereas less than 20% had an onset in may through august ( 5 ) . a factor that most likely explains the winter peaking of menarche in rural bangladesh is the increased food consumption during the harvest season . food availability and consumption generally increases during the winter harvest season in bangladesh , thereby improving nutritional status , a factor known to be associated with onset of menarche ( 5 ) . in a study by tetens et al . elsewhere in rural bangladesh , the mean energy intake increased from 6.5 mj per person per day in the lean season to 8.3 mj per person per day in the harvest season in female adolescents ( 24 ) . in the jivita study , the proportion of women consuming more than one egg per week increased from 37% in the lean season to 56% in the harvest season . likewise , 73% of the women consumed dark green - leafy vegetables at least once a week in the harvest season compared to 60% in the lean season ( data not shown ) . another potential explanation may be the psychological effects of reduced stress due to school vacation . during school season , adolescents have homework and regular examinations , including mid - term and final , which could contribute to the increased level of stress . in addition , the support for female education by the government of bangladesh , particularly for secondary school , is merit - based ; so , to receive continuous financial support from the government , the student needs to perform well in school . thus , a relaxing and less - stressful atmosphere during school vacation has been postulated to trigger the onset of menarche by influencing the hypothalamus ( 810 ) . the winter school vacation which follows the final examination in december may explain the high frequencies of menarche in winter , especially in december . however , we are limited in supporting this hypothesis because , to our knowledge , there is no evidence showing that female adolescents in bangladesh have reduced stress during school holidays , and we lack data to estimate the proportion of adolescents enrolled in school at the time of onset of menarche . the winter peak of onset of menarche in the present study is consistent with a previous finding in 1977 and may be related to increased intake of foods during the winter harvest season . , the date of menarche was self - reported , and the associated recall bias can not be ruled out despite the relatively short interval of recall . although efforts were made to help elicit the date of birth of all the enumerated female adolescents , approximately 12% were unable to report the months of their birth . assigning a random month of birth when only year was available ; as done in our study , may have limitations due to the seasonality of birth reported in bangladesh and other countries ( 25,26 ) . in this study , muac was measured after the onset of menarche . using the post - menarcheal muac measurement as a proxy for nutritional status before the onset of menarche is not ideal . in addition , the study sample comprised female adolescents , aged 1219 years , living in a defined study area . thus , our findings may not be generalizable to or comparable with other randomly - sampled populations of adolescents in bangladesh . in conclusion , the mean age at menarche of the adolescents in this rural area of bangladesh was slightly lower than the previous estimate of 13.0 years in 1996 ( 4 ) . although the small difference in age at menarche could be an artifact of different study sites and sample characteristics , it may also reflect the downward trend of onset of menarche taking place in rural bangladesh , as observed elsewhere . these findings need confirmation in random samples of married and unmarried adolescents in rural bangladesh , assessing age at menarche using pros - pective and probit analysis methods ( 27,28 ) . the negative association between the nutritional status and the onset of menarche in the present study was consistent with the previous findings that adolescents who are better - nourished tend to attain menarche at younger ages . it is likely that , in a rural setting in bangladesh , in addition to the strong cultural and social norms favouring early marriage , an early onset of menarche may also trigger premature marriage and first childbirth . this is associated with numerous adverse birth outcomes , such as premature birth , low birthweight , and neonatal and maternal mortality . meanwhile , it is accepted that maintaining optimal nutritional status during adolescence is important for future reproductive outcomes in adulthood . thus , while continuing efforts should be made to improve the nutritional status of female adolescents , educational activities , advocacy , and strong policies need to be in place at the same time to help delay marriage and first childbirth among female adolescents in the deve - loping world . the jivita project was conducted under a global research activity cooperative agreement between the johns hopkins university and the office of health and nutrition , us agency for international development , washington , dc ( ghs - a-00 - 03 - 00019 - 00 ) , and the bill & melinda gates foundation , seattle , washington , dc ( global control of micronutrient deficiency , grant no . . additional direct or in - kind support for this trial was provided by sight and life , basel , switzerland , the sight and life research institute , baltimore , md , nutrilite health institute ( nutrilite division , access business group , buena park , ca , usa ) , the canadian international development agency , and the national integrated population and health programme of the ministry of health and family welfare , government of bangladesh . barkat ullah , hasmot ali , shafiul alam , shahab uddin , and sayeda khatun sharifa , and the jivita field management team . additional appreciation goes to the field staff of the jivita project for their excellent work and dedication during data collection . the authors acknowledge keith west , principal investigator of the jivita trial , for his insightful comments and guidance throughout the study and rolf klemm for his helpful comments and support . special thanks are due to allan massie , maithilee mitra , ahasanul haque , and lee wu for computer programming and data - management support ; jonathan sugimoto and salahuddin ahmed for gis support ; and andre hackman for technical assistance .

menarche is an important milestone in the development of female adolescents . the study assessed the age at menarche using recall , its seasonality , and association with marital and nutritional status ( using midupper arm circumference [ muac ] ) among 3,923 female adolescents aged 1219 years in a rural area of bangladesh . at the time of assessment , most ( 88% ) adolescents had attained menarche at the mean ( standard deviation [ sd ] ) age of 12.8 ( 1.4 ) years . age of onset of menarche among married adolescents ( 13% ) occurred earlier than in those who were unmarried ( 12.61.3 years vs 12.91.4 years , p<0.01 ) . age at menarche was negatively associated with muac after adjusting for age and marital status ( =0.10 , p<0.01 ) . more than 50% of the adolescents had an onset of menarche during winter ( 2=634.97 ; p<0.001 ) , with peaks in december and january . in this rural population , the current age at menarche was found to be slightly lower than the previous estimates of 13.0 years in bangladesh . an early onset of menarche was associated with season and better nutritional status of the female adolescents and may be associated with early marriage .

INTRODUCTION MATERIALS AND METHODS Statistical analyses RESULTS DISCUSSION ACKNOWLEDGEMENTS

menarche is a vital maturational event of puberty in female adolescents . the timing of menarche is an important determinant of population size , reproductive performance , and other chronic outcomes , such as cancers of the reproductive organs ( 1 ) . early onset of menarche has been associated with premature marriage and first childbirth , especially in developing countries , and is a risk factor for breast - cancer , ovarian cancer , and other diseases ( 1,2 ) . during the past century , there has been a secular ( time - related ) trend towards an earlier onset of menarche in most developed countries , with a decline of 23 months per decade in europe and the united states ( 3 ) . the earlier onset of menarche has also been observed in some developing countries , such as bangladesh . the most recent study of unmarried female adolescents conducted in 1996 in a rural area of bangladesh reported an average age at menarche of 13.0 years ( 4 ) , which was substantially lower than the previous estimate of 15.8 years in 1976 ( 5 ) . the relatively high age at menarche in 1976 was linked to severe malnutrition caused by inflation , famine , and flooding following bangladesh 's war for independence in 1971 ( 5 ) . the association between the nutritional status and the onset of menarche has been well - documented ( 46 ) . female adolescents in bangladesh , particularly those in rural areas , are largely affected by undernutrition , with 48% and 59% being stunted ( height - for - age <3 percentile ) and thin ( body mass index [ bmi]-for - age < 5 percentile ) respectively , applying the national center for health statistics reference ( 7 ) . considering the high prevalence of undernutrition and a lack of significant improvement in nutritional status during recent years ( 7 ) , it is not clear whether the age at menarche has continued to decrease in rural bangladesh . a marked seasonal pattern of menarche has previously been reported such that the monthly occurrence of menarche is not uniformly distributed throughout the year but peaks show up in summer and winter ( 8,9 ) . the present analysis was conducted to determine the age at menarche using self - reported date of onset in a group of female adolescents aged 1219 years in a rural area of bangladesh and to examine its association with marital and nutritional status and season . we believe that the study of onset of menarche is important not only from an anthropological point of view but also because the mean menarcheal age has been accepted as a particularly sensitive indicator of the biosocial status of the population ( 12 ) . the mean age at menarche in rural bangladesh was last estimated more than a decade ago , and a number of biological , social and ecological factors have likely changed since then . we believe that it is opportune to re - assess the onset of menarche and its association with various factors , particularly marital status , considering the numerous negative consequences of marriage and pregnancy in adolescence , which may be triggered by early onset of menarche . this study was conducted in a smaller area ( 96 communities with 200 households in four unions of gaibandha district ) , which was part of the study area where a randomized community trial for assessing the impact of vitamin a and -carotene supplementation on maternal and infant survival called the jivita project was being conducted by our group . this rural and densely - populated ( 1,000 per sq km ) region is characterized by average to below - average socioeconomic status indicators , rates of health service - use , child health , and nutritional status compared to national statistics . at the outset , all the households in this area were visited by 96 local female workers from january to march 2005 to identify and list all married and unmarried adolescents aged less than 20 years . all enumerated adolescents were interviewed in the home to elicit dates of their birth , months and years of menarche , marital status , and pregnancy history using recall and to measure mid - upper arm circumference ( muac ) . various probes , such as age at school enrollment , differences in age between siblings , year of marriage , and tools , such as a local events calendar , were used for determining the year and month of birth . adolescents who were post - menarcheal at the time of enumeration were asked to recall the year and month of menarche . reference to age of school enrollment and the number of years spent in each class was used as recall probes as the majority of adolescents in the study area had received some schooling due to the government support for female education ( 14 ) . for example , the adolescents were asked in which grade they had started menstruating , along with the highest grade completed and its corresponding calendar year . family events , seasons , school examinations , and a local events calendar were used for probing the month of menarche . in addition , the mother or other female relatives living in the household were asked to help with the recall of the age at menarche . of the 5,582 female adolescents enumerated , 100 ( 1.8% ) refused to participate in the study , and 1,365 ( 24.4% ) were excluded either due to not meeting the age criteria of 1219 years or for not being able to provide years of their birth . further , 29 ( 0.5% ) adolescents who were unable to report months of their menarche and 165 ( 3.0% ) who had previously been pregnant were excluded because the adolescent cohort study enrolled only never - pregnant adolescents ( 13 ) . the resulting sample included in the analysis comprised 3,923 ( 70.3% ) married nulliparous and unmarried female adolescents aged 1219 years . of the 3,923 female adolescents , 483 ( 12.3% ) were unable to report months of their birth . thus , the mean age at menarche was assessed by assigning a random month of birth to this group using microsoft excel 2000 ( microsoft corp , redmond , wa , usa ) . as some households had more than one female adolescent aged 1219 years and age at menarche of those living in the same households could be correlated , we estimated the mean age at menarche adjusting for the clustering of girls of the same households . the differences in age at menarche , age , and muac between married and unmarried adolescents and that in muac and age between pre- and post - menarcheal adolescents were examined using a t - test . a multiple linear regression analysis was conducted with age at menarche as the dependent variable and marital status , age , and muac as the independent variables . to examine the seasonality of menarche , frequencies of onset by calendar month and season were computed . the uniformity of monthly and seasonal distribution of menarche was examined using a test ( 8,9 ) , accounting for the different number of days in each month . the observed frequencies of menarche for each month and season were compared against the expected uniform distribution [ 31/3653,452 ( total number of adolescents ) , 28/3653,452 , etc . ] the bangladesh medical research council and the institutional review board at the bloomberg school of public health , johns hopkins university , baltimore , md , usa , approved the study . of the 3,923 female adolescents , 483 ( 12.3% ) were unable to report months of their birth . thus , the mean age at menarche was assessed by assigning a random month of birth to this group using microsoft excel 2000 ( microsoft corp , redmond , wa , usa ) . as some households had more than one female adolescent aged 1219 years and age at menarche of those living in the same households could be correlated , we estimated the mean age at menarche adjusting for the clustering of girls of the same households . the differences in age at menarche , age , and muac between married and unmarried adolescents and that in muac and age between pre- and post - menarcheal adolescents were examined using a t - test . a multiple linear regression analysis was conducted with age at menarche as the dependent variable and marital status , age , and muac as the independent variables . to examine the seasonality of menarche , frequencies of onset by calendar month and season were computed . the uniformity of monthly and seasonal distribution of menarche was examined using a test ( 8,9 ) , accounting for the different number of days in each month . the observed frequencies of menarche for each month and season were compared against the expected uniform distribution [ 31/3653,452 ( total number of adolescents ) , 28/3653,452 , etc . ] the bangladesh medical research council and the institutional review board at the bloomberg school of public health , johns hopkins university , baltimore , md , usa , approved the study . the mean ( sd ) age and muac of the adolescents included in the analysis ( n=3,923 ) were 15.1 ( 1.8 ) years and 22.4 ( 2.3 ) cm respectively . only 13% of the adolescents were married at the time of interview , although most ( 88% ) were already menstruating . of those who had already attained menarche , the mean age of onset of menarche was 12.8 ( 1.4 ) years . a higher proportion of married vs unmarried female adolescents was post - menarcheal ( 99% vs 86% , p<0.01 ) , and married adolescents had an earlier onset of menarche than unmarried girls ( 12.61.3 years vs 12.91.4 years , p<0.01 ) , a difference that remained significant after adjusting for age and muac ( =-0.44 , p<0.01 ) ( table ) . the mean muac was significantly higher among post- vs pre - menarcheal adolescents ( 22.82.0 vs 19.51.9 cm , p<0.01 ) , and there was a negative linear association between age at menarche and muac , after adjusting for age and marital status ( =-0.11 , p<0.001 ) ( table ) . factors associated with mean age at menarche among adolescents in rural bangladesh r=0.20 , f=250.31 , p=<0.01 ; muac = mid - upper arm circumference the monthly occurrence of menarche was not uniformly distributed throughout the year ( = 885.97 , df=11 , p<0.001 ) but had peaks in december and january and troughs in august and september ( fig . ) more than half of the female adolescents reached menarche during the winter months whereas less than 20% had an onset in monsoon ( = 634.97 , df=2 , p<0.001 ) . frequency distribution of calendar month of onset of menarche among adolescent girls , aged 1219 years , in rural bangladesh ; expected frequencies for each month calculated as 31/3653,452 ( total number of adolescents , 28/3653,452 , 30/3653,452 , etc . ) the estimated mean age at menarche of 12.8 years reported from this study indicated a slightly earlier onset of menarche of the female adolescents in a rural area of bangladesh by 2 months compared to the mean of 13.0 ( 0.9 ) years that had been estimated in narayanganj district of bangladesh in 1996 ( 4 ) . while not directly tested , this apparent small difference in age at menarche compared to that found in a survey in 1996 may be attributable to differences in the timing of studies , location of study areas , and sample characteristics . while the present study included both married and unmarried female adolescents aged 1219 years , the previous study included only unmarried ones aged 1017 years ( 4 ) . on the other hand , one can conjecture that the mean age at menarche in rural bangladesh has lowered slightly over the past decade and that the downward trend in age of onset of menarche observed elsewhere ( 3 ) may also be occurring in rural bangladesh , albeit at a slower pace . in europe , a trend towards an earlier onset of menarche by 23 months per decade has been reported , with the smallest magnitude of decline of 1.1 months per decade observed in france ( 3 ) . analysis of the national health and nutrition examination survey ( nhanes ) in the usa indicated declines in age at menarche for all race / ethnic groups , with non - hispanic white females having a decline in the mean age at menarche from 13.3 years in the pre-1920 birth - cohort to 12.5 years in the 19801984 birth - cohort ( 16 ) . while the declining trend in age at menarche has been attributed to improvements in health and nutrition in developed countries , a large proportion of female adolescents in rural bangladesh is still affected by undernutrition as reflected by the high prevalence of stunting and thinness ( 7 ) . if real , factors associated with a potential declining trend in age at menarche in rural bangladesh remain to be elucidated . in the present study , the mean interval of recall was 2.5 years , and 63% of the adolescents had reached menarche within three years of the time of interview . previous studies that used a similar recall procedure reported a correlation coefficient of 0.83 and 0.67 between the actual and the recalled age at menarche when the mean interval of recall compared to occurrence of menarche was 4.8 years and 33 years respectively ( 18,19 ) . therefore , the recall errors are expected to be low in this study due to the relatively short interval of recall . the married adolescents reached menarche at an earlier age than the unmarried adolescents . in developing countries , such as bangladesh and pakistan , age at menarche is suggested to be positively associated with age at marriage and first childbirth ( 20 ) . the mean age at marriage in the jivita study area is 16.5 years despite the legal age of marriage being 18 years in bangladesh , and the first pregnancy ( conception ) occurs within an average of 13 months from marriage ( 13 ) . the inverse association between age at menarche and muac shown in this study corresponds with the previous findings that adolescents who are taller and heavier with a greater body - fat mass tend to attain menarche at younger ages ( 4,5 ) . the nutritional status of the adolescents was assessed by muac in this study whereas most previous studies measured weight , height , bmi , or skinfold thickness . in a study of rural senegalese female adolescents , the mean muac was shown to be higher among post- vs pre - menarcheal girls ( 23 ) , coinciding with the present finding . the winter peak of menarche found in our study corresponds with the previous findings from rural bangladesh that about half of adolescents attained menarche in winter whereas less than 20% had an onset in may through august ( 5 ) . food availability and consumption generally increases during the winter harvest season in bangladesh , thereby improving nutritional status , a factor known to be associated with onset of menarche ( 5 ) . elsewhere in rural bangladesh , the mean energy intake increased from 6.5 mj per person per day in the lean season to 8.3 mj per person per day in the harvest season in female adolescents ( 24 ) . in the jivita study , the proportion of women consuming more than one egg per week increased from 37% in the lean season to 56% in the harvest season . likewise , 73% of the women consumed dark green - leafy vegetables at least once a week in the harvest season compared to 60% in the lean season ( data not shown ) . in addition , the support for female education by the government of bangladesh , particularly for secondary school , is merit - based ; so , to receive continuous financial support from the government , the student needs to perform well in school . thus , a relaxing and less - stressful atmosphere during school vacation has been postulated to trigger the onset of menarche by influencing the hypothalamus ( 810 ) . the winter school vacation which follows the final examination in december may explain the high frequencies of menarche in winter , especially in december . however , we are limited in supporting this hypothesis because , to our knowledge , there is no evidence showing that female adolescents in bangladesh have reduced stress during school holidays , and we lack data to estimate the proportion of adolescents enrolled in school at the time of onset of menarche . the winter peak of onset of menarche in the present study is consistent with a previous finding in 1977 and may be related to increased intake of foods during the winter harvest season . , the date of menarche was self - reported , and the associated recall bias can not be ruled out despite the relatively short interval of recall . in this study , muac was measured after the onset of menarche . using the post - menarcheal muac measurement as a proxy for nutritional status before the onset of menarche is not ideal . in addition , the study sample comprised female adolescents , aged 1219 years , living in a defined study area . in conclusion , the mean age at menarche of the adolescents in this rural area of bangladesh was slightly lower than the previous estimate of 13.0 years in 1996 ( 4 ) . although the small difference in age at menarche could be an artifact of different study sites and sample characteristics , it may also reflect the downward trend of onset of menarche taking place in rural bangladesh , as observed elsewhere . these findings need confirmation in random samples of married and unmarried adolescents in rural bangladesh , assessing age at menarche using pros - pective and probit analysis methods ( 27,28 ) . the negative association between the nutritional status and the onset of menarche in the present study was consistent with the previous findings that adolescents who are better - nourished tend to attain menarche at younger ages . it is likely that , in a rural setting in bangladesh , in addition to the strong cultural and social norms favouring early marriage , an early onset of menarche may also trigger premature marriage and first childbirth . this is associated with numerous adverse birth outcomes , such as premature birth , low birthweight , and neonatal and maternal mortality . thus , while continuing efforts should be made to improve the nutritional status of female adolescents , educational activities , advocacy , and strong policies need to be in place at the same time to help delay marriage and first childbirth among female adolescents in the deve - loping world . the jivita project was conducted under a global research activity cooperative agreement between the johns hopkins university and the office of health and nutrition , us agency for international development , washington , dc ( ghs - a-00 - 03 - 00019 - 00 ) , and the bill & melinda gates foundation , seattle , washington , dc ( global control of micronutrient deficiency , grant no . additional direct or in - kind support for this trial was provided by sight and life , basel , switzerland , the sight and life research institute , baltimore , md , nutrilite health institute ( nutrilite division , access business group , buena park , ca , usa ) , the canadian international development agency , and the national integrated population and health programme of the ministry of health and family welfare , government of bangladesh . the authors acknowledge keith west , principal investigator of the jivita trial , for his insightful comments and guidance throughout the study and rolf klemm for his helpful comments and support .

sandflies - p. papatasi israel strain was reared in the biology of disease vectors laboratory at the department of entomology , kansas state university . flies were maintained on 30% sucrose solution at 27c and 70% humidity with 12 h light and dark cycles . for blood feeding , sandflies were allowed to feed approximately 30 min on a balb / c mouse anesthetised with 3 mg ketamine ( ketaset , fort dodge animal health , fort dodge , ia , usa ) and 0.12 mg xylazine ( anased , acorn inc , decatur , il , usa ) per mouse ( 100 mg / kg of ketamine and 4 mg / kg of xylazine ) . use of animals was preapproved by the kansas state university institutional animal care and use committee under protocols 2747 , 2748 and 2749 . infectious blood meals contained l. major amastigotes and were offered artificially , while simultaneously a control set of sandflies were fed on uninfected blood as previously described ( coutinho - abreu et al . at 20 h post - blood meal ( pbm ) all blood - fed flies were briefly anesthetised with co2 and examined under a dissecting microscope . fully fed flies ( i.e. , abdomen fully distended ) of similar size were selected for dissection . midguts were dissected in 30 l 1x phosphate buffered saline rnase free with eliminase ( fisher , scientific , pittsburgh , pa , usa ) treated tools and equipment . dissected midguts were then transferred to 50 l of rna later ( qiagen , valencia , ca , usa ) , homogenised with a hand - held homogeniser for approximately 20 s and placed at -80c . sequence analysis - ppkzl1 and ppkzl2 were previously identified from p. papatasi cdna midgut libraries ( ramalho - ortigo et al . 2007 ) . molecular weights and isoelectric points ( pi ) were predicted using the swiss institute of bioinformatics expasy tools ( gasteiger et al . 2003 ) . sequences similar to ppkzl1 and ppkzl2 were identified in national center for biotechnology information using blastp for the non - redundant protein database ( altschul et al . the conserved six cysteine domain in ppkzl1 and ppkzl2 was used for multiple sequence alignments ( msa ) with selected sequences from blast results . 2007 ) and manual edits were performed in jalview version 2 ( waterhouse et al . a lutzomyia longipalpis kazal2 contig ( 69116 ) was identified using blast searching for homologs of ppkzl2 in the l. longipalpis llon 0.1 preliminary genome assembly on the baylor college of medicine human genome sequencing center website ( hgsc.bcm.tmc.edu/project-species-i-lutzomyia_longipalpis.hgsc ) . the sequence was translated with swiss institute of bioinformatics expasy ( gasteiger et al . rna extraction and cdna synthesis - total rna was extracted from whole sample pools or individual dissected midguts using the rneasy mini kit ( qiagen ) and eluted in 40 l of rnase - free water . three rnas were obtained for each developmental stage from pools of 20 eggs , 10 l1 larvae and five each for stages l2 , l3 , l4 and pupae . extracted rna was treated with turbo dnase ( ambion , austin , tx , usa ) to eliminate any residual genomic dna . up to 100 ng of each rna was used for first strand cdna synthesis and was added to 3.3 m oligo - dt20 primer , 0.67 mm deoxynucleotide triphosphates and rnase - free water to total volume of 15 l . samples were incubated at 65c for 5 min and then placed on ice for 1 min . addition of 4 l of 5x superscript iii reverse transcriptase first - strand buffer , 5 mm dtt , 0.5 l rnaseout ( 40 units/l ) and 1 l of superscript iii reverse transcriptase ( 200 units/l ) ( invitrogen , carlsbad , ca , usa ) was followed with 1 h incubation at 50c . real - time polymerase chain reaction ( rt - pcr ) - ppkzl1 and ppkzl2 relative expression was analysed in non - blood - fed and blood - fed adult female sandflies . individual midguts were dissected from non - blood - fed flies ( 0 h ) and blood - fed flies at 24 h , 48 h and 72 h pbm . total rna was extracted from individual midguts and used for first - strand cdna synthesis . rt - pcr was carried out on an eppendorf mastercycler ep realplex in 8 l reactions . forward and reverse 0.3 m primers ( table ) were mixed with 4 l iq sybr green supermix ( biorad , hercules , ca , usa ) and added to 0.2 l cdna and 3.32 l molecular grade water ( invitrogen ) . all cdna samples were run in duplicate for ppkzl1 and ppkzl2 and in parallel for 40s ribosomal protein s3 ( genbank accession fg113203 ) . reactions were carried out 40 cycles of 95c/30 s , 58c/1 min and 72c/30 s , followed by 95c/15 s , 60c/15 s and a melt curve up to 95c/20 min . tablecomplete list of primersprimerprimer sequence 5'-3 ' forwardprimer sequence 5'-3 ' reverseannealing ( c)pcrppkzl859gcaccagcccaaaagacctcactgcaatctgatggcgc56.5pcrvr1020acaggagtccagggctggagagaaagtggcaccttccagggtcaagga49pcrppkzl2-r - hisgcaccagcccaaaagacchis tag -ctgcaatctgatggcgc60pcr ppkzl1_137agagcgttacctgtccttgccagcgaatactgaggttc58rt - pcrppkzl2_152aatgaatgtctgaaggcctgccttgggatttcacctccc58rt - pcrpp40s_s3_136ggacagaaatcatcatcatgccttttcagcgtacagctc58rt - pcra : his tag - tcagtggtgatggtgatgatg ; b : touchdown polymerase chain reaction ( pcr ) ; rt : real - time . a : his tag - tcagtggtgatggtgatgatg ; b : touchdown polymerase chain reaction ( pcr ) ; rt : real - time . expression levels of mrna were calculated with the comparative ct method as previously described ( coutinho - abreu et al . briefly , ct values were normalised to the expression of a non - regulated internal control gene , 40s ribosomal protein s3 and then normalised to a calibrator . calibrators for analysis of temporal , developmental and infected expression were mean averages of expression in 0 h , eggs and non - infected blood - fed samples respectively . comparative ct method : ct = { ct variant x sample } - { average ( ct calibrator samples ) } , where variant x equals time points or tissue type . mean fold change of at least five individual samples or three pools were graphed for each time point or tissue . distribution of the data was tested with the kolmogorov - smirnov test for normality and levene 's test for equality of variance . data was evaluated with one - way analysis of variance and a parametric t test with the bonferroni correction for multiple comparisons . for temporal expression profiles of l. major infected sandflies , statistical analysis used two - tailed unpaired t tests for parametric analysis and the two - tailed mann - whitney u test for nonparametric statistical comparisons . prism 5 software ( graphpad , la jolla , ca , usa ) was used for all graphing and statistical analysis . recombinant protein expression and purification - the mature ( minus signal peptide ) ppkzl2 cdna was amplified using the forward primer ppkzl859 and the reverse primer ppkzl2-r - his containing a 6x - his tag on its 3 ' end ( table ) , touchdown reverse transcriptase pcr was performed as follows , 95c/3 min , three cycles of 94c/1 min , 72c/1 min , three cycles of 94c/1 min , 68c/1 min , 72c/1 min , five cycles of 94c/1 min , 62c/1 min , 72c/1 min , 25 cycles of 94c/1 min , 60c 1 min , 72c 1 min , finished with 72c 5 min . two microlitres of the pcr product was separated on an agarose gel for analysis and to assess concentration . the mature ppkzl2 was cloned into vr1020-topo vector as described previously ( ramalho - ortigo et al . insert - containing clones were screened by pcr ( table ) and orientation was confirmed by sequencing . final concentration was 2.5 mg / ml and plasmid sequence was confirmed by sequencing . the recombinant rppkzl2 was expressed in cho - s free style cells , following transfection using 37.5 g of purified plasmid following the manufacturer 's protocol ( invitrogen ) . transfected cho supernatant was collected after 72 h of culture , concentrated using a 3 kda cut - off centricon filter ( milipore , billerica , ma , usa ) and purified by nickel - nitrilotriacetic acid chromatography with a gravity flow column . the column was washed with 15 ml of 20 mm sodium phosphate buffer-300 mm sodium chloride-20 mm imidazole , eluted with 5 ml 20 mm sodium phosphate buffer-300 mm sodium chloride-300 mm imidazole and the eluted rppkzl2 was concentrated to 1.5 g/l . two hundred and fifty nanograms of protein were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis using 4 - 12% reducing bis - tris nupage pre - cast gel purchased from invitrogen . the protein was transferred to nitrocellulose and incubated with anti - his antibody ( santa cruz , santa cruz , ca , usa ) overnight at 4c and followed by three washes of 10 min each in tris buffered saline buffer with 0.1% tween-20 ( tbs - t ) . the blot was incubated with anti - mouse antibody conjugated to alkaline phosphatase ( promega , madison , wi , usa ) diluted 1:10,000 in tbs - t for 1 h at room temperature and washed in tbs - t . the protein bands were visualised using the western blue substrate ( promega ) . inhibition assays - the inhibition activity of rppkzl2 was tested against human -thrombin and trypsin and bovine -chymotrypsin . increasing concentrations of rppkzl2 were pre - incubated with 0.05 m human -thrombin ( calbiochem , emd chemicals inc , gibbstown , nj , usa ) , 2 m trypsin ( sigma , st . louis , mo , usa ) or 0.25 m -chymotrypsin ( calbiochem , emd chemicals inc ) in 50 mm hepes-0.5% bsa , ph 7.3 for thrombin and in 50 mm tris - hcl , ph 8.0 for trypsin and -chymotrypsin . each enzyme and rppkzl2 combination was incubated for 15 min at 37c in a 96-well non - binding microtitre plate . chromogenic peptide substrate h - d - phenylalanyl - l - pipecolyl - larginine - p - nitroaniline dihydrochloride ( s-2238 ) ( chromogenix , diapharma , west chester township , oh , usa ) , na - benzoyl - d , l - arginine 4-nitroanilide hydrochloride ( bapna ) ( sigma ) or n - succinyl - l - alanyl - l - alanyl - l - prolyl - l - phenylalanine 4-nitroanilide ( suc - aapf - pna ) ( sigma ) was added at increasing concentrations for -thrombin , trypsin or -chymotrypsin respectively for a total reaction volume of 100 l . inhibiton of trypsin activity was measured for 3 nm , 30 nm and 300 nm rppkzl2 at increasing concentrations of bapna ( 25 m , 125 m , 250 m , 500 m and 1000 m ) . inhibition of -chymotrypsin activity was measured for 0.0005 nm , 0.005 nm and 0.05 nm rppkzl2 and inhibition of -thrombin was measured at 0.5 nm , 3 nm and 300 nm rppkzl2 at increasing concentrations 250 m , 500 m and 1000 m of suc - aapf - pna or s-2238 , respectively . the rate of proteinase hydrolysis of the chromogenic substrate was measured at 405 nm every 35 s during the reaction with a biotek synergy ht microplate reader ( biotek , winooski , vt , usa ) . each reaction was run in triplicate and each assay was repeated at least twice . graphs of initial velocity ( v ) vs. substrate concentration { s } were fit with the michaelis - menten equation to obtain the kinetic constant ( km ) and maximum velocity /(copeland 2000 ) . residual activity in the presence of different concentrations of rppkzl2 was calculated with apparent vmax values , residual activity = /(copeland 2005 ) . sequence analysis - both ppkzl1 and ppkzl2 code for six cysteine residues in a conserved arrangement characterised as a non - classical kazal - type domain . predicted molecular weights and isoelectric points for ppkzl1and ppkzl2 are estimated to be 6.4 kda and 5.22 pi and 7.6 kda and 6.10 pi respectively . in ppkzl1 an arginine residue is in the deduced p1 site , the predicted active site for kazal - type inhibitors ( fig . arginine has been shown to confer thrombin and trypsin inhibitory activities and tyrosine in the p1 commonly shows chymotrypsin inhibitory activity ( kanost 1999 ) . fig . ppkzl1 ( genbank i d : eu045342 ) ( a ) and ppkzl2 ( genbank i d : jx171681 ) ( b ) are both similar to putative proteins in l. longipalpis with kazal - type domains : llkzl1 ( genbank i d : abv60319 ) and llkzl2 ( contig 69116 ) . predicted signal peptides are underlined , asterisks mark predicted p1 residues , conserved cysteines are marked ( c ) and gaps are indicated by dashes . ppkzl1 has 81% similarity and 73% identity to a putative protein identified in the new world sandfly l. longipalpis , vector of leishmania infantum chagasi ( jochim et al . ppkzl1 has conserved sequence features previously described in non - classical kazal - type domains in blood - feeding and non - blooding insects such as p - x - c3-g - x4-t - y - x - n - x - c4 and g - x - c6 , with ( x ) representing various residues ( augustin et al . a msa with the top blast results for ppkzl1 was assembled as described in materials and methods section and displayed high conservation of arginine in the p1 site for this group of kazals ( supplementary data ) . ppkzl2 is also similar to another predicted protein in l. longipalpis ( ramalho - ortigo et al . 2007 ) , but to a lesser degree with only 44% identity and 53% similarity ( fig . while the amino acids in the predicted p1 site in the p. papatasi and l. longipalpis proteins differ , tyrosine and phenylalanine do share similar structural and chemical properties and have both been shown to inhibit chymotrypsin . the kazal - type domains in the ppkzl2 msa displayed large diversity in p1 residues ( supplementary data ) . non - classical kazal - type domain patterns are partially conserved in ppkzl2 including regions p - x - c3 and g - x - c6 , ( fig . 1b , supplementary data ) . ppkzl2 also has more residues between c3 and c4 shifting the location of the fifth cysteine closer to the c - terminus , which has been seen in other non - classical kazal - type domains ( hemmi et al . residues specific to the ppkzl2 msa include n - c5-e / q and a phenylalanine located four residues upstream of the fourth cysteine ( supplementary data ) . expression profiles - expression of ppkzl1 and ppkzl2 in the female midgut increased after blood feeding . temporal expression was analysed 0 h , 24 h , 48 h and 72 h pbm . ppkzl1 transcript expression was up - regulated at 24 h and 48 h pbm ( p < 0.05 , p < 0.001 ) ( fig . . expression of ppkzl2 was up - regulated 24 h , 48 h and 72 h pbm ( p < 0.01 , p < 0.001 , p < 0.05 ) ( fig . 2b ) . transcript levels were up - regulated at 24 h and continued to increase significantly at 48 h pbm ( p < 0.01 ) . ppkzl2 expression was then down - regulated by 72 h ( p < 0.001 ) with expression at 72 h decreasing to levels similar to 24 h expression . 2:ppkzl1 and ppkzl2 expression in adult females post - blood meal ( pbm ) . a : ppkzl1 is up - regulated 24 h and 48 h pbm with highest expression at 48 h pbm . by 72 h expression is down - regulated to levels similar to 0 h ; b : ppkzl2 is up - regulated 24 h , 48 h and 72 h pbm . values are the mean fold change of five or more individual midguts with standard error of the mean . * : p < 0.05 ; * * : p < 0.01 ; * * * : p < 0.001 . following results indicating that ppkzl1 and ppkzl2 expression is regulated following a blood meal , we then investigated if these transcripts are expressed in developmental ( non - blood feeding ) stages . expression profiles of developmental stages for ppkzl1 and ppkzl2 show both transcripts expressed during early development ( fig . 3 ) . both ppkzl1 and ppkzl2 are expressed in larval stages l1 , l2 , l3 , l4 and pupa at constant levels showing no significant differential regulation in expression between developmental stages . both ppkzl1 and ppkzl2 are expressed in whole male tissues , but expression was not detected in eggs for either kazal transcript ( supplementary data ) . ppkzl1 expression was not significantly different when compared between larval stages ; b : ppkzl2 was also expressed in all larval stages and pupae at similar expression levels . five or more individuals were pooled for each developmental stage and this was repeated for a total of three replicates . anova t test with the bonferroni correction for multi - comparisons was used for statistical analysis . ppkzl1 and ppkzl2 expression was further analysed at 24 h , 48 h and 72 h following an infective blood feeding with 5 x 10 l. major amastigotes per ml of blood . no significant difference in the mrna expression levels of ppkzl1 and ppkzl2 between non - infected vs. l. major infected flies were detected in these three time points ( fig . 4 ) . fig . 4:ppkzl1 and ppkzl2 expression in adult females infected with leishmania major . temporal expression profiles 24 h , 48 h and 72 h post - infective blood meal ( i ) ( ) and post - non - infected blood meal ( ni ) ( ) . eight individual midguts were assayed for each infected and non - infected time point . ppkzl1 and ppkzl2 expression was not significantly different 24 h , 48 h and 72 h i when compared to ni control groups . statistical analysis used two - tailed unpaired t tests and two - tailed mann - whitney u tests for parametric and nonparametric comparisons respectively ( p < 0.05 ) . inhibition assays - inhibition activity of rppkzl2 was tested for -thrombin , trypsin and -chymotrypsin enzymes . residual activity of enzymes in the presence of rppkzl2 was reduced to 9.4% for -chymotrypsin , 33.5% for -thrombin and 63.9% for trypsin both vmax and km decreased in all inhibition assays with increasing concentrations of rppkzl2 ( supplementary data ) . recombinant ppkzl2 inhibited -chymotrypsin at the nanomolar level and inhibited -thrombin and trypsin at micromolar levels . reactions were fit with michaelis - menten non - linear regression and apparent maximum velocity ( v max ) values were used to calculate residual activity . activity of 0.05 m -thrombin in the presence of rppkzl2 was reduced to 33.5% . reactions were run in triplicate and each graph represents one of two replicates of each experiment . kazal - type inhibitors are a diverse group of serine proteinase inhibitors with a wide range of roles in invertebrates . in blood - feeding triatomines , kazal - type inhibitors in the midgut 2007 , meiser et al . 2010 ) . here , we characterised two single domain non - classical kazal - type inhibitors from the sandfly p. papatasi . ppkzl1 and ppkzl2 mrna transcripts are expressed in non - blood - fed and blood - fed female midguts and expression is regulated by the blood meal with up - regulation at 24 h and 48 h pbm . the decrease in ppkzl1 and ppkzl2 expression detected around 72 h pbm correlates with the completion of blood meal digestion , which culminates with the midgut emptying between 72 - 144 h pbm . furthermore , the expression levels of both ppkzl1 and ppkzl2 remain constant between 72 - 144 h pbm ( supplementary data ) . such expression profiles of ppkzl1 and ppkzl2 are suggestive of a role in digestion for their respective proteins . in addition , as ppkzl1 and ppkzl2 also are expressed in all larval stages , pupae and males , inhibition during digestion is likely not specific to serine proteinases involved in the coagulation cascade , but rather serine proteinases engaged across life stages and sexes . the predicted ppkzl1 is similar to a single domain non - classical kazal - type inhibitor from aedes aegypti , aati ( ribeiro et al . interestingly , a recombinant aati was shown to inhibit trypsin and plasmin , with weak inhibition of thrombin activity ; the aati transcript also was shown to be expressed in larva , pupa , male and female tissues ( watanabe et al . ppkzl1 is also similar to the multi - domain kazal - type inhibitors infestin and dipetalogastin , identified in t. infestans ( campos et al . 2002 ) , and dipetalogaster maximus ( mende et al . 2004 ) , respectively , but with the highest identity to infestin 's domain-4 . this domain was found to strongly inhibit factor xiia , plasmin and trypsin , with no activity for thrombin ( campos et al . consistent with previous findings , ppkzl1 as a non - classical kazal - type domain displays a predicted active site residue that suggests it likely possess inhibitory activity for trypsin - like serine proteinases . ppkzl2 on the other hand is similar to kazal - type domains from dipteran , lepidopteran and hymenopteran species . though no functional characterisation for these kazal domains have been described , putative proteins were identified in expressed sequence tag and cdna libraries of immune - challenged insects ( bartholomay et al . inhibition activity of rppkzl2 was observed for -chymotrypsin , -thrombin and trypsin , in agreement with previous reports on single - domain kazal - type inhibitors having activity against multiple serine proteinases ( nirmala et al . the ability of ppkzl2 to inhibit serine proteinases in p. papatasi midgut is dependent upon the rate of inhibition and concentrations present in the midgut ( kanost & jiang 1996 ) and therefore in vivo activity may be enzyme specific . we previously characterised two chymotrypsin - like and four trypsin - like proteases from p. papatasi and demonstrated that chymotrypsin and trypsin activities in the midgut of this sandfly peak between 27 - 48 h pbm and by 72 h pbm no such activities were detected ( ramalho - ortigo et al . 2003 ) . also , as our results indicate , the peak in rna abundance for kazals in p. papatasi is 48 h pbm . these data , together with the observations that rppkzl2 inhibited both chymotrypsin and trypsin and expression of the mrna was also observed in non - blood - feeding life stages , suggest to us that ppkzl2 is more likely involved in regulating digestive proteases than blood fluidity within the midgut . knock down by injection of 127 ng of double stranded rna produced against each target did not affect mrna expression levels of ppkzl2 and ppkzl1 in the midgut of p. papatasi and therefore analysis of effects on blood meal digestion rate via haemoglobin levels in female midguts were not informative ( data unpublished ) . some kazals have been shown to have immune - like activity ; however there was no response in transcript expression of ppkzl1 and ppkzl2 during l. major infection . no effects were observed on ppkzl1 and ppkzl2 expression during l. major infection in the midgut at 24 h , 48 h or 72 h post - infective - blood meal . it has been described in sandflies that infection with leishmania leads to modulation of trypsin - like activity in the midgut during digestion , suggesting that modulation of trypsin activity allows the parasites to survive ( borovsky & schlein 1987 , sant'anna et al . this has been supported with data showing that rnai of a trypsin gene increased parasite numbers during infection ( sant'anna et al . the dynamics of serine proteinases and serine proteinase inhibitors in the midgut are not only crucial to sandfly metabolism and digestion , but may also affect leishmania development . further characterisation of the serine proteinase cascades and their inhibitors in p. papatasi may provide insight into the complex interactions that constitute vector competence .

sandflies ( diptera : psychodidae ) are important disease vectors of parasites of the genus leishmania , as well as bacteria and viruses . following studies of the midgut transcriptome of phlebotomus papatasi , the principal vector of leishmania major , two non - classical kazal - type serine proteinase inhibitors were identified ( ppkzl1 and ppkzl2 ) . analyses of expression profiles indicated that ppkzl1 and ppkzl2 transcripts are both regulated by blood - feeding in the midgut of p. papatasi and are also expressed in males , larva and pupa . we expressed a recombinant ppkzl2 in a mammalian expression system ( cho - s free style cells ) that was applied to in vitro studies to assess serine proteinase inhibition . recombinant ppkzl2 inhibited -chymotrypsin to 9.4% residual activity and also inhibited -thrombin and trypsin to 33.5% and 63.9% residual activity , suggesting that native ppkzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut . early stages of leishmania are susceptible to killing by digestive proteinases in the sandfly midgut . thus , characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of leishmania .

MATERIALS AND METHODS RESULTS DISCUSSION

sandflies - p. papatasi israel strain was reared in the biology of disease vectors laboratory at the department of entomology , kansas state university . flies were maintained on 30% sucrose solution at 27c and 70% humidity with 12 h light and dark cycles . for blood feeding , sandflies were allowed to feed approximately 30 min on a balb / c mouse anesthetised with 3 mg ketamine ( ketaset , fort dodge animal health , fort dodge , ia , usa ) and 0.12 mg xylazine ( anased , acorn inc , decatur , il , usa ) per mouse ( 100 mg / kg of ketamine and 4 mg / kg of xylazine ) . at 20 h post - blood meal ( pbm ) all blood - fed flies were briefly anesthetised with co2 and examined under a dissecting microscope . sequence analysis - ppkzl1 and ppkzl2 were previously identified from p. papatasi cdna midgut libraries ( ramalho - ortigo et al . sequences similar to ppkzl1 and ppkzl2 were identified in national center for biotechnology information using blastp for the non - redundant protein database ( altschul et al . the conserved six cysteine domain in ppkzl1 and ppkzl2 was used for multiple sequence alignments ( msa ) with selected sequences from blast results . a lutzomyia longipalpis kazal2 contig ( 69116 ) was identified using blast searching for homologs of ppkzl2 in the l. longipalpis llon 0.1 preliminary genome assembly on the baylor college of medicine human genome sequencing center website ( hgsc.bcm.tmc.edu/project-species-i-lutzomyia_longipalpis.hgsc ) . real - time polymerase chain reaction ( rt - pcr ) - ppkzl1 and ppkzl2 relative expression was analysed in non - blood - fed and blood - fed adult female sandflies . individual midguts were dissected from non - blood - fed flies ( 0 h ) and blood - fed flies at 24 h , 48 h and 72 h pbm . all cdna samples were run in duplicate for ppkzl1 and ppkzl2 and in parallel for 40s ribosomal protein s3 ( genbank accession fg113203 ) . briefly , ct values were normalised to the expression of a non - regulated internal control gene , 40s ribosomal protein s3 and then normalised to a calibrator . calibrators for analysis of temporal , developmental and infected expression were mean averages of expression in 0 h , eggs and non - infected blood - fed samples respectively . comparative ct method : ct = { ct variant x sample } - { average ( ct calibrator samples ) } , where variant x equals time points or tissue type . distribution of the data was tested with the kolmogorov - smirnov test for normality and levene 's test for equality of variance . for temporal expression profiles of l. major infected sandflies , statistical analysis used two - tailed unpaired t tests for parametric analysis and the two - tailed mann - whitney u test for nonparametric statistical comparisons . two microlitres of the pcr product was separated on an agarose gel for analysis and to assess concentration . final concentration was 2.5 mg / ml and plasmid sequence was confirmed by sequencing . the recombinant rppkzl2 was expressed in cho - s free style cells , following transfection using 37.5 g of purified plasmid following the manufacturer 's protocol ( invitrogen ) . the blot was incubated with anti - mouse antibody conjugated to alkaline phosphatase ( promega , madison , wi , usa ) diluted 1:10,000 in tbs - t for 1 h at room temperature and washed in tbs - t . the protein bands were visualised using the western blue substrate ( promega ) . inhibition assays - the inhibition activity of rppkzl2 was tested against human -thrombin and trypsin and bovine -chymotrypsin . increasing concentrations of rppkzl2 were pre - incubated with 0.05 m human -thrombin ( calbiochem , emd chemicals inc , gibbstown , nj , usa ) , 2 m trypsin ( sigma , st . louis , mo , usa ) or 0.25 m -chymotrypsin ( calbiochem , emd chemicals inc ) in 50 mm hepes-0.5% bsa , ph 7.3 for thrombin and in 50 mm tris - hcl , ph 8.0 for trypsin and -chymotrypsin . each enzyme and rppkzl2 combination was incubated for 15 min at 37c in a 96-well non - binding microtitre plate . the rate of proteinase hydrolysis of the chromogenic substrate was measured at 405 nm every 35 s during the reaction with a biotek synergy ht microplate reader ( biotek , winooski , vt , usa ) . graphs of initial velocity ( v ) vs. substrate concentration { s } were fit with the michaelis - menten equation to obtain the kinetic constant ( km ) and maximum velocity /(copeland 2000 ) . residual activity in the presence of different concentrations of rppkzl2 was calculated with apparent vmax values , residual activity = /(copeland 2005 ) . sequence analysis - both ppkzl1 and ppkzl2 code for six cysteine residues in a conserved arrangement characterised as a non - classical kazal - type domain . in ppkzl1 an arginine residue is in the deduced p1 site , the predicted active site for kazal - type inhibitors ( fig . arginine has been shown to confer thrombin and trypsin inhibitory activities and tyrosine in the p1 commonly shows chymotrypsin inhibitory activity ( kanost 1999 ) . ppkzl1 ( genbank i d : eu045342 ) ( a ) and ppkzl2 ( genbank i d : jx171681 ) ( b ) are both similar to putative proteins in l. longipalpis with kazal - type domains : llkzl1 ( genbank i d : abv60319 ) and llkzl2 ( contig 69116 ) . ppkzl1 has 81% similarity and 73% identity to a putative protein identified in the new world sandfly l. longipalpis , vector of leishmania infantum chagasi ( jochim et al . ppkzl1 has conserved sequence features previously described in non - classical kazal - type domains in blood - feeding and non - blooding insects such as p - x - c3-g - x4-t - y - x - n - x - c4 and g - x - c6 , with ( x ) representing various residues ( augustin et al . a msa with the top blast results for ppkzl1 was assembled as described in materials and methods section and displayed high conservation of arginine in the p1 site for this group of kazals ( supplementary data ) . ppkzl2 is also similar to another predicted protein in l. longipalpis ( ramalho - ortigo et al . while the amino acids in the predicted p1 site in the p. papatasi and l. longipalpis proteins differ , tyrosine and phenylalanine do share similar structural and chemical properties and have both been shown to inhibit chymotrypsin . the kazal - type domains in the ppkzl2 msa displayed large diversity in p1 residues ( supplementary data ) . non - classical kazal - type domain patterns are partially conserved in ppkzl2 including regions p - x - c3 and g - x - c6 , ( fig . ppkzl2 also has more residues between c3 and c4 shifting the location of the fifth cysteine closer to the c - terminus , which has been seen in other non - classical kazal - type domains ( hemmi et al . residues specific to the ppkzl2 msa include n - c5-e / q and a phenylalanine located four residues upstream of the fourth cysteine ( supplementary data ) . expression profiles - expression of ppkzl1 and ppkzl2 in the female midgut increased after blood feeding . ppkzl2 expression was then down - regulated by 72 h ( p < 0.001 ) with expression at 72 h decreasing to levels similar to 24 h expression . 2:ppkzl1 and ppkzl2 expression in adult females post - blood meal ( pbm ) . by 72 h expression is down - regulated to levels similar to 0 h ; b : ppkzl2 is up - regulated 24 h , 48 h and 72 h pbm . values are the mean fold change of five or more individual midguts with standard error of the mean . following results indicating that ppkzl1 and ppkzl2 expression is regulated following a blood meal , we then investigated if these transcripts are expressed in developmental ( non - blood feeding ) stages . expression profiles of developmental stages for ppkzl1 and ppkzl2 show both transcripts expressed during early development ( fig . both ppkzl1 and ppkzl2 are expressed in larval stages l1 , l2 , l3 , l4 and pupa at constant levels showing no significant differential regulation in expression between developmental stages . both ppkzl1 and ppkzl2 are expressed in whole male tissues , but expression was not detected in eggs for either kazal transcript ( supplementary data ) . ppkzl1 expression was not significantly different when compared between larval stages ; b : ppkzl2 was also expressed in all larval stages and pupae at similar expression levels . anova t test with the bonferroni correction for multi - comparisons was used for statistical analysis . ppkzl1 and ppkzl2 expression was further analysed at 24 h , 48 h and 72 h following an infective blood feeding with 5 x 10 l. major amastigotes per ml of blood . no significant difference in the mrna expression levels of ppkzl1 and ppkzl2 between non - infected vs. l. major infected flies were detected in these three time points ( fig . 4 ) . 4:ppkzl1 and ppkzl2 expression in adult females infected with leishmania major . temporal expression profiles 24 h , 48 h and 72 h post - infective blood meal ( i ) ( ) and post - non - infected blood meal ( ni ) ( ) . eight individual midguts were assayed for each infected and non - infected time point . ppkzl1 and ppkzl2 expression was not significantly different 24 h , 48 h and 72 h i when compared to ni control groups . residual activity of enzymes in the presence of rppkzl2 was reduced to 9.4% for -chymotrypsin , 33.5% for -thrombin and 63.9% for trypsin both vmax and km decreased in all inhibition assays with increasing concentrations of rppkzl2 ( supplementary data ) . recombinant ppkzl2 inhibited -chymotrypsin at the nanomolar level and inhibited -thrombin and trypsin at micromolar levels . reactions were fit with michaelis - menten non - linear regression and apparent maximum velocity ( v max ) values were used to calculate residual activity . activity of 0.05 m -thrombin in the presence of rppkzl2 was reduced to 33.5% . reactions were run in triplicate and each graph represents one of two replicates of each experiment . kazal - type inhibitors are a diverse group of serine proteinase inhibitors with a wide range of roles in invertebrates . in blood - feeding triatomines , kazal - type inhibitors in the midgut 2007 , meiser et al . 2010 ) . here , we characterised two single domain non - classical kazal - type inhibitors from the sandfly p. papatasi . ppkzl1 and ppkzl2 mrna transcripts are expressed in non - blood - fed and blood - fed female midguts and expression is regulated by the blood meal with up - regulation at 24 h and 48 h pbm . the decrease in ppkzl1 and ppkzl2 expression detected around 72 h pbm correlates with the completion of blood meal digestion , which culminates with the midgut emptying between 72 - 144 h pbm . furthermore , the expression levels of both ppkzl1 and ppkzl2 remain constant between 72 - 144 h pbm ( supplementary data ) . such expression profiles of ppkzl1 and ppkzl2 are suggestive of a role in digestion for their respective proteins . in addition , as ppkzl1 and ppkzl2 also are expressed in all larval stages , pupae and males , inhibition during digestion is likely not specific to serine proteinases involved in the coagulation cascade , but rather serine proteinases engaged across life stages and sexes . the predicted ppkzl1 is similar to a single domain non - classical kazal - type inhibitor from aedes aegypti , aati ( ribeiro et al . interestingly , a recombinant aati was shown to inhibit trypsin and plasmin , with weak inhibition of thrombin activity ; the aati transcript also was shown to be expressed in larva , pupa , male and female tissues ( watanabe et al . ppkzl1 is also similar to the multi - domain kazal - type inhibitors infestin and dipetalogastin , identified in t. infestans ( campos et al . this domain was found to strongly inhibit factor xiia , plasmin and trypsin , with no activity for thrombin ( campos et al . consistent with previous findings , ppkzl1 as a non - classical kazal - type domain displays a predicted active site residue that suggests it likely possess inhibitory activity for trypsin - like serine proteinases . ppkzl2 on the other hand is similar to kazal - type domains from dipteran , lepidopteran and hymenopteran species . though no functional characterisation for these kazal domains have been described , putative proteins were identified in expressed sequence tag and cdna libraries of immune - challenged insects ( bartholomay et al . inhibition activity of rppkzl2 was observed for -chymotrypsin , -thrombin and trypsin , in agreement with previous reports on single - domain kazal - type inhibitors having activity against multiple serine proteinases ( nirmala et al . the ability of ppkzl2 to inhibit serine proteinases in p. papatasi midgut is dependent upon the rate of inhibition and concentrations present in the midgut ( kanost & jiang 1996 ) and therefore in vivo activity may be enzyme specific . we previously characterised two chymotrypsin - like and four trypsin - like proteases from p. papatasi and demonstrated that chymotrypsin and trypsin activities in the midgut of this sandfly peak between 27 - 48 h pbm and by 72 h pbm no such activities were detected ( ramalho - ortigo et al . also , as our results indicate , the peak in rna abundance for kazals in p. papatasi is 48 h pbm . these data , together with the observations that rppkzl2 inhibited both chymotrypsin and trypsin and expression of the mrna was also observed in non - blood - feeding life stages , suggest to us that ppkzl2 is more likely involved in regulating digestive proteases than blood fluidity within the midgut . knock down by injection of 127 ng of double stranded rna produced against each target did not affect mrna expression levels of ppkzl2 and ppkzl1 in the midgut of p. papatasi and therefore analysis of effects on blood meal digestion rate via haemoglobin levels in female midguts were not informative ( data unpublished ) . some kazals have been shown to have immune - like activity ; however there was no response in transcript expression of ppkzl1 and ppkzl2 during l. major infection . no effects were observed on ppkzl1 and ppkzl2 expression during l. major infection in the midgut at 24 h , 48 h or 72 h post - infective - blood meal . it has been described in sandflies that infection with leishmania leads to modulation of trypsin - like activity in the midgut during digestion , suggesting that modulation of trypsin activity allows the parasites to survive ( borovsky & schlein 1987 , sant'anna et al . the dynamics of serine proteinases and serine proteinase inhibitors in the midgut are not only crucial to sandfly metabolism and digestion , but may also affect leishmania development . further characterisation of the serine proteinase cascades and their inhibitors in p. papatasi may provide insight into the complex interactions that constitute vector competence .

venous leg ulcers ( vlus ) pose a serious clinical dilemma and an economic burden on health services . they are frequently associated with morbidity , pain , and decreased quality of life in affected patients . about 60% of patients with vlus will experience pain related to their wounds ( hofman et al 1997 ) , which may be constant or intermittent ( ryan et al 2003 ) . the pain can cause depression and a feeling of constant tiredness ( price et al 2007 ) and may also interfere with the healing process ( soon and acton , 2006 ) . these patients may have highly exuding ulcers that could lead to wound malodour and its associated social stigma , resulting in further detrimental psychological effects . vlus also provide an ideal environment for the growth of pathogenic bacteria , with wound colonisation / infection prevalent in immunocompromised patients . vlus are associated with significant treatment costs ( bandages , dressings , and adjunctive therapies ) and nursing resource . about 1%2% of the whole population ( anderson 2006 ) and 3%5% of the population over 65 years of age ( mekkes et al 2003 ) will suffer from a leg ulcer during their lifetime . however , in a typical western population where the average age is steadily increasing , the burden placed upon the health economy by vlus looks set to increase proportionally . back in the 1960s , george winter published his landmark paper in which he demonstrated that , contrary to the belief at the time that wounds should be allowed to dry out and form scabs to promote healing , wounds heal faster if kept moist ( winter et al 1962 ) . since then , the use of cheap gauze dressings and bandages has given way to more technically advanced dressings , such as foams and hydrofibres , which interact with and manage the wound environment . the use of these now established dressings in combination with the gold standard high compression therapy is generally effective in the management of vlus , although up to 20% of vlus fail to heal despite being treated with this regime ( white 2006 ) . the reasons for the lack of response to treatment of hard - to - heal vlus have not yet been fully elucidated , although scientific and clinical research indicates that , instead of progressing through the four distinct but overlapping phases of healing ( hemostasis , inflammation , proliferation , and remodeling ) , these wounds become stuck in a prolonged inflammatory phase ( timmons 2006 ) . control of the processes within the phases of wound healing is complex and involves the interaction of several different cell types ( eg , neutrophils , lymphocytes , macrophages , fibroblasts ) , regulatory mediators ( eg , cytokines , growth factors ) , extracellular matrix ( ecm ) components ( eg , fibronectin , fibrin , collagen , elastin , laminin , proteoglycans , glycosaminoglycans ) , proteases and their inhibitors ( eg , matrix metalloproteinases [ mmps ] and tissue inhibitors of metalloproteinases [ timps ] ) ( schultz et al 2005 ) . early in the wound healing process there is a requirement for the synthesis and deposition of ecm proteins such as fibrin and fibronectin ( hodde and johnson 2007 ) and a sub - set of matrix proteins that are expressed transiently during the wound healing process , eg , galectins , osteopontin , sparc , syndecans , tenascins , thrombospondins , and vitronectin ( agren and werthen 2007 ) . all of these matrix proteins form a provisional wound matrix which provides a scaffold that directs cells into the wound as well as stimulating them to proliferate , differentiate and synthesise new ecm to facilitate the proliferation and remodeling phases of the healing process ( schultz et al 2005 ) . in many chronic wounds , however , the normal healing process is disrupted by extensive tissue damage accompanied by biochemical and cellular imbalances , or by an underlying pathological state ( eg , venous insufficiency ) that can impair or even prevent healing . for example , in the case of hard - to - heal vlus , venous hypertension causes disturbed microcirculation and pathological changes to capillaries , which ultimately locks the condition in a self - amplifying cascade with persistent elevated levels of pro - inflammatory cytokines and proteases that appear to degrade the ecm components , growth factors and receptors that are essential for healing ( agren et al 2000 ; schultz et al 2005 ) the ecm is a vital component of the healing process ; intact or fragmented ecm molecules play a central role in modulating cells through transduction of a variety of signaling mechanisms ( agren et al 2000 ) . additionally , ecm plays a role in angiogenesis , recruitment of circulating progenitor cells , rapid scaffold degradation and constructive remodeling of damaged or missing tissues ( badylak 2002 ) . in studies investigating the mechanisms that lead to venous leg ulceration , it has been demonstrated that intrinsic ecm degradation processes , prevalent in this disease state , are caused by the mmp family of enzymes ( herouy et al 2000 ; bogaczewicz et al 2005 ) . specifically , it has been shown that fibronectin is absent in the base of nonhealing ulcers ( herrick et al 1992 ) although fibroblasts in such wounds synthesize fibronectin normally ( herrick et al 1996 ) . it is hypothesized , therefore , that the reduced levels of fibronectin in nonhealing ulcers is due to excessive degradation of fibronectin by proteases in the ulcers , rather than decreased synthesis ( agren and werthen 2007 ) . on this basis , there is a clear need for new and advanced interventions that can interact with hard - to - heal vlus that are locked in the inflammatory phase of the healing process and progress them to the subsequent proliferative stage of the process . a specific focus of attention for the development of advanced wound therapies should be ( and has been in recent years ) the ecm , either by inducing its synthesis through growth factors , preventing its damage with sacrificial proteins or replacing it with autologous / homologous ecm proteins or their like . one mechanism by which this can be achieved is to provide hard - to - heal ulcers with surrogate ecms which , although not homologous to the patient s own ecm , will provide transient structures that allow cellular adhesion and facilitate tissue regeneration by advancing the wounds to the proliferative phase of the healing process . amelogenin has been identified as an ecm biocompatible protein that can be used as a surrogate scaffold protein . at physiological conditions , amelogenin self - assembles into globular aggregates up to micron - sizes . when applied to the wound bed it provides a temporary matrix for cell attachment and promotes wound healing amelogenin has been used successfully to treat patients with hard - to - heal vlus and other chronic wound types . amelogenin has been used in periodontal applications for some time and there a number of experimental and clinical studies supporting its use in this application . amelogenin has been used as a therapy for the regeneration of alveolar bone by inducing the formation of acellular extrinsic fiber cementum ( kim et al 2005 ) . this protein has also been used to aid in soft tissue root coverage where it has been shown to enhance cellular activity during healing and improve periodontal regeneration ( giannobile and somerman 2003 ; young 2003 ) . more recently , it has been shown that the combination of amelogenin and platelet - derived growth factor - bb ( pdgf - bb ) stimulated proliferation of primary human periodontal ligament fibroblasts and caused enhanced wound fill in an in vitro model ( chong et al 2006 ) . experimental studies designed to elucidate the mechanism by which amelogenin works have shown that it interacts with a variety of cells and chemical messengers involved in the wound healing process . the studies indicate that amelogenin can reduce levels of pro - inflammatory cytokines ( myhre et al 2006 ) . . this cell synthesizes matrix components ( eg , collagen ) that are designed to fill the wound cavity and a variety of cell signals that modulate many different cellular responses such as angiogenesis and epithelialization . in experimental studies , amelogenin has been shown to : augment fibroblast - driven collagen matrix remodeling , increase dermal contraction and fibroblast proliferation ( grayson et al 2006 ) increase synthesis of growth factors , eg , transforming growth factor-1 ( grayson et al 2006 ) and vascular endothelial growth factor ( mirastschijski et al 2004 ) convert chronic fibroblasts into acute fibroblast phenotypes , and the restitution of proliferation and chemokine expression favoring an acute inflammatory response . amelogenin has also been shown to be effective in inducing an angiogenic response in a variety of in vitro models ( yuan et al 2003 ; schlueter et al 2007 ; gren and kleinman 2007 ) . angiogenesis , a key component of the healing process , is significantly reduced in the chronic wound ( drinkwater et al 2002 ; ulrich et al 2005 ) , thereby affecting the healing response . the fact that amelogenin can be used as a temporary ecm protein scaffold provides a sound basis for its use in the treatment of wounds , particularly chronic wounds that are deficient in normal ecm components . it was , therefore , proposed that amelogenin could be used to treat hard - to - heal vlus . initially , a preliminary clinical study was undertaken to ascertain the optimum number of applications of amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) that are required to be applied to a vlu to produce a healing response . the study compared once weekly applications of amelogenin in conjunction with high compression therapy to the hard - to - heal vlus ( duration > 6 months ) of patients over 3 , 6 and 12 week periods ( romanelli et al 2006 ) the wounds were assessed every second week up to week 8 , at final visit ( week 12 ) , and at follow - up visit ( 12 weeks after the final visit ) . the results showed that patients who received the 12-weeks treatment had a larger ulcer reduction ( 72% ) compared with patients who received the 3 or 6-weeks ( 22 and 48% respectively ) treatment with amelogenin ( figure 1 and table 1 ) . percentage change in vlu size following 3 , 6 and 12 weeks of treatment with amelogenin . reproduced with permission from romanelli m , ellervee t , jarve h , et al 2006 . amelogenins ( xelma ) in hard - to - heal venous leg ulcers , an open regime investigation [ poster ] . percentage change in vlu size following 3 , 6 and 12 weeks of treatment with amelogenin . copyright 2006 . reproduced with permission from romanelli m , ellervee t , jarve h , et al 2006 . amelogenins ( xelma ) in hard - to - heal venous leg ulcers , an open regime investigation [ poster ] . european wound management association conference , prague , czech republic the results of this preliminary study showed that hard - to - heal vlus elicited a positive healing response to a defined period ( ie , 12 weeks ) treatment with amelogenin . using this information , a single - blinded , randomized , multi - centre study involving 117 patients was undertaken to assess the effect of amelogenin on hard - to - heal vlus ( vowden et al 2006 ) . to satisfy the inclusion criteria for the study , patients had to have a vlu with a size between 5 and 25 cm and duration of at least six months that had been treated with controlled compression therapy for at least one month prior to enrolment . patients were randomised to receive either amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) plus high compression ( n = 62 ) or a control treatment consisting of a placebo gel ( the amelogenin carrier vehicle ) plus high compression ( n = 61 ) . the amelogenin was applied weekly under secondary dressings for up to a maximum of 12 weeks . although no statistical differences could be identified between the groups as a whole , the percentage wound size reduction was greater in the group treated with amelogenin . a more detailed sub - group analysis was undertaken on patients with ulcers of greater than 10 cm at baseline and for ulcers of duration greater than 6 months . the results of this analysis demonstrated that there was a difference in percentage wound reduction in the group treated with amelogenin ( 33.8% vs 25.6% , respectively ) . it was highlighted that this difference was greatest for the group of patients ( n = 61 ) with the larger ulcers ( > 10 cm ) ( amelogenin 25% vs control 7.9% ) ( figure 2 ) . median wound size reduction in the itt population and sub - group itts following treatment with amelogenin and control . reproduced with permission from vowden p , romanelli m , peter r , et al 2006 . the effect of amelogenins ( xelma ) on hard - to - heal venous leg ulcers . overall the results of this study showed that amelogenin was well tolerated by the patients , with a trend towards less pain and a reduction in the volume of exudate produced by the ulcers in favor of the group treated with amelogenin . this study highlighted the problems associated with undertaking clinical trials involving vlus , the main ones being the wide variations in wound demographics ( eg , sizes and ages ) and their response to treatments . in addition , there was a significant amount of variation between the pre - study treatment regimens employed at different centers , a phenomenon which if not accounted for by allowing a run - in period , may alter the outcome of a clinical trial . as such , the authors concluded that amelogenin could be clinically useful in the treatment of patients with ulcers that could be classified as hard - to - heal ( eg , of duration longer than 6 months and a size greater than 10 cm ) . another clinical trial was undertaken using the lessons learnt from the previous study : this was an open , randomized , comparative , parallel group , multi - centre study involving patients with hard - to - heal vlus ( vowden et al 2007 ) . the inclusion criteria incorporated ulcer size of greater than 10 cm and duration of longer than 6 months , both of which have previously been reported in the literature as prognostic indicators of venous ulcers that are unlikely to respond to treatment and may be classed as hard - to - heal ( european wound management association 2002 ) . the primary objective of this study was to compare the results of 12 consecutive weeks of treatment with amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) plus high compression versus compression therapy alone in hard - to - heal vlus . percentage reduction of wound size from baseline , the percentage of improved ulcers , and parameters such as pain related to the disease , pain at dressing changes and the amount and nature of exudate were evaluated . eligibility for inclusion included adult , mobile patients with hard - to - heal vlus that had been treated with compression therapy for at least 1 month prior to screening . the ulcers had to be at least 6 months old , with a surface area at inclusion of at least 10 cm , but not exceeding 30 cm , and not demonstrating excessive exudate or signs of infection . at the end of the run in period additional criteria for eligibility , eg , change in wound area ( increase / decrease ) of greater than or equal to 50% and a wound area between 8 and 36 cm were applied . in total , 83 patients were randomized to receive either amelogenin plus high compression bandaging ( n = 42 ) or high compression bandaging ( control ) alone ( n = 41 ) . all participants received high compression bandaging therapy one month prior to , during the investigational period of 3-weeks run - in and throughout the 12 weeks of active treatment the results of this study demonstrated that the amelogenin group showed a greater percentage reduction in ulcer size ( mean 33.11% ) compared to the control group ( mean 11.07% ) from baseline to the last visit ( p = 0.03 ) . the number of ulcers showing an improvement was significantly greater ( p = 0.01 ) in the amelogenin treated group than the control group . statistically significant differences in favor of the amelogenin group were also found the proportion of patients with none or low levels of exudate ( p = 0.01 ) . pain was evaluated in this study as related to the disease or to the ulcer . it was evaluated as part of the protocol by interviewing the patients who were asked to rate their pain on a scale with 11 steps ; from 0 = no pain to 10 = unbearable pain ( hartrick et al 2003 ) . the results showed that a reduction in pain related to the disease and a reduction in pain at dressing change were more apparent in the amelogenin treated group . statistical analysis showed that the amelogenin group had significantly ( p = 0.01 ) greater ulcer pain reduction . the mean and 95% ci for the difference between the amelogenin and the control group at final visit was 1.59 ( 2.84 0.34 ) . pain is considered to be a significant problem in relation to patients with vlus , and has been highlighted as cause for major concern when treating patients ( price et al 2007 ) . the european wound management association ( 2002 ) has developed a position document that provides clinical recommendations to assess and manage wound pain , especially at dressing change . pain management has therefore become a major part of wound care with many organizations and care providers incorporating pain management into standards , guidelines and clinical practice ( price et al 1997 ; young 2007 ) . thus the fact that amelogenin therapy , in a way as yet undetermined , was shown to significantly reduce the pain that patients with hard - to - heal vlus experienced is noteworthy and perhaps requires further investigation . as well as the study participants having hard - to - heal wounds , the population evaluated represents possibly the worst case scenario patients , eg , some patients within the amelogenin group had very old ulcers ( 10 years or more ) . this highlights the potential advantages of using advanced therapies for the treatment of ulcers within this patient population . the safety of amelogenin has been established by completing the extensive testing that is required for medical devices . the safety profile of amelogenin is underlined by the results of this study with few adverse events reported , and no significant difference between the treatment and control groups . a series of case studies has been presented in which a number of patients with hard - to - heal vlus were treated with amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) therapy ( huldt - nystrom et al 2007 ) . the healing of these wounds was evaluated by comparing the wound area at baseline ( before treatment ) and upon completion of treatment . in this study , the amelogenin was applied to a clean wound bed , on a weekly basis , up to a maximum of 12 weeks in conjunction with standard compression therapy . some of these patients were treated with compression for four weeks prior to being included in the evaluations , in order to ensure that ulcers were truly nonhealing . the results showed that the success rate with amelogenin therapy was high , with approximately 80% of the ulcers either healing ( 60% ) or showing a reduction in ulcer size ( 20% ) after treatment . only two ulcers ( 10% ) remained unchanged and two ulcers deteriorated ( 10% ) during the treatment period . pseudomonas colonization / infection was present in some wounds after treatment had been started , but this was treated with appropriate antibiotic therapy , and did not affect the successful healing outcome of the wounds . a small study in the united kingdom ( acton 2007 ) has demonstrated similar results in that amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) stimulated a healing response . even if the wounds did not progress to complete healing , they were significantly healthier than before the amelogenin treatment had started and were able to go on to be grafted . in addition , an improvement in quality of life was observed in the patients who received amelogenin therapy . another study undertaken in the united kingdom evaluated amelogenin for the treatment of patients ( n = 17 ) with a variety of complex hard - to - heal ulcers , including rheumatoid ulcers ( n = 2 ) , wounds complicated by rheumatoid arthritis ( n = 3 ) , neuropathic foot ulcers ( n = 4 ) , venous ulcers ( n = 4 ) , and a single ulcer of mixed etiology . the results from this study followed a similar trend to that seen in the treatment of other hard - to - heal ulcers , in that an early reduction in wound pain and exudate were apparent . overall , six wounds healed after a mean of 8 applications of ( xelma , molnlycke health care , gothenburg , sweden ) ( range 316 applications ) , six patients showed an improvement of a greater than 50% reduction in ulcer size , and were continuing to receive amelogenin therapy . only two patients were discontinued due to infection and wound deterioration ( vowden et al 2007a ) . in a case study series involving eight patients with a total of 10 vlus ( mean duration of 9.3 years ) , amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) was applied weekly and covered with a secondary dressing . treatment duration with amelogenin was 12 weeks and patients were followed up for a period of 24 weeks . an overall healing rate of 50% was reported , including one patient with a wound duration of 65 years . the other ulcers were in a healing state at 24 weeks , with a mean reduction in size of 60% compared with baseline values . exudate levels and wound odor also reduced over the treatment period ( hampton et al 2007 ) . amelogenin has recently been evaluated in a study consisting of a mixed population of patients with both diabetic foot ulcers ( n = 5 ) and vlus ( n = 5 ) . both sets of patients demonstrated initiation of healing with a high percentage ( 80% ) of ulcers healing completely ( meuleneire 2007 ) . diabetic foot ulcers have a different aetiology to that of vlus , they are a significant complication of diabetes mellitus and often precede lower - extremity amputation . the most frequent underlying aetiologies are neuropathy , trauma , deformity , high plantar pressures , and peripheral arterial disease . the primary treatment is usually offloading to reduce pressure to the sensitive areas and then management of the wound with appropriate dressings . infection and high levels of wound exudate may be complicating factors in the treatment of these wounds , but the early indications are that amelogenin therapy , in conjunction with appropriate wound management techniques , can be beneficial . pyoderma gangrenosum ( pg ) is an inflammatory ulcerative condition of unknown etiology , although an autoimmune mechanism including immune complex - mediated neutrophilic vascular reactions has been suggested . pg is frequently associated with various diseases , but up to 50% of cases are idiopathic . it is a disease that causes tissue to become necrotic , causing deep ulcers that usually occur on the legs . there are two main types of ulcers that occur : the normal ulcerative form which occurs on the legs , and an atypical form that is more superficial and occurs on the hands and other parts of the body . though the etiology is not well understood , the disease is thought to be due to immune system dysfunction and , in particular , improper functioning of neutrophils . at least half of all pg patients also suffer from illnesses that affect their systemic function . classical ulcerative pg is characterized by the appearance of nodules with pustules that enlarge and lead to chronic ulcers violaceous and undermined borders ( dini et al 2007 ) amelogenin therapy has been used to treat two female patients with recalcitrant pg of the lower leg lasting an average of 11 months ( dini et al 2007a ) . the treatment was applied weekly under an occlusive dressing for a maximum of 8 weeks in conjunction with systemic immunosuppressive therapy before and during the topical treatment . the lesions improved , showing advances in granulation tissue formation and wound size reduction ( figures 3a - c ) . additionally , pain control was reported and the therapy was well - tolerated with no adverse effects . the clinical evidence suggests that in order for advanced therapies like amelogenin to be cost effective in for example the treatment of vlus , then suitable patients must be carefully identified and according to prognostic indicators of delayed healing such as wound size ( larger than 10 cm ) and wound age ( duration of greater than 6 months ) . additionally , if wounds do not show a decrease in size of 40% or more during 4 weeks of compression therapy , then these wounds can be identified as hard - to - heal ( phillips et al 2000 ) and consideration should be given to their treatment with advanced therapies such as amelogenin . this is because it treats the underlying cause of venous hypertension and enables blood to be returned from the lower limbs , thereby reducing oedema and the consequences of stasis . infection in the wound must be treated prior to application of amelogenin , although it has been seen that concurrent treatment of infection with antibacterial therapy is not detrimental to the amelogenin protein as a therapeutic agent . additionally , wounds that exudate highly do not appear to benefit as well from amelogenin therapy as wounds that have low to moderate levels of exudate , implicit in this therefore is the requirement that exudate should be managed with compression and appropriate dressings that can absorb the fluid . this tissue impedes the growth of healthy granulation tissue and re - epithelisation and therefore must be removed . it is also thought that the presence of such dead tissue may prevent the amelogenin protein integrating into the fabric of the wound bed and thus providing the temporary scaffolding to which cells can attach and subsequently stimulate the healing process . qualitatively , vlus treated with amelogenin appear to heal much better than with high compression alone , with no evidence of hypertrophic scarring or excessive wound contracture . the fact that amelogenin can be used as a temporary ecm protein scaffold provides a sound basis for its use in the treatment of wounds , particularly chronic wounds that are deficient in normal ecm components . it was , therefore , proposed that amelogenin could be used to treat hard - to - heal vlus . initially , a preliminary clinical study was undertaken to ascertain the optimum number of applications of amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) that are required to be applied to a vlu to produce a healing response . the study compared once weekly applications of amelogenin in conjunction with high compression therapy to the hard - to - heal vlus ( duration > 6 months ) of patients over 3 , 6 and 12 week periods ( romanelli et al 2006 ) the wounds were assessed every second week up to week 8 , at final visit ( week 12 ) , and at follow - up visit ( 12 weeks after the final visit ) . the results showed that patients who received the 12-weeks treatment had a larger ulcer reduction ( 72% ) compared with patients who received the 3 or 6-weeks ( 22 and 48% respectively ) treatment with amelogenin ( figure 1 and table 1 ) . percentage change in vlu size following 3 , 6 and 12 weeks of treatment with amelogenin . reproduced with permission from romanelli m , ellervee t , jarve h , et al 2006 . amelogenins ( xelma ) in hard - to - heal venous leg ulcers , an open regime investigation [ poster ] . percentage change in vlu size following 3 , 6 and 12 weeks of treatment with amelogenin . copyright 2006 . reproduced with permission from romanelli m , ellervee t , jarve h , et al 2006 . amelogenins ( xelma ) in hard - to - heal venous leg ulcers , an open regime investigation [ poster ] . european wound management association conference , prague , czech republic the results of this preliminary study showed that hard - to - heal vlus elicited a positive healing response to a defined period ( ie , 12 weeks ) treatment with amelogenin . using this information , a single - blinded , randomized , multi - centre study involving 117 patients was undertaken to assess the effect of amelogenin on hard - to - heal vlus ( vowden et al 2006 ) . to satisfy the inclusion criteria for the study , patients had to have a vlu with a size between 5 and 25 cm and duration of at least six months that had been treated with controlled compression therapy for at least one month prior to enrolment . patients were randomised to receive either amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) plus high compression ( n = 62 ) or a control treatment consisting of a placebo gel ( the amelogenin carrier vehicle ) plus high compression ( n = 61 ) . the amelogenin was applied weekly under secondary dressings for up to a maximum of 12 weeks . although no statistical differences could be identified between the groups as a whole , the percentage wound size reduction was greater in the group treated with amelogenin . a more detailed sub - group analysis was undertaken on patients with ulcers of greater than 10 cm at baseline and for ulcers of duration greater than 6 months . the results of this analysis demonstrated that there was a difference in percentage wound reduction in the group treated with amelogenin ( 33.8% vs 25.6% , respectively ) . it was highlighted that this difference was greatest for the group of patients ( n = 61 ) with the larger ulcers ( > 10 cm ) ( amelogenin 25% vs control 7.9% ) ( figure 2 ) . median wound size reduction in the itt population and sub - group itts following treatment with amelogenin and control . reproduced with permission from vowden p , romanelli m , peter r , et al 2006 . the effect of amelogenins ( xelma ) on hard - to - heal venous leg ulcers . overall the results of this study showed that amelogenin was well tolerated by the patients , with a trend towards less pain and a reduction in the volume of exudate produced by the ulcers in favor of the group treated with amelogenin . this study highlighted the problems associated with undertaking clinical trials involving vlus , the main ones being the wide variations in wound demographics ( eg , sizes and ages ) and their response to treatments . in addition , there was a significant amount of variation between the pre - study treatment regimens employed at different centers , a phenomenon which if not accounted for by allowing a run - in period , may alter the outcome of a clinical trial . as such , the authors concluded that amelogenin could be clinically useful in the treatment of patients with ulcers that could be classified as hard - to - heal ( eg , of duration longer than 6 months and a size greater than 10 cm ) . another clinical trial was undertaken using the lessons learnt from the previous study : this was an open , randomized , comparative , parallel group , multi - centre study involving patients with hard - to - heal vlus ( vowden et al 2007 ) . the inclusion criteria incorporated ulcer size of greater than 10 cm and duration of longer than 6 months , both of which have previously been reported in the literature as prognostic indicators of venous ulcers that are unlikely to respond to treatment and may be classed as hard - to - heal ( european wound management association 2002 ) . the primary objective of this study was to compare the results of 12 consecutive weeks of treatment with amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) plus high compression versus compression therapy alone in hard - to - heal vlus . percentage reduction of wound size from baseline , the percentage of improved ulcers , and parameters such as pain related to the disease , pain at dressing changes and the amount and nature of exudate were evaluated . eligibility for inclusion included adult , mobile patients with hard - to - heal vlus that had been treated with compression therapy for at least 1 month prior to screening . the ulcers had to be at least 6 months old , with a surface area at inclusion of at least 10 cm , but not exceeding 30 cm , and not demonstrating excessive exudate or signs of infection . at the end of the run in period additional criteria for eligibility , eg , change in wound area ( increase / decrease ) of greater than or equal to 50% and a wound area between 8 and 36 cm were applied . in total , 83 patients were randomized to receive either amelogenin plus high compression bandaging ( n = 42 ) or high compression bandaging ( control ) alone ( n = 41 ) . all participants received high compression bandaging therapy one month prior to , during the investigational period of 3-weeks run - in and throughout the 12 weeks of active treatment the results of this study demonstrated that the amelogenin group showed a greater percentage reduction in ulcer size ( mean 33.11% ) compared to the control group ( mean 11.07% ) from baseline to the last visit ( p = 0.03 ) . the number of ulcers showing an improvement was significantly greater ( p = 0.01 ) in the amelogenin treated group than the control group . statistically significant differences in favor of the amelogenin group were also found the proportion of patients with none or low levels of exudate ( p = 0.01 ) . pain was evaluated in this study as related to the disease or to the ulcer . it was evaluated as part of the protocol by interviewing the patients who were asked to rate their pain on a scale with 11 steps ; from 0 = no pain to 10 = unbearable pain ( hartrick et al 2003 ) . the results showed that a reduction in pain related to the disease and a reduction in pain at dressing change were more apparent in the amelogenin treated group . statistical analysis showed that the amelogenin group had significantly ( p = 0.01 ) greater ulcer pain reduction . the mean and 95% ci for the difference between the amelogenin and the control group at final visit was 1.59 ( 2.84 0.34 ) . pain is considered to be a significant problem in relation to patients with vlus , and has been highlighted as cause for major concern when treating patients ( price et al 2007 ) . the european wound management association ( 2002 ) has developed a position document that provides clinical recommendations to assess and manage wound pain , especially at dressing change . pain management has therefore become a major part of wound care with many organizations and care providers incorporating pain management into standards , guidelines and clinical practice ( price et al 1997 ; young 2007 ) . thus the fact that amelogenin therapy , in a way as yet undetermined , was shown to significantly reduce the pain that patients with hard - to - heal vlus experienced is noteworthy and perhaps requires further investigation . as well as the study participants having hard - to - heal wounds , the population evaluated represents possibly the worst case scenario patients , eg , some patients within the amelogenin group had very old ulcers ( 10 years or more ) . this highlights the potential advantages of using advanced therapies for the treatment of ulcers within this patient population . the safety of amelogenin has been established by completing the extensive testing that is required for medical devices . the safety profile of amelogenin is underlined by the results of this study with few adverse events reported , and no significant difference between the treatment and control groups . a series of case studies has been presented in which a number of patients with hard - to - heal vlus were treated with amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) therapy ( huldt - nystrom et al 2007 ) . the healing of these wounds was evaluated by comparing the wound area at baseline ( before treatment ) and upon completion of treatment . in this study , the amelogenin was applied to a clean wound bed , on a weekly basis , up to a maximum of 12 weeks in conjunction with standard compression therapy . some of these patients were treated with compression for four weeks prior to being included in the evaluations , in order to ensure that ulcers were truly nonhealing . the results showed that the success rate with amelogenin therapy was high , with approximately 80% of the ulcers either healing ( 60% ) or showing a reduction in ulcer size ( 20% ) after treatment . only two ulcers ( 10% ) remained unchanged and two ulcers deteriorated ( 10% ) during the treatment period . pseudomonas colonization / infection was present in some wounds after treatment had been started , but this was treated with appropriate antibiotic therapy , and did not affect the successful healing outcome of the wounds . a small study in the united kingdom ( acton 2007 ) has demonstrated similar results in that amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) stimulated a healing response . even if the wounds did not progress to complete healing , they were significantly healthier than before the amelogenin treatment had started and were able to go on to be grafted . in addition , an improvement in quality of life was observed in the patients who received amelogenin therapy . another study undertaken in the united kingdom evaluated amelogenin for the treatment of patients ( n = 17 ) with a variety of complex hard - to - heal ulcers , including rheumatoid ulcers ( n = 2 ) , wounds complicated by rheumatoid arthritis ( n = 3 ) , neuropathic foot ulcers ( n = 4 ) , venous ulcers ( n = 4 ) , and a single ulcer of mixed etiology . the results from this study followed a similar trend to that seen in the treatment of other hard - to - heal ulcers , in that an early reduction in wound pain and exudate were apparent . overall , six wounds healed after a mean of 8 applications of ( xelma , molnlycke health care , gothenburg , sweden ) ( range 316 applications ) , six patients showed an improvement of a greater than 50% reduction in ulcer size , and were continuing to receive amelogenin therapy . only two patients were discontinued due to infection and wound deterioration ( vowden et al 2007a ) . in a case study series involving eight patients with a total of 10 vlus ( mean duration of 9.3 years ) , amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) was applied weekly and covered with a secondary dressing . treatment duration with amelogenin was 12 weeks and patients were followed up for a period of 24 weeks . an overall healing rate of 50% was reported , including one patient with a wound duration of 65 years . the other ulcers were in a healing state at 24 weeks , with a mean reduction in size of 60% compared with baseline values . exudate levels and wound odor also reduced over the treatment period ( hampton et al 2007 ) . amelogenin has recently been evaluated in a study consisting of a mixed population of patients with both diabetic foot ulcers ( n = 5 ) and vlus ( n = 5 ) . both sets of patients demonstrated initiation of healing with a high percentage ( 80% ) of ulcers healing completely ( meuleneire 2007 ) . diabetic foot ulcers have a different aetiology to that of vlus , they are a significant complication of diabetes mellitus and often precede lower - extremity amputation . the most frequent underlying aetiologies are neuropathy , trauma , deformity , high plantar pressures , and peripheral arterial disease . the primary treatment is usually offloading to reduce pressure to the sensitive areas and then management of the wound with appropriate dressings . infection and high levels of wound exudate may be complicating factors in the treatment of these wounds , but the early indications are that amelogenin therapy , in conjunction with appropriate wound management techniques , can be beneficial . pyoderma gangrenosum ( pg ) is an inflammatory ulcerative condition of unknown etiology , although an autoimmune mechanism including immune complex - mediated neutrophilic vascular reactions has been suggested . pg is frequently associated with various diseases , but up to 50% of cases are idiopathic . it is a disease that causes tissue to become necrotic , causing deep ulcers that usually occur on the legs . there are two main types of ulcers that occur : the normal ulcerative form which occurs on the legs , and an atypical form that is more superficial and occurs on the hands and other parts of the body . though the etiology is not well understood , the disease is thought to be due to immune system dysfunction and , in particular , improper functioning of neutrophils . at least half of all pg patients also suffer from illnesses that affect their systemic function . classical ulcerative pg is characterized by the appearance of nodules with pustules that enlarge and lead to chronic ulcers violaceous and undermined borders ( dini et al 2007 ) amelogenin therapy has been used to treat two female patients with recalcitrant pg of the lower leg lasting an average of 11 months ( dini et al 2007a ) . the treatment was applied weekly under an occlusive dressing for a maximum of 8 weeks in conjunction with systemic immunosuppressive therapy before and during the topical treatment . the lesions improved , showing advances in granulation tissue formation and wound size reduction ( figures 3a - c ) . additionally , pain control was reported and the therapy was well - tolerated with no adverse effects . the clinical evidence suggests that in order for advanced therapies like amelogenin to be cost effective in for example the treatment of vlus , then suitable patients must be carefully identified and according to prognostic indicators of delayed healing such as wound size ( larger than 10 cm ) and wound age ( duration of greater than 6 months ) . additionally , if wounds do not show a decrease in size of 40% or more during 4 weeks of compression therapy , then these wounds can be identified as hard - to - heal ( phillips et al 2000 ) and consideration should be given to their treatment with advanced therapies such as amelogenin . this is because it treats the underlying cause of venous hypertension and enables blood to be returned from the lower limbs , thereby reducing oedema and the consequences of stasis . infection in the wound must be treated prior to application of amelogenin , although it has been seen that concurrent treatment of infection with antibacterial therapy is not detrimental to the amelogenin protein as a therapeutic agent . additionally , wounds that exudate highly do not appear to benefit as well from amelogenin therapy as wounds that have low to moderate levels of exudate , implicit in this therefore is the requirement that exudate should be managed with compression and appropriate dressings that can absorb the fluid . this tissue impedes the growth of healthy granulation tissue and re - epithelisation and therefore must be removed . it is also thought that the presence of such dead tissue may prevent the amelogenin protein integrating into the fabric of the wound bed and thus providing the temporary scaffolding to which cells can attach and subsequently stimulate the healing process . qualitatively , vlus treated with amelogenin appear to heal much better than with high compression alone , with no evidence of hypertrophic scarring or excessive wound contracture . there is a raft of experimental evidence that supports a mechanism for amelogenin in facilitating the wound healing process . for example , it appears that amelogenin provides a temporary scaffold to allow cellular adherence of fibroblasts and epithelial cells . this in turn will stimulate various functions of cells with regards to the wound healing process , eg , migration ( into the wound ) , proliferation ( increasing the number of cells and filling the wound ) , and synthesis of mediators ( growth factors and cytokines ) that can regulate processes that are relevant to wound healing such as angiogenesis . this review highlights that a number of well controlled trials have established that amelogenin treatment has a statistically beneficial effect in the clinical environment . it has been shown that amelogenin can significantly reduce ulcer size and also significantly increase the number of patients showing a greater than 50% reduction in wound size , compared with comparator controls . pain is a problem in patients with vlus , but it has been shown that amelogenin therapy can reduce the level of pain experienced by patients with hard - to - heal vlus , compared with comparator controls . it is interesting to conjecture whether this is simply due to healing of the wound or whether amelogenin has a direct remedial effect upon pain , future studies may elucidate this mechanism .

amelogenins are extracellular matrix proteins that , under physiological conditions , self - assemble into globular aggregates up to micron - sizes . studies with periodontal fibroblasts indicate that attachment to these structures increases the endogenous secretion of multiple growth factors and cell proliferation . pre - clinical and clinical studies indicate that cutaneous wounds benefit from treatment with amelogenins . a randomized controlled trial ( rct ) involving patients with hard - to - heal venous leg ulcers ( vlus ) ( ie , ulcers with a surface area 10 cm2 and duration of 6 months ) showed that the application of amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) as an adjunct treatment to compression results in significant reduction in ulcer size , improvement in the state of ulcers , reduced pain , and a larger proportion of ulcers with low levels of exudate , compared with treatment with compression alone . amelogenin therapy was also shown to be safe to use in that there were no significant differences in adverse events noted between patients treated with amelogenin plus compression and those treated with compression alone . case study evaluations indicate that the benefits of amelogenin therapy demonstrated in the rct are being repeated in real life situations and that amelogenin therapy may also have a role to play in the treatment of other wound types such as diabetic foot ulcers .

Introduction In vitro studies Clinical evaluations Venous ulcers Diabetic ulcers Pyoderma gangrenosum Conclusions

venous leg ulcers ( vlus ) pose a serious clinical dilemma and an economic burden on health services . the reasons for the lack of response to treatment of hard - to - heal vlus have not yet been fully elucidated , although scientific and clinical research indicates that , instead of progressing through the four distinct but overlapping phases of healing ( hemostasis , inflammation , proliferation , and remodeling ) , these wounds become stuck in a prolonged inflammatory phase ( timmons 2006 ) . early in the wound healing process there is a requirement for the synthesis and deposition of ecm proteins such as fibrin and fibronectin ( hodde and johnson 2007 ) and a sub - set of matrix proteins that are expressed transiently during the wound healing process , eg , galectins , osteopontin , sparc , syndecans , tenascins , thrombospondins , and vitronectin ( agren and werthen 2007 ) . for example , in the case of hard - to - heal vlus , venous hypertension causes disturbed microcirculation and pathological changes to capillaries , which ultimately locks the condition in a self - amplifying cascade with persistent elevated levels of pro - inflammatory cytokines and proteases that appear to degrade the ecm components , growth factors and receptors that are essential for healing ( agren et al 2000 ; schultz et al 2005 ) the ecm is a vital component of the healing process ; intact or fragmented ecm molecules play a central role in modulating cells through transduction of a variety of signaling mechanisms ( agren et al 2000 ) . on this basis , there is a clear need for new and advanced interventions that can interact with hard - to - heal vlus that are locked in the inflammatory phase of the healing process and progress them to the subsequent proliferative stage of the process . one mechanism by which this can be achieved is to provide hard - to - heal ulcers with surrogate ecms which , although not homologous to the patient s own ecm , will provide transient structures that allow cellular adhesion and facilitate tissue regeneration by advancing the wounds to the proliferative phase of the healing process . at physiological conditions , amelogenin self - assembles into globular aggregates up to micron - sizes . when applied to the wound bed it provides a temporary matrix for cell attachment and promotes wound healing amelogenin has been used successfully to treat patients with hard - to - heal vlus and other chronic wound types . the fact that amelogenin can be used as a temporary ecm protein scaffold provides a sound basis for its use in the treatment of wounds , particularly chronic wounds that are deficient in normal ecm components . it was , therefore , proposed that amelogenin could be used to treat hard - to - heal vlus . initially , a preliminary clinical study was undertaken to ascertain the optimum number of applications of amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) that are required to be applied to a vlu to produce a healing response . the study compared once weekly applications of amelogenin in conjunction with high compression therapy to the hard - to - heal vlus ( duration > 6 months ) of patients over 3 , 6 and 12 week periods ( romanelli et al 2006 ) the wounds were assessed every second week up to week 8 , at final visit ( week 12 ) , and at follow - up visit ( 12 weeks after the final visit ) . the results showed that patients who received the 12-weeks treatment had a larger ulcer reduction ( 72% ) compared with patients who received the 3 or 6-weeks ( 22 and 48% respectively ) treatment with amelogenin ( figure 1 and table 1 ) . amelogenins ( xelma ) in hard - to - heal venous leg ulcers , an open regime investigation [ poster ] . amelogenins ( xelma ) in hard - to - heal venous leg ulcers , an open regime investigation [ poster ] . european wound management association conference , prague , czech republic the results of this preliminary study showed that hard - to - heal vlus elicited a positive healing response to a defined period ( ie , 12 weeks ) treatment with amelogenin . using this information , a single - blinded , randomized , multi - centre study involving 117 patients was undertaken to assess the effect of amelogenin on hard - to - heal vlus ( vowden et al 2006 ) . to satisfy the inclusion criteria for the study , patients had to have a vlu with a size between 5 and 25 cm and duration of at least six months that had been treated with controlled compression therapy for at least one month prior to enrolment . patients were randomised to receive either amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) plus high compression ( n = 62 ) or a control treatment consisting of a placebo gel ( the amelogenin carrier vehicle ) plus high compression ( n = 61 ) . the results of this analysis demonstrated that there was a difference in percentage wound reduction in the group treated with amelogenin ( 33.8% vs 25.6% , respectively ) . the effect of amelogenins ( xelma ) on hard - to - heal venous leg ulcers . overall the results of this study showed that amelogenin was well tolerated by the patients , with a trend towards less pain and a reduction in the volume of exudate produced by the ulcers in favor of the group treated with amelogenin . as such , the authors concluded that amelogenin could be clinically useful in the treatment of patients with ulcers that could be classified as hard - to - heal ( eg , of duration longer than 6 months and a size greater than 10 cm ) . another clinical trial was undertaken using the lessons learnt from the previous study : this was an open , randomized , comparative , parallel group , multi - centre study involving patients with hard - to - heal vlus ( vowden et al 2007 ) . the inclusion criteria incorporated ulcer size of greater than 10 cm and duration of longer than 6 months , both of which have previously been reported in the literature as prognostic indicators of venous ulcers that are unlikely to respond to treatment and may be classed as hard - to - heal ( european wound management association 2002 ) . the primary objective of this study was to compare the results of 12 consecutive weeks of treatment with amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) plus high compression versus compression therapy alone in hard - to - heal vlus . eligibility for inclusion included adult , mobile patients with hard - to - heal vlus that had been treated with compression therapy for at least 1 month prior to screening . the ulcers had to be at least 6 months old , with a surface area at inclusion of at least 10 cm , but not exceeding 30 cm , and not demonstrating excessive exudate or signs of infection . statistically significant differences in favor of the amelogenin group were also found the proportion of patients with none or low levels of exudate ( p = 0.01 ) . thus the fact that amelogenin therapy , in a way as yet undetermined , was shown to significantly reduce the pain that patients with hard - to - heal vlus experienced is noteworthy and perhaps requires further investigation . as well as the study participants having hard - to - heal wounds , the population evaluated represents possibly the worst case scenario patients , eg , some patients within the amelogenin group had very old ulcers ( 10 years or more ) . the safety profile of amelogenin is underlined by the results of this study with few adverse events reported , and no significant difference between the treatment and control groups . a series of case studies has been presented in which a number of patients with hard - to - heal vlus were treated with amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) therapy ( huldt - nystrom et al 2007 ) . the results showed that the success rate with amelogenin therapy was high , with approximately 80% of the ulcers either healing ( 60% ) or showing a reduction in ulcer size ( 20% ) after treatment . a small study in the united kingdom ( acton 2007 ) has demonstrated similar results in that amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) stimulated a healing response . another study undertaken in the united kingdom evaluated amelogenin for the treatment of patients ( n = 17 ) with a variety of complex hard - to - heal ulcers , including rheumatoid ulcers ( n = 2 ) , wounds complicated by rheumatoid arthritis ( n = 3 ) , neuropathic foot ulcers ( n = 4 ) , venous ulcers ( n = 4 ) , and a single ulcer of mixed etiology . the results from this study followed a similar trend to that seen in the treatment of other hard - to - heal ulcers , in that an early reduction in wound pain and exudate were apparent . overall , six wounds healed after a mean of 8 applications of ( xelma , molnlycke health care , gothenburg , sweden ) ( range 316 applications ) , six patients showed an improvement of a greater than 50% reduction in ulcer size , and were continuing to receive amelogenin therapy . in a case study series involving eight patients with a total of 10 vlus ( mean duration of 9.3 years ) , amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) was applied weekly and covered with a secondary dressing . infection and high levels of wound exudate may be complicating factors in the treatment of these wounds , but the early indications are that amelogenin therapy , in conjunction with appropriate wound management techniques , can be beneficial . the clinical evidence suggests that in order for advanced therapies like amelogenin to be cost effective in for example the treatment of vlus , then suitable patients must be carefully identified and according to prognostic indicators of delayed healing such as wound size ( larger than 10 cm ) and wound age ( duration of greater than 6 months ) . additionally , if wounds do not show a decrease in size of 40% or more during 4 weeks of compression therapy , then these wounds can be identified as hard - to - heal ( phillips et al 2000 ) and consideration should be given to their treatment with advanced therapies such as amelogenin . additionally , wounds that exudate highly do not appear to benefit as well from amelogenin therapy as wounds that have low to moderate levels of exudate , implicit in this therefore is the requirement that exudate should be managed with compression and appropriate dressings that can absorb the fluid . the fact that amelogenin can be used as a temporary ecm protein scaffold provides a sound basis for its use in the treatment of wounds , particularly chronic wounds that are deficient in normal ecm components . it was , therefore , proposed that amelogenin could be used to treat hard - to - heal vlus . initially , a preliminary clinical study was undertaken to ascertain the optimum number of applications of amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) that are required to be applied to a vlu to produce a healing response . the study compared once weekly applications of amelogenin in conjunction with high compression therapy to the hard - to - heal vlus ( duration > 6 months ) of patients over 3 , 6 and 12 week periods ( romanelli et al 2006 ) the wounds were assessed every second week up to week 8 , at final visit ( week 12 ) , and at follow - up visit ( 12 weeks after the final visit ) . the results showed that patients who received the 12-weeks treatment had a larger ulcer reduction ( 72% ) compared with patients who received the 3 or 6-weeks ( 22 and 48% respectively ) treatment with amelogenin ( figure 1 and table 1 ) . amelogenins ( xelma ) in hard - to - heal venous leg ulcers , an open regime investigation [ poster ] . amelogenins ( xelma ) in hard - to - heal venous leg ulcers , an open regime investigation [ poster ] . european wound management association conference , prague , czech republic the results of this preliminary study showed that hard - to - heal vlus elicited a positive healing response to a defined period ( ie , 12 weeks ) treatment with amelogenin . using this information , a single - blinded , randomized , multi - centre study involving 117 patients was undertaken to assess the effect of amelogenin on hard - to - heal vlus ( vowden et al 2006 ) . to satisfy the inclusion criteria for the study , patients had to have a vlu with a size between 5 and 25 cm and duration of at least six months that had been treated with controlled compression therapy for at least one month prior to enrolment . patients were randomised to receive either amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) plus high compression ( n = 62 ) or a control treatment consisting of a placebo gel ( the amelogenin carrier vehicle ) plus high compression ( n = 61 ) . the results of this analysis demonstrated that there was a difference in percentage wound reduction in the group treated with amelogenin ( 33.8% vs 25.6% , respectively ) . the effect of amelogenins ( xelma ) on hard - to - heal venous leg ulcers . overall the results of this study showed that amelogenin was well tolerated by the patients , with a trend towards less pain and a reduction in the volume of exudate produced by the ulcers in favor of the group treated with amelogenin . as such , the authors concluded that amelogenin could be clinically useful in the treatment of patients with ulcers that could be classified as hard - to - heal ( eg , of duration longer than 6 months and a size greater than 10 cm ) . another clinical trial was undertaken using the lessons learnt from the previous study : this was an open , randomized , comparative , parallel group , multi - centre study involving patients with hard - to - heal vlus ( vowden et al 2007 ) . the inclusion criteria incorporated ulcer size of greater than 10 cm and duration of longer than 6 months , both of which have previously been reported in the literature as prognostic indicators of venous ulcers that are unlikely to respond to treatment and may be classed as hard - to - heal ( european wound management association 2002 ) . the primary objective of this study was to compare the results of 12 consecutive weeks of treatment with amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) plus high compression versus compression therapy alone in hard - to - heal vlus . eligibility for inclusion included adult , mobile patients with hard - to - heal vlus that had been treated with compression therapy for at least 1 month prior to screening . the ulcers had to be at least 6 months old , with a surface area at inclusion of at least 10 cm , but not exceeding 30 cm , and not demonstrating excessive exudate or signs of infection . statistically significant differences in favor of the amelogenin group were also found the proportion of patients with none or low levels of exudate ( p = 0.01 ) . thus the fact that amelogenin therapy , in a way as yet undetermined , was shown to significantly reduce the pain that patients with hard - to - heal vlus experienced is noteworthy and perhaps requires further investigation . as well as the study participants having hard - to - heal wounds , the population evaluated represents possibly the worst case scenario patients , eg , some patients within the amelogenin group had very old ulcers ( 10 years or more ) . the safety profile of amelogenin is underlined by the results of this study with few adverse events reported , and no significant difference between the treatment and control groups . a series of case studies has been presented in which a number of patients with hard - to - heal vlus were treated with amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) therapy ( huldt - nystrom et al 2007 ) . the results showed that the success rate with amelogenin therapy was high , with approximately 80% of the ulcers either healing ( 60% ) or showing a reduction in ulcer size ( 20% ) after treatment . a small study in the united kingdom ( acton 2007 ) has demonstrated similar results in that amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) stimulated a healing response . another study undertaken in the united kingdom evaluated amelogenin for the treatment of patients ( n = 17 ) with a variety of complex hard - to - heal ulcers , including rheumatoid ulcers ( n = 2 ) , wounds complicated by rheumatoid arthritis ( n = 3 ) , neuropathic foot ulcers ( n = 4 ) , venous ulcers ( n = 4 ) , and a single ulcer of mixed etiology . the results from this study followed a similar trend to that seen in the treatment of other hard - to - heal ulcers , in that an early reduction in wound pain and exudate were apparent . overall , six wounds healed after a mean of 8 applications of ( xelma , molnlycke health care , gothenburg , sweden ) ( range 316 applications ) , six patients showed an improvement of a greater than 50% reduction in ulcer size , and were continuing to receive amelogenin therapy . in a case study series involving eight patients with a total of 10 vlus ( mean duration of 9.3 years ) , amelogenin ( xelma , molnlycke health care , gothenburg , sweden ) was applied weekly and covered with a secondary dressing . infection and high levels of wound exudate may be complicating factors in the treatment of these wounds , but the early indications are that amelogenin therapy , in conjunction with appropriate wound management techniques , can be beneficial . the clinical evidence suggests that in order for advanced therapies like amelogenin to be cost effective in for example the treatment of vlus , then suitable patients must be carefully identified and according to prognostic indicators of delayed healing such as wound size ( larger than 10 cm ) and wound age ( duration of greater than 6 months ) . additionally , if wounds do not show a decrease in size of 40% or more during 4 weeks of compression therapy , then these wounds can be identified as hard - to - heal ( phillips et al 2000 ) and consideration should be given to their treatment with advanced therapies such as amelogenin . additionally , wounds that exudate highly do not appear to benefit as well from amelogenin therapy as wounds that have low to moderate levels of exudate , implicit in this therefore is the requirement that exudate should be managed with compression and appropriate dressings that can absorb the fluid . it has been shown that amelogenin can significantly reduce ulcer size and also significantly increase the number of patients showing a greater than 50% reduction in wound size , compared with comparator controls . pain is a problem in patients with vlus , but it has been shown that amelogenin therapy can reduce the level of pain experienced by patients with hard - to - heal vlus , compared with comparator controls .

the vertebrate innate immune system provides protection against a wide range of pathogenic microorganisms , and defensins are an important component of this response as well as having a role in adaptive immunity . in mammals , the defensins can be divided into the - and -defensin subfamilies on the basis of differences in the spacing of six , conserved cysteine residues . the -defensins are produced by neutrophils and intestinal paneth cells , whereas the -defensins are mainly produced by epithelial cells in contact with the environment . the functions of human -defensins seem to be disrupted in cystic fibrosis and inflammatory skin lesions such as psoriasis . a fuller knowledge of the human complement of -defensins may therefore be useful in understanding human disease as well as in the design of novel , synthetic antimicrobial peptides . the known human -defensin genes show a conserved two - exon structure : the first exon encodes a signal peptide whereas the second exon encodes a short propiece and the mature defensin peptide with a characteristic six - cysteine motif and many basic amino - acid residues . the -defensin genes are present at five syntenic loci in the human and mouse genomes , with the main locus on human chromosome 8p22 - 23 and mouse chromosome 8a3 . all four , full - length , human -defensins that are present in the public databases are from 8p22 - 23 ( genbank sequence accession numbers are human -defensin 1 or defb1 , q09753 ; defb4 ( formerly defb2 ) , o15263 ; defb103 ( formerly defb3 ) , np_061131 ; defb104 ( formerly defb4 ) , cac85520 ) , but there are substantial differences in their coding sequences , expression patterns and antimicrobial activities . defb1 is constitutively expressed in many tissues ( respiratory tract , kidney , urogenital and oral cavity epithelia ) whereas defb4 is expressed in response to bacterial infection or proinflammatory agonists in respiratory tract epithelial cells , and epidermal and gingival keratinocytes . both defb1 and defb4 proteins have salt - sensitive , bactericidal activity against a spectrum of gram - positive and gram - negative enteric , urinary tract , and respiratory bacteria in vitro . defb103 is expressed in epithelial cells , adult heart , skeletal muscle , placenta and fetal thymus , it has broad - spectrum antimicrobial activity under conditions of low salt and ( unusually among -defensins ) it retains activity against staphylococcus aureus even in physiological saline . defb104 achieves highest expression in the testis ( with lower levels in gastric antrum , neutrophils , uterus , thyroid , lung and kidney ) and was found to be inducible in the respiratory epithelium upon exposure to pseudomonas aeruginosa or streptococcus pneumoniae . the evolution of various genes involved in the vertebrate immune system has involved duplication followed by selection to provide responses to a wide range of pathogens , with well documented examples in immunoglobulin and major histocompatibility complex genes . hughes and yeager studied the evolution of -defensins and found evidence for duplication followed by diversification driven by positive selection . similar phenomena were also implicated in the evolution of bovine -defensins and amphibian antimicrobial peptides . here we describe a combined strategy to identify further -defensin genes in the draft human genome sequence using computational techniques and verification using reverse transcription - pcr . four full - length , novel genes ( defb105 , defb106 , defb107 , defb108 ) and a related pseudogene defb109p are reported , as well as their expression patterns and evidence for their evolution by duplication and positive selection . all tblastx matches to human bacterial artificial chromosome ( bac ) clone sequences were in the 8p22-p23 region in a subsection of fpc contig ctg45 ( 1 april freeze washu accession map layout files ) bounded by the bac clones rp11 - 161b1 ( ac079018 ) and scb-177k12 ( af252831 ) and consisting of 53 bacs in total . these 53 bacs were deemed to represent the human -defensin gene family locus and were masked for repetitive sequences using repeatmasker . this locus was the subject of a more sensitive search for the presence of novel human -defensins , using a hidden markov model constructed from an alignment of the genbank -defensin sequences mentioned above . as well as the known , full - length human -defensins and the related epididymis - specific spag11 ( formerly ep2 ) gene , two novel -defensin genes , defb105 and defb106 , were identified in this search and were then incorporated into the previous hidden markov model . further searches with this revised model identified a further three genes : defb107 , defb108 and defb109p . none of these five genes was found in the embl sequence database ( 24 june 2002 release ) or in the ensembl genomic annotation database ( version 6.28.1 ) . the novel gene defb109p appears to be a pseudogene as it contains a premature stop codon within its first exon , as observed in three independently sequenced , overlapping 8p22-p23 bac sequences ( accession numbers ac068974 , ac087203 and af252830 ) . given the absence of premature stop codons in the other four genes , despite considerable divergence among them ( they encode only 18 - 28% identical amino - acid residues ) , it is unlikely that they too are pseudogenes . many putative final exon fragments from novel mouse -defensins were recently reported by schutte et al . . they used computational techniques to identify sequences matching a central portion of the mature defensin peptide including the six - cysteine motif characteristic of -defensins . at best , this method could only identify incomplete final exons encoding this region of the peptide . no attempt was made to delineate precisely the boundaries of final exons or to identify first exons by these authors . in the present study we restrict our attention to complete genes , present in a bac - clone - based map of the region and verified as encoding real transcripts by rt - pcr . our full - length novel genes correspond to five final exon fragments ( defb5 , defb6 , defb7 , defb8 and defb9 ) reported by schutte et al . and we have adopted the official hugo human gene nomenclature committee names for these fragments : defb105 , defb106 , defb107 , defb108 and defb109p ( amended from defb109 ) respectively . rt - pcr amplification confirmed the presence of the computationally predicted , functional genes , but sequencing of these products and subsequent alignment to genomic sequence also uncovered several instances where the actual gene structures differed from those that were predicted . in two cases , defb106 and defb107 , splice sites other than those predicted were used and it was found that the predicted coding sequences had been respectively 36 base - pairs ( bp ) longer and 9 bp shorter than the actual coding sequences . in the case of defb105 rt - pcr verified the two predicted exons but also uncovered an additional , 42-bp intervening exon . the rt - pcr analysis also produced interesting results with respect to the coding sequence of defb108 . in four independent , sequenced rt - pcr products from one mrna sample , the amplified defb108 sequence was consistently found to differ from the computationally predicted sequence from the human public draft sequence at three nucleotide sites , but was otherwise identical . the three differences observed between predicted and amplified sequences respectively were an a to g at nucleotide 62 ( which causes a conservative amino - acid change from lysine to arginine ) , a t to c at nucleotide 111 ( which is a synonymous change ) and a c to t at nucleotide 120 ( also synonymous ) . this indicates novel human polymorphisms in defb108 and is consistent with observations of high degrees of polymorphism for other human -defensins ( see also additional data files 1 - 6 for the novel gene sequences confirmed by rt - pcr and the computationally predicted sequence of defb109p ) . figure 1a depicts the relative positions and orientations of the four novel genes , the novel pseudogene defb109p and four known genes in the vicinity : defb2 - 4 and the related epididymis - specific spag11 . a phylogenetic tree relating functional human -defensins ( figure 1b ) reflects the spatial distribution of these genes , with the cluster of genes in figure 1a appearing to form a clade separate from defb1 . the simplest explanation for the origin of this cluster is a series of local duplication events followed by substantial divergence . in addition it seems that the four novel functional genes are more closely related to defb104 than to defb4 or defb103 . three of these genes , defb106 , defb105 and defb108 , encode mature peptides exhibiting the same spacing of conserved cysteine residues as seen in defb104 , although in defb105 there is an extra cysteine residue ( amino acid 43 ) towards the amino - terminal end of the mature peptide . defb105 also encodes an unusually long propiece peptide region in the second of its three exons . strikingly , the defb107 protein lacks the first canonical -defensin cysteine altogether and instead has a serine residue at the same position ( figure 2 ) . the changes in the number of cysteine residues seen in defb105 and defb107 are likely to have important functional consequences . the predicted mature peptides for the four novel , functional -defensins presented here have a similar proportion of cationic residues to human defb104 , with higher proportions of anionic residues ( 10 - 13% ) than are seen in human defb1 , defb4 and defb103 ( less than 4% ) . indeed the pi of defb107 and defb108 are 6.74 and 6.89 respectively , whereas all other -defensins described to date are cationic . this relative increase in anionic residues is expected to affect function , as the action of defensins initially involves interactions between the cationic mature defensin peptides and anionic membrane lipids . expression analysis for the novel , functional human genes was carried out by rt - pcr on a panel of human rna samples and the novel gene pcr products were confirmed by hybridization to an internal probe ( figure 3 ) . expression of all four novel genes was readily detected in testis . a longer exposure period revealed low levels of expression of defb108 in the liver ( figure 3 , left - hand panel of defb108 ) . six genes highly similar ( 85 - 98% identical at the amino - acid level ) to three of the novel human -defensins ( defb105 , defb106 and defb107 ) as well as to defb4 , defb103 and defb104 , were found within two olive baboon ( papio cynocephalus anubis ) draft genomic sequences ( genbank accession numbers ac116558 and ac116559 ) using blast . full - length sequences were obtained for each baboon gene except the putative defb4 ortholog ( which lacks a first exon because of gaps in the draft genomic sequences ) ; in all other cases the exonic structure of the putative baboon ortholog was identical to that of the human gene ( figure 2 ) . it is very likely that these sequences originate from the baboon locus orthologous to the human region under study , but without more complete sequence or mapping data for the baboon genome it is impossible to be certain . these novel baboon gene sequences ( see additional data files for the accession numbers ) , together with the published sequence for olive baboon -defensin 1 ( aak61474 ) and the full - length human -defensins formed the basis for our evolutionary analyses . figure 4 shows dn ( number of nonsynonymous substitutions per nonsynonymous site ) plotted against ds ( number of synonymous substitutions per synonymous site ) for comparisons between the first exon ( which encodes the signal peptide ) and second exon ( which encodes the mature defensin ) from all human and baboon genes ( the full dn , ds and dn - ds estimates for all first and second exon comparisons are available as additional data file 7 ) . exceeds dn ( figure 4a ) and this excess is statistically significant in almost every case according to two - tailed z - test results for all human and baboon genes , but there were no significant excesses of ds according to the more rigorous fisher 's exact test ( data not shown ) . thus the rates of substitution in first exons indicate that they are evolving approximately neutrally , perhaps under weak purifying selection . the pattern seen in the second exon comparisons is quite different ( figure 4b ) . in the second exons dn often exceeds ds , and even in these short sequences , for certain comparisons this excess reaches statistical significance . significant excesses of dn over ds are seen between defb1 and defb104 and between defb103 and defb107 , with similar effects seen among the second exons of orthologous baboon genes ( table 1 ) using the method of nei and gojobori . moreover , in these comparisons ds tends to be rather low relative to the rest of the data set ( mean ds = 0.464 ) . similar results are obtained using this method either modified to take account of the transition - to - transversion ratio r or using the jukes - cantor correction , although the unmodified method is thought to be a more reliable basis for the detection of positive selection . if we assume that synonymous substitutions ( which are selectively neutral or nearly so ) have accumulated regularly with time , such a pattern of substitution suggests that duplication was followed by rapid nonsynonymous change that subsequently decelerated . most comparisons involving the second exons of defb103 , defb104 and defb107 with the second exons of other genes in the dataset show excesses of dn over ds , but these excesses fail to reach significance by either of the tests used ( data not shown ) . although some of the comparisons shown in figure 4b indicate an excess of ds over dn , this is never significant by either of the two statistical tests used . there is no detectable similarity between the introns of the genes under study except between the five putatively orthologous pairs of human and baboon genes where intronic sequence is available ( defb103 , defb104 , defb105 , defb106 and defb107 ) : each pair shares 74 - 91% identity over 80 - 99% of their lengths in spite of many small indel events . in every case these pairs of orthologous introns show a substitution rate that is not significantly different from the value of ds obtained for the coding sequences . in particular , the orthologous intron sequence comparisons for defb103 , defb104 and defb107 give substitution - rate estimates of 0.1066 0.010 , 0.0816 0.005 and 0.0666 0.004 respectively , which are consistent with the ds estimates for the coding sequences of these pairs : 0.1666 0.077 , 0.0856 0.058 and 0.0536 0.049 . thus , the excess of nonsynonymous substitutions observed for the second exons of these genes is not attributable to artificially low ds estimates as a result of sampling error , but is caused by a real increase in dn relative to ds , which is the pattern expected to be generated by positive selection . averages for the ratio of radical to conservative amino - acid changes , pr / pc calculated over 47 mammalian genes were reported as 0.81 and 0.49 for charge and the miyata - yasunaga ( polarity and volume ) classification respectively . the equivalent values from table 1 , for comparisons between the second exons of genes showing evidence of positive selection , are all greater than these averages . furthermore table 1 shows that there has been a higher rate of change with respect to charge than with respect to polarity and volume ( miyata - yasunaga amino - acid classification ) . that is , where there is evidence of positive selection , most nonsynonymous changes have tended to change the charges of the residues encoded but have tended to conserve the polarities and volumes of those residues . likelihood ratio tests ( lrts ) , as implemented by the paml package also indicate the operation of positive selection at sites within second exons . these tests indicate whether data ( the substitutions inferred from an alignment ) are best explained by one of two models of = dn / ds . since is a measure of selective pressure on proteins , these models can be used to assess the evidence for variable selective pressures among sites . in a test for positive selection ( the presence of sites at which > 1 ) two statistical distributions are compared : a null model that uses a distribution that does not allow for sites with > 1 and another model which does allow such sites . three pairs of site - specific likelihood models were compared that assume variable selective pressure ( as determined by the value of ) among sites but no variation among sequences in the dataset : m0 ( one - ratio ) and m3 ( discrete ) , m1 ( neutral ) and m2 ( selection ) , and m7 ( beta ) and m8 ( beta + ) . the discrete model ( m3 ) with two site classes suggested that 41% of second - exon sites are under positive selection with = 1.67 and identified nine amino - acid sites under positive selection at the 95% cutoff . m3 was a significantly better fit to the data than the one - ratio model m0 ; the lrt statistic is 2l = -1061.79-(-1004.12 ) = 115.32 , and p < 0.001 with two degrees of freedom . however the m0-m3 comparison is essentially a test of variability in the ratio among sites and does not constitute a rigorous test of positive selection . model m1 ( neutral ) assumes two site classes with = 0 and 1 = 1 fixed and with the proportions p and p1 estimated . model m2 ( selection ) adds a third site class with the ratio 2 estimated , it suggests that about 47% of sites are under positive selection with 2 = 53.17 and identified 12 amino - acid sites under positive selection at the 95% cutoff . the two models can be compared using an lrt as follows , 2l = -1030.51 -(-1022.03 ) = 16.97 ; p < 0.001 with 2 df . the beta distribution is limited to values between 0 and 1 , providing the most flexible null hypothesis for testing positive selection . model m8 ( beta + ) adds another site class to m7 ( beta ) , with the ratio estimated from the data . however , the difference between m7 and m8 is not statistically significant , as indicated by the lrt : 2l = -1006.44 -(-1006.44 ) = 0 . nine particular sites were implicated ( in both m2 and m3 models ) as being under positive selection with greater than 95% confidence : positions 43 , 44 , 48 , 52 , 56 , 57 , 69 , 70 and 73 in figure 2a . all these positions are close or adjacent to a conserved cysteine residue and so it is possible they are important in determining -defensin structure . in summary , the paml analysis indicates that varies significantly between sites , and in two separate lrts the parameters estimated suggest a substantial proportion of sites are under positive selection . in spite of this , the most stringent test ( m7 versus m8 ) does not indicate a significant difference from neutrality . this result may be attributable to the extremely short lengths ( 33 codons aligned omitting positions with gaps ) of the sequences aligned , such an effect was seen when analyzing short ( 130 codons ) lysozyme sequences in the same way . this study represents the most detailed study of a human -defensin cluster to date , including the full - length sequences of four novel genes ( defb105 , defb106 , defb107 , defb108 ) , and a novel pseudogene ( defb109p ) , their expression patterns and sequences for the baboon orthologs of six genes from this cluster . the 8p22 - 23 defensin locus appears to have evolved by successive rounds of duplication followed by substantial divergence , to produce a diverse cluster of paralogous genes defined by these four novel genes and four known -defensin genes ( defb4 , defb103 and defb104 ) . divergence has been most rapid within the second exons of these genes , which encode the mature -defensin peptide , with many comparisons between paralogous genes showing an excess of nonsynonymous over synonymous substitutions . statistically significant evidence of elevated nonsynonymous change is seen by two methods in the second exons , indicating the action of positive selection . by contrast , comparisons between the first exons of genes from this cluster , which encode a signal peptide , show an excess of synonymous substitutions consistent with neutral evolution or weak purifying selection . the duplication and subsequent positive selection of these genes predates human - baboon divergence more than 23 million years ago and is consistent with observations that defb1 has undergone very little change during the evolution of primates . the positive selection observed has tended to change the charges of residues encoded more than other qualities such as residue polarity or volume . as seems to be the case with other antimicrobial peptides , such as mhc receptors , immunoglobulins and -defensins [ 6 - 8 ] , this selection may be a response to the rapid evolution of pathogens . in this study the computationally predicted gene structures were found by laboratory work to deviate from the actual structures in three out of five novel genes . these errors in the predictions arose in spite of the fact that the predictions were based on all the -defensin protein - sequence data available and involved three completed bac sequences rather than unfinished , gapped sequence . this has implications for purely computational approaches to novel gene discovery such as that of schutte et al . . defb104 and defb108 have no detectable orthologs in the mouse genome and therefore appear to have arisen by duplication since the divergence of rodents and primates ; alternatively , there could have been a loss of these genes in the rodent lineage . they are also the best candidates for primate - specific -defensins as they lack orthologs within all other known mammalian defensins , although our knowledge of mammalian defensins is currently incomplete . as we have shown , the evolution of the defb104 mature peptide has been driven by positive selection since its emergence , which is consistent with its novel antimicrobial properties . all defensins were thought to exist as monomers stabilized by three disulfide bridges between their three pairs of conserved cysteines . however , when compared with other known human -defensins ( defb1 , defb4 and defb103 ) , defb104 was found to have a different number of residues between its second and third , and between its fourth and fifth cysteine residues . furthermore , defb104 was found to have bactericidal activity against pseudomonas aeruginosa that was more than sixfold stronger than for any other -defensin . three of the novel genes described here ( defb105 , defb106 and defb108 ) encode mature peptides exhibiting the same spacing of conserved cysteine residues as seen in defb104 as well as sharing similar expression patterns , with highest expression in the testes . the functional divergence of -defensin genes appears to have continued following human - baboon divergence , as exemplified by defb107 which displays a serine residue instead of the first canonical cysteine seen in the baboon ortholog . it is notable that a novel mouse -defensin gene ( defr1 ) , which also lacks the first canonical cysteine , has potent antimicrobial activity against a spectrum of pathogens . in addition a polymorphism in the defb1 gene which alters the first canonical cysteine to a serine residue has been shown to produce a peptide which is as active against the microorganisms tested as the usual form . the unusual amino - acid composition of the proteins encoded by the novel genes presented here suggests that they may possess novel functions , indeed the cationic nature of -defensins is lost in defb107 and defb108 . as shown recently , there may be more subtle consequences of variation in -defensin protein sequences , affecting dimerization as well as net charge and disulfide bridges . it is worth noting that recent research has identified additional functions for -defensins that link the innate and adaptive immune response . both human and mouse -defensins have been shown to be chemotactic for immature dendritic cells and memory t cells via the ccr6 chemokine receptor . the mouse -defensin defb2 has been shown to act directly on immature dendritic cells as an endogenous ligand for toll - like receptor 4 ( tlr-4 ) , inducing upregulation of co - stimulatory molecules and dendritic cell maturation . these events , in turn , trigger robust , type-1 polarized adaptive immune responses in vivo , which suggests that -defensins may have an important role in immunosurveillance against pathogens . the expression of the novel antimicrobial peptides reported here in the human male reproductive tract is also of interest . recent work has shown that the male urogenital tracts in mammals express a broad range of - and -defensins , with human defb1 expressed in testicular biopsies , seminal plasma and ejaculated spermatozoa . together with our results demonstrating that defb105 , defb106 , defb107 and defb108 are predominantly expressed in the testes , these data suggest that the male reproductive tract has a complex innate defense mechanism . the 8p22 - 23 -defensin locus has evolved by duplication and subsequent divergence , to produce a diverse cluster of paralogous genes defined by four novel genes ( defb105 , defb106 , defb107 , defb108 ) , a novel pseudogene ( defb109p ) , and three known genes ( defb4 , defb103 and defb104 ) . we present full - length sequences for the four novel genes , their expression patterns and the predicted sequences for the baboon orthologs of six genes from this cluster . although comparisons among first - exon sequences of these human genes show little variation , the second - exon sequences that encode the mature -defensin peptides show substantial divergence . evolutionary analyses suggest that the divergence seen in second exons has involved positive selection disproportionately favoring alterations in the charge of amino - acid residues . , np_038784 , np_062702 , np_109659 , np_473415 , np_631966 , cac44635 , np_631965 , cad26894 , cad26895 ) , -defensin 13 ( np_631969 ) , -defensin 15 ( np_631970 ) , -defensin 35 ( np_631970 ) , defensin - related peptide ( aj344114 ) . human : defb1 ( q09753 ) , defb4 ( o15263 ) , defb103 ( np_061131 ) , defb104 ( cac85520 ) . rat : rattus norvegicus -defensin 1 ( np_113998 ) , -defensin 2 ( o88514 ) . cow : bos taurus -defensin 1 ( p46159 ) , -defensin 2 ( p46160 ) , -defensin 3 ( p46161 ) , -defensin 4 ( p46162 ) , -defensin 5 ( p46163 ) , -defensin 6 ( p46164 ) , -defensin 7 ( p46165 ) , -defensin 8 ( p46166 ) , -defensin 9 ( p46167 ) , -defensin 10 ( p46168 ) , -defensin 11 ( p46169 ) , -defensin 12 ( p46170 ) , -defensin 13 ( p46171 ) , tracheal antimicrobial peptide ( p25068 ) , lingual antimicrobial peptide ( q28880 ) , enteric -defensin ( o02775 ) , -defensin c7 ( o18815 ) . pig : sus scrofa -defensin 1 ( o62697 ) . goat : capra hircus -defensin 1 ( o97946 ) , -defensin 2 ( caa08905 ) . rhesus monkey : macaca mulatta -defensin 1 ( o18794 ) , -defensin 2 ( aak26259 ) . chimpanzee : pan troglodytes -defensin 1 ( aaf04110 ) , -defensin 2 ( aaf20154 ) , -defensin 3 ( aak61549 ) . these sequences were used as tblastx ( version 2.1.1 with default settings ) queries against the htg ( high - throughput genomic ) section of the embl database ( 15 july 2001 release ) . hidden markov models were constructed using hmmer ( version 2.1.1 ) to process clustalw ( version 1.82 with default settings ) multiple sequence alignments . these models were searched against genomic sequence using wise2 ( version 2 - 1 - 20c with the human gene model option ) . clustalw alignments of diverse second - exon sequences were corrected using the patterns of gaps seen in the corresponding protein - sequence alignments that were more highly conserved . all phylogenetic trees were constructed by the neighbour - joining method based on the proportion of amino - acid sites at which sequences compared were different and omitting alignment gaps . the trees constructed were rooted with chicken gallinacin 1 ( gggal1 ; p46156 ) and the reliability of each branch was assessed using 1,000 bootstrap replications . in pairwise comparisons between nucleotide sequences , the number of synonymous substitutions per synonymous site ( ds ) and the number of nonsynonymous substitutions per nonsynonymous site ( dn ) were estimated using the method of nei and gojobori , modified to take account of the transition - to - transversion ratio r . in addition , the jukes - cantor correction was applied to account for multiple substitutions at the same site . standard errors for ds and dn were calculated using 1,000 bootstrap replicates . in the first test ds and dn and their respective variances are used in a two - tailed z - test to test the null hypothesis that dn - ds = 0 . in the second test , fisher 's exact test is used to test the null hypothesis that the proportions of synonymous and nonsynonymous differences are the same . additional tests for the presence of sites under positive selection were carried out using the paml package , which uses likelihood ratio tests ( lrt ) to compare models of the variation in dn / ds ratio between sites . the six models recommended by anisimova et al . were tested : m0 ( one - ratio ) , m1 ( neutral ) , m2 ( selection ) , m3 ( discrete ) , m7 ( beta ) , and m8 ( beta+ ) . intron sequences were aligned using dialign ( version 2.1 ) and the numbers of substitutions between them were estimated using kimura 's two - parameter method . all phylogenetic trees , distance calculations and codon - based tests of selection were carried out using mega2 . estimates of the proportions of radical and conservative nonsynonymous substitutions , along with their standard errors , were made using the hon - new program in an extension of earlier methods for the measurement of conservative and radical substitution rates . the radical or conservative nature of nonsynonymous substitutions was assessed with respect to charge and to the polarity and volume of the amino acids ( the miyata - yasunaga amino - acid classification ) . a range of human rna samples was purchased from stratagene ( amsterdam , the netherlands ) ( stomach , vulva , ovary , kidney , placenta , thyroid , lung , skeletal , uterus , breast , liver , skin , colon , heart and cervix ) and human testis rna ( bd biosciences clontech , oxford ) . cdna synthesis was carried out using a first - strand cdna synthesis kit ( roche , lewes , uk ) according to the instructions , using random hexameric oligonucleotides . pcrs were carried out , using 5 ml of the resultant cdna according to the following procedures : 94c for 1 min followed by 35 cycles of 94c for 30 sec , 55c for 30 sec and 72c for 1 min , and a final round of extension for 5 min . products were analyzed on a 4% nusieve agarose gel ( fmc bioproducts , rockland , me , usa ) by electrophoresis and also cloned using pgem - t easy vector system i ( promega , southampton ) . amplification of glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) was carried out in parallel with conditions as for the other amplifications , but with an annealing temperature of 56c . rt - pcr products were hybridized with a radiolabeled , internal oligonucleotide probe designed to each novel sequence to confirm the presence of the correctly amplified product . purified plasmid dna was sequenced from both strands with abi prism drhodamine terminator cycle sequencing ready reaction kit ( pe applied biosystems , warrington ) . primers and internal probes were designed using the primer3 primer design program from the whitehead institute , center for genome research . the sequences for the primers and internal probes used were as follows : defb105 : 5 ' primer : tctatttgctatgttcttcattttgg , internal oligo : ttcaactgccatcaggtgag , 3 ' primer : gcagcagagaaagttcagcc ; defb106 : 5 ' primer : cgtgctcttctttctgaccc , internal oligo : tacagggaaggtgatcggag , 3 ' primer : gttcttcatttttcccgcaa ; defb107 : 5 ' primer : ttttggctgctctcattcttc , internal oligo : tcactgtgaagccgaatgtc , 3 ' primer : tgcagcaaaatggtgctaat ; defb108 : 5 ' primer : tgctgtcctcttcttcacca , internal oligo : gccaagttctaccagccaag , 3 ' primer : cggctatttaaacatctccca . the novel human gene sequences for defb105 , defb106 , defb107 , defb108 and defb109p , as confirmed by rt - pcr , have been deposited in genbank under sequence accession numbers af540977 , af540978 , af540979 , af540980 and af540981 respectively . the putative baboon orthologs of the human defb4 ( second exon only ) , defb103 , defb104 , defb105 , defb106 and defb107 genes have been also been deposited in genbank under sequence accession numbers bk000556 , bk000557 , bk000558 , bk000559 , bk000560 and bk000561 respectively . , np_038784 , np_062702 , np_109659 , np_473415 , np_631966 , cac44635 , np_631965 , cad26894 , cad26895 ) , -defensin 13 ( np_631969 ) , -defensin 15 ( np_631970 ) , -defensin 35 ( np_631970 ) , defensin - related peptide ( aj344114 ) . human : defb1 ( q09753 ) , defb4 ( o15263 ) , defb103 ( np_061131 ) , defb104 ( cac85520 ) . rat : rattus norvegicus -defensin 1 ( np_113998 ) , -defensin 2 ( o88514 ) . cow : bos taurus -defensin 1 ( p46159 ) , -defensin 2 ( p46160 ) , -defensin 3 ( p46161 ) , -defensin 4 ( p46162 ) , -defensin 5 ( p46163 ) , -defensin 6 ( p46164 ) , -defensin 7 ( p46165 ) , -defensin 8 ( p46166 ) , -defensin 9 ( p46167 ) , -defensin 10 ( p46168 ) , -defensin 11 ( p46169 ) , -defensin 12 ( p46170 ) , -defensin 13 ( p46171 ) , tracheal antimicrobial peptide ( p25068 ) , lingual antimicrobial peptide ( q28880 ) , enteric -defensin ( o02775 ) , -defensin c7 ( o18815 ) . pig : sus scrofa -defensin 1 ( o62697 ) . goat : capra hircus -defensin 1 ( o97946 ) , -defensin 2 ( caa08905 ) . rhesus monkey : macaca mulatta -defensin 1 ( o18794 ) , -defensin 2 ( aak26259 ) . chimpanzee : pan troglodytes -defensin 1 ( aaf04110 ) , -defensin 2 ( aaf20154 ) , -defensin 3 ( aak61549 ) . these sequences were used as tblastx ( version 2.1.1 with default settings ) queries against the htg ( high - throughput genomic ) section of the embl database ( 15 july 2001 release ) . hidden markov models were constructed using hmmer ( version 2.1.1 ) to process clustalw ( version 1.82 with default settings ) multiple sequence alignments . these models were searched against genomic sequence using wise2 ( version 2 - 1 - 20c with the human gene model option ) . clustalw alignments of diverse second - exon sequences were corrected using the patterns of gaps seen in the corresponding protein - sequence alignments that were more highly conserved . all phylogenetic trees were constructed by the neighbour - joining method based on the proportion of amino - acid sites at which sequences compared were different and omitting alignment gaps . the trees constructed were rooted with chicken gallinacin 1 ( gggal1 ; p46156 ) and the reliability of each branch was assessed using 1,000 bootstrap replications . in pairwise comparisons between nucleotide sequences , the number of synonymous substitutions per synonymous site ( ds ) and the number of nonsynonymous substitutions per nonsynonymous site ( dn ) were estimated using the method of nei and gojobori , modified to take account of the transition - to - transversion ratio r . in addition , the jukes - cantor correction was applied to account for multiple substitutions at the same site . standard errors for ds and dn were calculated using 1,000 bootstrap replicates . in the first test ds and dn and their respective variances are used in a two - tailed z - test to test the null hypothesis that dn - ds = 0 . in the second test , fisher 's exact test is used to test the null hypothesis that the proportions of synonymous and nonsynonymous differences are the same . additional tests for the presence of sites under positive selection were carried out using the paml package , which uses likelihood ratio tests ( lrt ) to compare models of the variation in dn / ds ratio between sites . the six models recommended by anisimova et al . were tested : m0 ( one - ratio ) , m1 ( neutral ) , m2 ( selection ) , m3 ( discrete ) , m7 ( beta ) , and m8 ( beta+ ) . intron sequences were aligned using dialign ( version 2.1 ) and the numbers of substitutions between them were estimated using kimura 's two - parameter method . all phylogenetic trees , distance calculations and codon - based tests of selection were carried out using mega2 . estimates of the proportions of radical and conservative nonsynonymous substitutions , along with their standard errors , were made using the hon - new program in an extension of earlier methods for the measurement of conservative and radical substitution rates . the radical or conservative nature of nonsynonymous substitutions was assessed with respect to charge and to the polarity and volume of the amino acids ( the miyata - yasunaga amino - acid classification ) . a range of human rna samples was purchased from stratagene ( amsterdam , the netherlands ) ( stomach , vulva , ovary , kidney , placenta , thyroid , lung , skeletal , uterus , breast , liver , skin , colon , heart and cervix ) and human testis rna ( bd biosciences clontech , oxford ) . cdna synthesis was carried out using a first - strand cdna synthesis kit ( roche , lewes , uk ) according to the instructions , using random hexameric oligonucleotides . pcrs were carried out , using 5 ml of the resultant cdna according to the following procedures : 94c for 1 min followed by 35 cycles of 94c for 30 sec , 55c for 30 sec and 72c for 1 min , and a final round of extension for 5 min . products were analyzed on a 4% nusieve agarose gel ( fmc bioproducts , rockland , me , usa ) by electrophoresis and also cloned using pgem - t easy vector system i ( promega , southampton ) . amplification of glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) was carried out in parallel with conditions as for the other amplifications , but with an annealing temperature of 56c . rt - pcr products were hybridized with a radiolabeled , internal oligonucleotide probe designed to each novel sequence to confirm the presence of the correctly amplified product . purified plasmid dna was sequenced from both strands with abi prism drhodamine terminator cycle sequencing ready reaction kit ( pe applied biosystems , warrington ) . primers and internal probes were designed using the primer3 primer design program from the whitehead institute , center for genome research . the sequences for the primers and internal probes used were as follows : defb105 : 5 ' primer : tctatttgctatgttcttcattttgg , internal oligo : ttcaactgccatcaggtgag , 3 ' primer : gcagcagagaaagttcagcc ; defb106 : 5 ' primer : cgtgctcttctttctgaccc , internal oligo : tacagggaaggtgatcggag , 3 ' primer : gttcttcatttttcccgcaa ; defb107 : 5 ' primer : ttttggctgctctcattcttc , internal oligo : tcactgtgaagccgaatgtc , 3 ' primer : tgcagcaaaatggtgctaat ; defb108 : 5 ' primer : tgctgtcctcttcttcacca , internal oligo : gccaagttctaccagccaag , 3 ' primer : cggctatttaaacatctccca . the novel human gene sequences for defb105 , defb106 , defb107 , defb108 and defb109p , as confirmed by rt - pcr , have been deposited in genbank under sequence accession numbers af540977 , af540978 , af540979 , af540980 and af540981 respectively . the putative baboon orthologs of the human defb4 ( second exon only ) , defb103 , defb104 , defb105 , defb106 and defb107 genes have been also been deposited in genbank under sequence accession numbers bk000556 , bk000557 , bk000558 , bk000559 , bk000560 and bk000561 respectively . the following files are available with this article : the dna sequences for olive baboon -defensins ( additional data file 1 ) , the dna sequences for human -defensins ( additional data file 2 ) , the aligned baboon and human dna sequences ( additional data file 3 ) , the aligned first - exon baboon and human dna sequences ( additional data file 4 ) , the aligned second - exon baboon and human dna sequences ( additional data file 5 ) and the dna sequences for baboon and human introns ( additional data file 6 ) and an excel file listing the full dn , ds and dn - ds estimates for all first and second exon comparisons shown in figures 4a and b ( additional data file 7 ) . the dna sequences for olive baboon beta - defensins click here for additional data file the dna sequences for human beta - defensins click here for additional data file the aligned baboon and human dna sequences in msf format click here for additional data file the aligned first - exon baboon and human dna sequences in msf format click here for additional data file the aligned second - exon baboon and human dna sequences in msf format click here for additional data file the dna sequences for baboon and human introns click here for additional data file the full dn , ds and dn - ds estimates for all first and second exon comparisons shown in figures 4a and b click here for additional data file we would also like to thank gillian morrison for her initial involvement in this project . ( a ) the genomic organization of novel human -defensin genes defb105 , defb106 , defb107 , defb108 and defb109p on 8p22-p23 . the horizontal lines represent the three bac clones in which all novel genes were found . exons are represented as triangles with the vertical side representing the position of the exon . exons above the horizontal lines are transcribed from the strand represented by the original bac clone sequence entries whereas those below are transcribed in the opposite direction from the complementary strand . those genes marked with an asterisk were found to have orthologs in baboon genomic sequences ( ac116558 and ac116559 ) . ( b ) a phylogenetic tree of functional human -defensins using the prepropeptide sequences encoded by the genes shown in ( a ) . the phylogenetic tree was rooted with chicken gallinacin 1 ( gggal1 ; p46156 ) and the reliability of each branch was assessed using 1,000 bootstrap replications . ( a ) alignment and ( b ) phylogenetic tree of human and baboon -defensin protein sequences . the tree was rooted with chicken gallinacin 1 ( gggal1 ; p46156 ) and the reliability of each branch was assessed using 1,000 bootstrap replications . the alignment shows the same sequences with the estimated locations of the signal peptide and mature peptide regions ; the intervening region is the propiece . the long arrow indicates the position of first introns : in each case except defb105 the intron splits the codon that encodes the residue immediately before the arrow . the shading represents the degree of conservation at each position in the alignment , taking into account similar physicochemical properties of residues . rt - pcr analysis of novel human gene expression carried out on a panel of human rna samples . the tissues are indicated with a plus ( + ) and a minus ( - ) reverse transcriptase reaction shown for each sample . graphs of the number of synonymous substitutions per synonymous site ( ds ) against the number of nonsynonymous substitutions per nonsynonymous site ( dn ) for comparisons between ( a ) the first exons and ( b ) second exons of all genes in the dataset . in each case results of comparisons between the second exons of human and baboon genes demonstrating positive selection * estimates ( 6se ) of the number of synonymous sites ( s ) , number of nonsynonymous sites , numbers of synonymous substitutions ( s ) , numbers of nonsynonymous substitutions ( n ) , the number of synonymous substitutions per synonymous site ( ds ) and the number of nonsynonymous substitutions per nonsynonymous site ( dn ) . the result of a two - tailed z - test of dn - ds = 0 . rates of radical ( pr ) and conservative ( pc ) changes in amino - acid properties , with the ratio of radical to conservative changes ( pr / pc ) for residues categorized in terms of their charges . rates of radical ( pr ) and conservative ( pc ) changes in amino - acid properties , with the ratio of radical to conservative changes ( pr / pc ) for residues categorized in terms of the miyata - yasunaga classification ( m - y ; a combination of polarity and volume ) .

peptides such as -defensins are an important component in innate immunity . a combination of laboratory and computational techniques have been used to characterize the main human -defensin locus on chromosome 8p22-p23 . the genomic structures and expression patterns of four novel human -defensin genes and a related pseudogene reveal prior duplication events and selection .

Background Results Discussion Conclusions Materials and methods Identification of novel genes Evolutionary analyses RT-PCR analysis of gene expression Additional data files Supplementary Material Acknowledgements Figures and Tables

the vertebrate innate immune system provides protection against a wide range of pathogenic microorganisms , and defensins are an important component of this response as well as having a role in adaptive immunity . the -defensins are produced by neutrophils and intestinal paneth cells , whereas the -defensins are mainly produced by epithelial cells in contact with the environment . the functions of human -defensins seem to be disrupted in cystic fibrosis and inflammatory skin lesions such as psoriasis . the known human -defensin genes show a conserved two - exon structure : the first exon encodes a signal peptide whereas the second exon encodes a short propiece and the mature defensin peptide with a characteristic six - cysteine motif and many basic amino - acid residues . the -defensin genes are present at five syntenic loci in the human and mouse genomes , with the main locus on human chromosome 8p22 - 23 and mouse chromosome 8a3 . all four , full - length , human -defensins that are present in the public databases are from 8p22 - 23 ( genbank sequence accession numbers are human -defensin 1 or defb1 , q09753 ; defb4 ( formerly defb2 ) , o15263 ; defb103 ( formerly defb3 ) , np_061131 ; defb104 ( formerly defb4 ) , cac85520 ) , but there are substantial differences in their coding sequences , expression patterns and antimicrobial activities . here we describe a combined strategy to identify further -defensin genes in the draft human genome sequence using computational techniques and verification using reverse transcription - pcr . four full - length , novel genes ( defb105 , defb106 , defb107 , defb108 ) and a related pseudogene defb109p are reported , as well as their expression patterns and evidence for their evolution by duplication and positive selection . these 53 bacs were deemed to represent the human -defensin gene family locus and were masked for repetitive sequences using repeatmasker . this locus was the subject of a more sensitive search for the presence of novel human -defensins , using a hidden markov model constructed from an alignment of the genbank -defensin sequences mentioned above . as well as the known , full - length human -defensins and the related epididymis - specific spag11 ( formerly ep2 ) gene , two novel -defensin genes , defb105 and defb106 , were identified in this search and were then incorporated into the previous hidden markov model . the novel gene defb109p appears to be a pseudogene as it contains a premature stop codon within its first exon , as observed in three independently sequenced , overlapping 8p22-p23 bac sequences ( accession numbers ac068974 , ac087203 and af252830 ) . given the absence of premature stop codons in the other four genes , despite considerable divergence among them ( they encode only 18 - 28% identical amino - acid residues ) , it is unlikely that they too are pseudogenes . they used computational techniques to identify sequences matching a central portion of the mature defensin peptide including the six - cysteine motif characteristic of -defensins . at best , this method could only identify incomplete final exons encoding this region of the peptide . in two cases , defb106 and defb107 , splice sites other than those predicted were used and it was found that the predicted coding sequences had been respectively 36 base - pairs ( bp ) longer and 9 bp shorter than the actual coding sequences . the rt - pcr analysis also produced interesting results with respect to the coding sequence of defb108 . the three differences observed between predicted and amplified sequences respectively were an a to g at nucleotide 62 ( which causes a conservative amino - acid change from lysine to arginine ) , a t to c at nucleotide 111 ( which is a synonymous change ) and a c to t at nucleotide 120 ( also synonymous ) . this indicates novel human polymorphisms in defb108 and is consistent with observations of high degrees of polymorphism for other human -defensins ( see also additional data files 1 - 6 for the novel gene sequences confirmed by rt - pcr and the computationally predicted sequence of defb109p ) . figure 1a depicts the relative positions and orientations of the four novel genes , the novel pseudogene defb109p and four known genes in the vicinity : defb2 - 4 and the related epididymis - specific spag11 . a phylogenetic tree relating functional human -defensins ( figure 1b ) reflects the spatial distribution of these genes , with the cluster of genes in figure 1a appearing to form a clade separate from defb1 . the simplest explanation for the origin of this cluster is a series of local duplication events followed by substantial divergence . in addition it seems that the four novel functional genes are more closely related to defb104 than to defb4 or defb103 . the predicted mature peptides for the four novel , functional -defensins presented here have a similar proportion of cationic residues to human defb104 , with higher proportions of anionic residues ( 10 - 13% ) than are seen in human defb1 , defb4 and defb103 ( less than 4% ) . expression of all four novel genes was readily detected in testis . six genes highly similar ( 85 - 98% identical at the amino - acid level ) to three of the novel human -defensins ( defb105 , defb106 and defb107 ) as well as to defb4 , defb103 and defb104 , were found within two olive baboon ( papio cynocephalus anubis ) draft genomic sequences ( genbank accession numbers ac116558 and ac116559 ) using blast . figure 4 shows dn ( number of nonsynonymous substitutions per nonsynonymous site ) plotted against ds ( number of synonymous substitutions per synonymous site ) for comparisons between the first exon ( which encodes the signal peptide ) and second exon ( which encodes the mature defensin ) from all human and baboon genes ( the full dn , ds and dn - ds estimates for all first and second exon comparisons are available as additional data file 7 ) . exceeds dn ( figure 4a ) and this excess is statistically significant in almost every case according to two - tailed z - test results for all human and baboon genes , but there were no significant excesses of ds according to the more rigorous fisher 's exact test ( data not shown ) . thus the rates of substitution in first exons indicate that they are evolving approximately neutrally , perhaps under weak purifying selection . in the second exons dn often exceeds ds , and even in these short sequences , for certain comparisons this excess reaches statistical significance . significant excesses of dn over ds are seen between defb1 and defb104 and between defb103 and defb107 , with similar effects seen among the second exons of orthologous baboon genes ( table 1 ) using the method of nei and gojobori . moreover , in these comparisons ds tends to be rather low relative to the rest of the data set ( mean ds = 0.464 ) . although some of the comparisons shown in figure 4b indicate an excess of ds over dn , this is never significant by either of the two statistical tests used . in particular , the orthologous intron sequence comparisons for defb103 , defb104 and defb107 give substitution - rate estimates of 0.1066 0.010 , 0.0816 0.005 and 0.0666 0.004 respectively , which are consistent with the ds estimates for the coding sequences of these pairs : 0.1666 0.077 , 0.0856 0.058 and 0.0536 0.049 . since is a measure of selective pressure on proteins , these models can be used to assess the evidence for variable selective pressures among sites . model m1 ( neutral ) assumes two site classes with = 0 and 1 = 1 fixed and with the proportions p and p1 estimated . however , the difference between m7 and m8 is not statistically significant , as indicated by the lrt : 2l = -1006.44 -(-1006.44 ) = 0 . all these positions are close or adjacent to a conserved cysteine residue and so it is possible they are important in determining -defensin structure . this result may be attributable to the extremely short lengths ( 33 codons aligned omitting positions with gaps ) of the sequences aligned , such an effect was seen when analyzing short ( 130 codons ) lysozyme sequences in the same way . this study represents the most detailed study of a human -defensin cluster to date , including the full - length sequences of four novel genes ( defb105 , defb106 , defb107 , defb108 ) , and a novel pseudogene ( defb109p ) , their expression patterns and sequences for the baboon orthologs of six genes from this cluster . the 8p22 - 23 defensin locus appears to have evolved by successive rounds of duplication followed by substantial divergence , to produce a diverse cluster of paralogous genes defined by these four novel genes and four known -defensin genes ( defb4 , defb103 and defb104 ) . the positive selection observed has tended to change the charges of residues encoded more than other qualities such as residue polarity or volume . as seems to be the case with other antimicrobial peptides , such as mhc receptors , immunoglobulins and -defensins [ 6 - 8 ] , this selection may be a response to the rapid evolution of pathogens . this has implications for purely computational approaches to novel gene discovery such as that of schutte et al . defb104 and defb108 have no detectable orthologs in the mouse genome and therefore appear to have arisen by duplication since the divergence of rodents and primates ; alternatively , there could have been a loss of these genes in the rodent lineage . however , when compared with other known human -defensins ( defb1 , defb4 and defb103 ) , defb104 was found to have a different number of residues between its second and third , and between its fourth and fifth cysteine residues . three of the novel genes described here ( defb105 , defb106 and defb108 ) encode mature peptides exhibiting the same spacing of conserved cysteine residues as seen in defb104 as well as sharing similar expression patterns , with highest expression in the testes . the functional divergence of -defensin genes appears to have continued following human - baboon divergence , as exemplified by defb107 which displays a serine residue instead of the first canonical cysteine seen in the baboon ortholog . it is notable that a novel mouse -defensin gene ( defr1 ) , which also lacks the first canonical cysteine , has potent antimicrobial activity against a spectrum of pathogens . both human and mouse -defensins have been shown to be chemotactic for immature dendritic cells and memory t cells via the ccr6 chemokine receptor . these events , in turn , trigger robust , type-1 polarized adaptive immune responses in vivo , which suggests that -defensins may have an important role in immunosurveillance against pathogens . together with our results demonstrating that defb105 , defb106 , defb107 and defb108 are predominantly expressed in the testes , these data suggest that the male reproductive tract has a complex innate defense mechanism . the 8p22 - 23 -defensin locus has evolved by duplication and subsequent divergence , to produce a diverse cluster of paralogous genes defined by four novel genes ( defb105 , defb106 , defb107 , defb108 ) , a novel pseudogene ( defb109p ) , and three known genes ( defb4 , defb103 and defb104 ) . we present full - length sequences for the four novel genes , their expression patterns and the predicted sequences for the baboon orthologs of six genes from this cluster . evolutionary analyses suggest that the divergence seen in second exons has involved positive selection disproportionately favoring alterations in the charge of amino - acid residues . clustalw alignments of diverse second - exon sequences were corrected using the patterns of gaps seen in the corresponding protein - sequence alignments that were more highly conserved . in addition , the jukes - cantor correction was applied to account for multiple substitutions at the same site . standard errors for ds and dn were calculated using 1,000 bootstrap replicates . in the second test , fisher 's exact test is used to test the null hypothesis that the proportions of synonymous and nonsynonymous differences are the same . additional tests for the presence of sites under positive selection were carried out using the paml package , which uses likelihood ratio tests ( lrt ) to compare models of the variation in dn / ds ratio between sites . a range of human rna samples was purchased from stratagene ( amsterdam , the netherlands ) ( stomach , vulva , ovary , kidney , placenta , thyroid , lung , skeletal , uterus , breast , liver , skin , colon , heart and cervix ) and human testis rna ( bd biosciences clontech , oxford ) . pcrs were carried out , using 5 ml of the resultant cdna according to the following procedures : 94c for 1 min followed by 35 cycles of 94c for 30 sec , 55c for 30 sec and 72c for 1 min , and a final round of extension for 5 min . amplification of glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) was carried out in parallel with conditions as for the other amplifications , but with an annealing temperature of 56c . the novel human gene sequences for defb105 , defb106 , defb107 , defb108 and defb109p , as confirmed by rt - pcr , have been deposited in genbank under sequence accession numbers af540977 , af540978 , af540979 , af540980 and af540981 respectively . the putative baboon orthologs of the human defb4 ( second exon only ) , defb103 , defb104 , defb105 , defb106 and defb107 genes have been also been deposited in genbank under sequence accession numbers bk000556 , bk000557 , bk000558 , bk000559 , bk000560 and bk000561 respectively . clustalw alignments of diverse second - exon sequences were corrected using the patterns of gaps seen in the corresponding protein - sequence alignments that were more highly conserved . all phylogenetic trees were constructed by the neighbour - joining method based on the proportion of amino - acid sites at which sequences compared were different and omitting alignment gaps . in the first test ds and dn and their respective variances are used in a two - tailed z - test to test the null hypothesis that dn - ds = 0 . in the second test , fisher 's exact test is used to test the null hypothesis that the proportions of synonymous and nonsynonymous differences are the same . the six models recommended by anisimova et al . all phylogenetic trees , distance calculations and codon - based tests of selection were carried out using mega2 . the radical or conservative nature of nonsynonymous substitutions was assessed with respect to charge and to the polarity and volume of the amino acids ( the miyata - yasunaga amino - acid classification ) . pcrs were carried out , using 5 ml of the resultant cdna according to the following procedures : 94c for 1 min followed by 35 cycles of 94c for 30 sec , 55c for 30 sec and 72c for 1 min , and a final round of extension for 5 min . purified plasmid dna was sequenced from both strands with abi prism drhodamine terminator cycle sequencing ready reaction kit ( pe applied biosystems , warrington ) . the sequences for the primers and internal probes used were as follows : defb105 : 5 ' primer : tctatttgctatgttcttcattttgg , internal oligo : ttcaactgccatcaggtgag , 3 ' primer : gcagcagagaaagttcagcc ; defb106 : 5 ' primer : cgtgctcttctttctgaccc , internal oligo : tacagggaaggtgatcggag , 3 ' primer : gttcttcatttttcccgcaa ; defb107 : 5 ' primer : ttttggctgctctcattcttc , internal oligo : tcactgtgaagccgaatgtc , 3 ' primer : tgcagcaaaatggtgctaat ; defb108 : 5 ' primer : tgctgtcctcttcttcacca , internal oligo : gccaagttctaccagccaag , 3 ' primer : cggctatttaaacatctccca . the novel human gene sequences for defb105 , defb106 , defb107 , defb108 and defb109p , as confirmed by rt - pcr , have been deposited in genbank under sequence accession numbers af540977 , af540978 , af540979 , af540980 and af540981 respectively . the putative baboon orthologs of the human defb4 ( second exon only ) , defb103 , defb104 , defb105 , defb106 and defb107 genes have been also been deposited in genbank under sequence accession numbers bk000556 , bk000557 , bk000558 , bk000559 , bk000560 and bk000561 respectively . ( a ) the genomic organization of novel human -defensin genes defb105 , defb106 , defb107 , defb108 and defb109p on 8p22-p23 . the phylogenetic tree was rooted with chicken gallinacin 1 ( gggal1 ; p46156 ) and the reliability of each branch was assessed using 1,000 bootstrap replications . the shading represents the degree of conservation at each position in the alignment , taking into account similar physicochemical properties of residues . rt - pcr analysis of novel human gene expression carried out on a panel of human rna samples . the tissues are indicated with a plus ( + ) and a minus ( - ) reverse transcriptase reaction shown for each sample . in each case results of comparisons between the second exons of human and baboon genes demonstrating positive selection * estimates ( 6se ) of the number of synonymous sites ( s ) , number of nonsynonymous sites , numbers of synonymous substitutions ( s ) , numbers of nonsynonymous substitutions ( n ) , the number of synonymous substitutions per synonymous site ( ds ) and the number of nonsynonymous substitutions per nonsynonymous site ( dn ) . rates of radical ( pr ) and conservative ( pc ) changes in amino - acid properties , with the ratio of radical to conservative changes ( pr / pc ) for residues categorized in terms of the miyata - yasunaga classification ( m - y ; a combination of polarity and volume ) .

establishing whether an infant was stillborn , or was born alive and subsequently died , is a difficult but important aspect of forensic medicine . traditional methods for assessing lung aeration include the lung flotation technique , an invasive test requiring evisceration of the lungs and observing whether they float or sink when placed into water ; floating lungs are traditionally deemed to contain air , indicating breathing before neonatal demise . however , there is uncertainty regarding the reliability of this test , with various factors potentially contributing to both false positive and false negative findings [ 2 , 3 ] . there is a paucity of published studies on the accuracy of the lung flotation test , with quoted accuracy ranging from 37 to 95 % [ 3 , 4 ] . post - mortem imaging is becoming widely accepted as an important component of the examination after death [ 5 , 6 ] , with excellent diagnostic agreement between perinatal and paediatric post - mortem magnetic resonance imaging ( pmmr ) and autopsy findings . part of the challenge of post - mortem ( pm ) imaging is the correct interpretation of imaging findings , and two small case series ( 11 cases in total ) have described the use of post - mortem computed tomography ( pmct ) to assess lung aeration in infant deaths [ 8 , 9 ] . however , pmmr provides much better soft tissue detail than pmct in foetuses , stillbirths and neonates and is likely to become the preferred imaging technique in this patient population [ 7 , 10 ] . in this study , we investigate the accuracy of pmmr imaging characteristics to distinguish between liveborn and stillborn infants to establish a non - invasive alternative or adjunct to the lung flotation test , which could be more acceptable to parents who decline an invasive autopsy , and to more accurately interpret routine pmmr imaging . we retrospectively reviewed our hospital database for cases of late foetal death , termination of pregnancy , and infant deaths in the first 3 months of life , who had undergone pmmr as part of their post - mortem assessment in the last 5 years . at our large specialist paediatric hospital , we perform over 100 foetal and paediatric pmmr studies per year as part of the clinical autopsy . this study did not require further specific institutional approval , as all parents had consented to a clinical pre - autopsy pmmr as part of our institution s clinical post - mortem assessment , and use of routinely collected post - mortem data is approved by the local research ethics committee . bodies were stored in a mortuary at 4 c , and pmmr was generally performed out of hours , causing least disturbance to clinical services . our inclusion criteria identified two groups : group 1 : live births defined as infants who were born alive and witnessed spontaneously breathing before death , and group 2 : non - live births defined as either late foetal terminations of pregnancy for abnormality following fetocide ( over 24-week gestation ) without documented evidence of breathing and stillbirths who had been born dead . we excluded all foetuses below 24-week gestation , as they were likely to have incomplete lung development , and also excluded cases with lung abnormalities at autopsy . pre - autopsy whole - body pmmr was acquired using a 1.5 t mr scanner ( avanto , siemens medical solutions , erlangen , germany ) . sequences acquired included whole - body 3-d t2-weighted turbo spin echo ( tse , tr 3500 ms , te 276 ms , voxel size 0.8 0.8 0.8 mm , two averages ) , 3-d t1-weighted volumetric interpolated breath - hold examination ( vibe ; tr 5.9 ms , te 2.4 ms , flip angle 25 , voxel size 0.8 0.8 0.8 mm , eight averages ) , and 3-d constructive interference in the steady state ( ciss ) sequence ( tr 9.2 ms , te 4.6 ms , flip angle 70 , voxel size 0.6 0.6 0.6 mm , four averages ) . the vibe sequence was chosen , as it was particularly sensitive to the presence of gas due to the associated chemical shift artefact . pmmr findings were reviewed by two radiologists with 3- ( jb ) and 7-year ( oa ) clinical radiology experience ( 0- and 2-year pmmr reporting experience , respectively ) , using the osirix platform ( osirix foundation , geneva , switzerland ) , blinded to the clinical history and autopsy findings . a consensus decision was reached regarding the presence of any air ( very low signal intensity ) identified on both t1-weighted and t2-weighted sequences in the following six anatomical locations : in ( 1 ) airways , ( 2 ) lung parenchyma , ( 3 ) gastrointestinal tract , ( 4 ) right heart cavities , ( 5 ) left heart cavities , and ( 6 ) hepatobiliary system . the presence of post - mortem pleural fluid collections was also recorded but predicted to be consistent across the two groups ( a control measure ) , as this was assumed to be unrelated to breathing . conventional autopsies were performed by one of several experienced specialized perinatal / paediatric pathologists in accordance with national guidelines and were reported blinded to the pmmr findings . autopsy findings were reviewed for this study to exclude any cases which had abnormal lung pathology . we used the contemporaneous clinical history and perinatal notes as the gold standard for assessing breathing status . primary outcomes were sensitivity , specificity , positive predictive value ( ppv ) and negative predictive value ( npv ) where pmmr ( the index test ) was compared with clinical history ( the gold standard ) , with 95 % confidence intervals ( cis ) . concordance was defined as the sum of true positives and true negatives divided by all cases . pre - autopsy whole - body pmmr was acquired using a 1.5 t mr scanner ( avanto , siemens medical solutions , erlangen , germany ) . sequences acquired included whole - body 3-d t2-weighted turbo spin echo ( tse , tr 3500 ms , te 276 ms , voxel size 0.8 0.8 0.8 mm , two averages ) , 3-d t1-weighted volumetric interpolated breath - hold examination ( vibe ; tr 5.9 ms , te 2.4 ms , flip angle 25 , voxel size 0.8 0.8 0.8 mm , eight averages ) , and 3-d constructive interference in the steady state ( ciss ) sequence ( tr 9.2 ms , te 4.6 ms , flip angle 70 , voxel size 0.6 0.6 0.6 mm , four averages ) . the vibe sequence was chosen , as it was particularly sensitive to the presence of gas due to the associated chemical shift artefact . pmmr findings were reviewed by two radiologists with 3- ( jb ) and 7-year ( oa ) clinical radiology experience ( 0- and 2-year pmmr reporting experience , respectively ) , using the osirix platform ( osirix foundation , geneva , switzerland ) , blinded to the clinical history and autopsy findings . a consensus decision was reached regarding the presence of any air ( very low signal intensity ) identified on both t1-weighted and t2-weighted sequences in the following six anatomical locations : in ( 1 ) airways , ( 2 ) lung parenchyma , ( 3 ) gastrointestinal tract , ( 4 ) right heart cavities , ( 5 ) left heart cavities , and ( 6 ) hepatobiliary system . the presence of post - mortem pleural fluid collections was also recorded but predicted to be consistent across the two groups ( a control measure ) , as this was assumed to be unrelated to breathing . conventional autopsies were performed by one of several experienced specialized perinatal / paediatric pathologists in accordance with national guidelines and were reported blinded to the pmmr findings . autopsy findings were reviewed for this study to exclude any cases which had abnormal lung pathology . we used the contemporaneous clinical history and perinatal notes as the gold standard for assessing breathing status . primary outcomes were sensitivity , specificity , positive predictive value ( ppv ) and negative predictive value ( npv ) where pmmr ( the index test ) was compared with clinical history ( the gold standard ) , with 95 % confidence intervals ( cis ) . concordance was defined as the sum of true positives and true negatives divided by all cases . we found 42 cases in our database which met our strict inclusion criteria from our hospital database of over 500 cases ( 20072012 ) . group 1 live births comprised 19 infant deaths ( mean post - natal age at death 3.0 6.6 weeks ) . sixteen breathed spontaneously at birth and had lived for up to 11 weeks prior to death , which was attributed to a variety of causes including trauma and sudden unexpected death in infancy ( sudi ) . three further cases had very low apgar scores at birth but underwent resuscitation with a short period of supported life prior to death at 2448 h of age . group 2 non - live births comprised 23 foetal terminations or stillbirths ( mean age 32.6 10.2-week gestation ) of which 17 were late foetal terminations ( known to have not breathed and not undergone resuscitation ) and six stillbirths without spontaneous breathing . gas in almost all locations , including airways , lungs , or gi tract , was identified more commonly in live births than stillbirths ( table 1).table 1diagnostic accuracy of imaging indices for live birthindexfp / tpfn / tnsensitivity ( % ) specificity ( % ) ppv ( % ) npv ( % ) concordance ( % ) gas in airway7/163/1684.2 % ( 62.4 , 94.5)*69.6 % ( 49.1 , 84.4)69.6 % ( 49.1 , 84.4)84.2 % ( 62.4 , 94.5)76.2 % ( 61.5 , 86.5)lung aeration1/172/2289.5 % ( 68.6 , 97.1)95.6 % ( 79.0 , 99.2)94.4 % ( 74.2 , 99.0)91.7 % ( 74.2 , 97.7)92.9 % ( 81.0 , 97.5)gas in gi tract5/190/18100 % ( 83.2 , 100)78.3 % ( 58.1 , 90.3)79.2 % ( 59.5 , 90.8)100 % ( 82.4 , 100)88.1 % ( 75.0 , 94.8)absence of r ht gas9/145/1473.7 % ( 51.2 , 88.2)60.9 % ( 40.8 , 77.8)60.9 % ( 40.8 , 77.8)73.7 % ( 51.2 , 88.2)66.7 % ( 51.6 , 79.0)absence of l ht gas8/172/1589.5 % ( 68.6 , 97.1)65.2 % ( 44.9 , 81.2)68.0 % ( 48.4 , 82.8)88.2 % ( 65.7 , 96.7)76.2 % ( 61.5 , 86.5)absence of hepatobiliary gas12/163/1184.2 % ( 62.4 , 94.5)47.8 % ( 29.2 , 67.0)57.1 % ( 39.1 , 73.5)78.6 % ( 52.4 , 92.4)64.3 % ( 49.2 , 77.0)bilateral effusions11/109/1252.6 % ( 31.7 , 72.7)52.2 % ( 33.0 , 70.8)47.6 % ( 28.3 , 67.6)57.1 % ( 36.6 , 75.5)52.4 % ( 37.7 , 66.6)a positive test result was defined as the presence of any aeration in the given location fp false positive , tp true positive , fn false negative , tn true negative , r ht right heart ( atrium or ventricle ) , l ht left heart ( atrium or ventricle)*all percentages are given 95 % confidence interval diagnostic accuracy of imaging indices for live birth a positive test result was defined as the presence of any aeration in the given location fp false positive , tp true positive , fn false negative , tn true negative , r ht right heart ( atrium or ventricle ) , l ht left heart ( atrium or ventricle ) * all percentages are given 95 % confidence interval lung aeration on pmmr was the best indicator of live birth , with highest concordance between pmmr and autopsy of 92.9 % ( 95 % ci 81.0 , 97.5 ) . lung aeration was the most specific marker of live birth , with specificity of 95.6 % ( 79.0 , 99.2 ) and ppv of 94.4 % ( 74.2 , 99.0 ) , whereas gas in the gi tract was the most sensitive at 100 % ( 86.2 , 100 ) and highest npv 100 % ( 82.4 , 100 ) ( fig . 1example of agreement between pmmr and autopsy . a coronal t2-weighted pmmr image in a 2-week - old neonate who fell from the pram and died . it demonstrates air within the airways and lungs ( dark ) and gas within the stomach ( dark ) , as would be expected in an infant who had breathed before death ( true positive in this study ) . b coronal t2-weighted pmmr image in a 30-week - old gestation foetus who underwent termination for foetal hydrops . there were no signs of life at delivery , no resuscitation implemented , and pmmr demonstrated no gas in the lungs ( grey lungs ) , nor gi tract ( not shown ) , as would be expected ( true negative in this study ) example of agreement between pmmr and autopsy . a coronal t2-weighted pmmr image in a 2-week - old neonate who fell from the pram and died . it demonstrates air within the airways and lungs ( dark ) and gas within the stomach ( dark ) , as would be expected in an infant who had breathed before death ( true positive in this study ) . b coronal t2-weighted pmmr image in a 30-week - old gestation foetus who underwent termination for foetal hydrops . there were no signs of life at delivery , no resuscitation implemented , and pmmr demonstrated no gas in the lungs ( grey lungs ) , nor gi tract ( not shown ) , as would be expected ( true negative in this study ) intracardiac gas and hepatobiliary gas were more commonly seen in stillbirths than in live births , but , together with the presence of pleural effusions , were not reliable indicators of live birth versus stillbirth status . for lung aeration , a single false positive diagnosis was made where apparently well - aerated lungs were seen in a stillborn infant ( fig . 2 ) . the clinical history recorded no witnessed spontaneous breathing at 38-week gestation ; however , this foetus may have undergone resuscitation attempts and the air within the lungs may be related to mechanical ventilation.fig . a faint foetal heart rate was thought to be detected prior to emergency caesarean section , but there was evidence of intrauterine death and maceration at delivery . t2-weighted pmmr imaging shows that the lung bases contain air ( dark ) on both coronal ( a ) and axial images ( b ) and gas within the hepatobiliary system ( c ) . we suggest that these findings may be related to initial resuscitation rather than spontaneous breathing example of false positive pmmr . a faint foetal heart rate was thought to be detected prior to emergency caesarean section , but there was evidence of intrauterine death and maceration at delivery . t2-weighted pmmr imaging shows that the lung bases contain air ( dark ) on both coronal ( a ) and axial images ( b ) and gas within the hepatobiliary system ( c ) . we suggest that these findings may be related to initial resuscitation rather than spontaneous breathing two false negative diagnoses on pmmr were made , where neonates had documented spontaneous breaths , but no gas was seen in the lung parenchyma ( fig . 3 ) . both were neonates who died from perinatal asphyxia and ischaemic brain injury in the first week of life.fig . coronal ( a ) and axial ( b ) t2-weighted pmmr imaging of the lungs in a 2-day - old neonate who died of ischaemic brain injury . the baby had no heart rate at birth and very poor apgar score and was resuscitated and mechanically ventilated but care was withdrawn . despite the ventilation , the lungs did not contain air on pmmr . interestingly , we also did not see gas in the airway ( not shown ) example of false negative pmmr . coronal ( a ) and axial ( b ) t2-weighted pmmr imaging of the lungs in a 2-day - old neonate who died of ischaemic brain injury . the baby had no heart rate at birth and very poor apgar score and was resuscitated and mechanically ventilated but care was withdrawn . despite the ventilation , the lungs did not contain air on pmmr . interestingly , we also did not see gas in the airway ( not shown ) as it was possible that the use of both lung and gi tract aeration or either lung or gi tract aeration together could improve the diagnostic accuracy of pmmr , we repeated the statistical analysis for both of these measures . both of these analyses gave the same results as those for lung aeration only and did not improve the diagnostic accuracy . we found 42 cases in our database which met our strict inclusion criteria from our hospital database of over 500 cases ( 20072012 ) . group 1 live births comprised 19 infant deaths ( mean post - natal age at death 3.0 6.6 weeks ) . sixteen breathed spontaneously at birth and had lived for up to 11 weeks prior to death , which was attributed to a variety of causes including trauma and sudden unexpected death in infancy ( sudi ) . three further cases had very low apgar scores at birth but underwent resuscitation with a short period of supported life prior to death at 2448 h of age . group 2 non - live births comprised 23 foetal terminations or stillbirths ( mean age 32.6 10.2-week gestation ) of which 17 were late foetal terminations ( known to have not breathed and not undergone resuscitation ) and six stillbirths without spontaneous breathing . gas in almost all locations , including airways , lungs , or gi tract , was identified more commonly in live births than stillbirths ( table 1).table 1diagnostic accuracy of imaging indices for live birthindexfp / tpfn / tnsensitivity ( % ) specificity ( % ) ppv ( % ) npv ( % ) concordance ( % ) gas in airway7/163/1684.2 % ( 62.4 , 94.5)*69.6 % ( 49.1 , 84.4)69.6 % ( 49.1 , 84.4)84.2 % ( 62.4 , 94.5)76.2 % ( 61.5 , 86.5)lung aeration1/172/2289.5 % ( 68.6 , 97.1)95.6 % ( 79.0 , 99.2)94.4 % ( 74.2 , 99.0)91.7 % ( 74.2 , 97.7)92.9 % ( 81.0 , 97.5)gas in gi tract5/190/18100 % ( 83.2 , 100)78.3 % ( 58.1 , 90.3)79.2 % ( 59.5 , 90.8)100 % ( 82.4 , 100)88.1 % ( 75.0 , 94.8)absence of r ht gas9/145/1473.7 % ( 51.2 , 88.2)60.9 % ( 40.8 , 77.8)60.9 % ( 40.8 , 77.8)73.7 % ( 51.2 , 88.2)66.7 % ( 51.6 , 79.0)absence of l ht gas8/172/1589.5 % ( 68.6 , 97.1)65.2 % ( 44.9 , 81.2)68.0 % ( 48.4 , 82.8)88.2 % ( 65.7 , 96.7)76.2 % ( 61.5 , 86.5)absence of hepatobiliary gas12/163/1184.2 % ( 62.4 , 94.5)47.8 % ( 29.2 , 67.0)57.1 % ( 39.1 , 73.5)78.6 % ( 52.4 , 92.4)64.3 % ( 49.2 , 77.0)bilateral effusions11/109/1252.6 % ( 31.7 , 72.7)52.2 % ( 33.0 , 70.8)47.6 % ( 28.3 , 67.6)57.1 % ( 36.6 , 75.5)52.4 % ( 37.7 , 66.6)a positive test result was defined as the presence of any aeration in the given location fp false positive , tp true positive , fn false negative , tn true negative , r ht right heart ( atrium or ventricle ) , l ht left heart ( atrium or ventricle)*all percentages are given 95 % confidence interval diagnostic accuracy of imaging indices for live birth a positive test result was defined as the presence of any aeration in the given location fp false positive , tp true positive , fn false negative , tn true negative , r ht right heart ( atrium or ventricle ) , l ht left heart ( atrium or ventricle ) * all percentages are given 95 % confidence interval lung aeration on pmmr was the best indicator of live birth , with highest concordance between pmmr and autopsy of 92.9 % ( 95 % ci 81.0 , 97.5 ) . lung aeration was the most specific marker of live birth , with specificity of 95.6 % ( 79.0 , 99.2 ) and ppv of 94.4 % ( 74.2 , 99.0 ) , whereas gas in the gi tract was the most sensitive at 100 % ( 86.2 , 100 ) and highest npv 100 % ( 82.4 , 100 ) ( fig . 1example of agreement between pmmr and autopsy . a coronal t2-weighted pmmr image in a 2-week - old neonate who fell from the pram and died . it demonstrates air within the airways and lungs ( dark ) and gas within the stomach ( dark ) , as would be expected in an infant who had breathed before death ( true positive in this study ) . b coronal t2-weighted pmmr image in a 30-week - old gestation foetus who underwent termination for foetal hydrops . there were no signs of life at delivery , no resuscitation implemented , and pmmr demonstrated no gas in the lungs ( grey lungs ) , nor gi tract ( not shown ) , as would be expected ( true negative in this study ) example of agreement between pmmr and autopsy . a coronal t2-weighted pmmr image in a 2-week - old neonate who fell from the pram and died . it demonstrates air within the airways and lungs ( dark ) and gas within the stomach ( dark ) , as would be expected in an infant who had breathed before death ( true positive in this study ) . b coronal t2-weighted pmmr image in a 30-week - old gestation foetus who underwent termination for foetal hydrops . there were no signs of life at delivery , no resuscitation implemented , and pmmr demonstrated no gas in the lungs ( grey lungs ) , nor gi tract ( not shown ) , as would be expected ( true negative in this study ) intracardiac gas and hepatobiliary gas were more commonly seen in stillbirths than in live births , but , together with the presence of pleural effusions , were not reliable indicators of live birth versus stillbirth status . for lung aeration , a single false positive diagnosis was made where apparently well - aerated lungs were seen in a stillborn infant ( fig . 2 ) . the clinical history recorded no witnessed spontaneous breathing at 38-week gestation ; however , this foetus may have undergone resuscitation attempts and the air within the lungs may be related to mechanical ventilation.fig . a faint foetal heart rate was thought to be detected prior to emergency caesarean section , but there was evidence of intrauterine death and maceration at delivery . t2-weighted pmmr imaging shows that the lung bases contain air ( dark ) on both coronal ( a ) and axial images ( b ) and gas within the hepatobiliary system ( c ) . we suggest that these findings may be related to initial resuscitation rather than spontaneous breathing example of false positive pmmr . a faint foetal heart rate was thought to be detected prior to emergency caesarean section , but there was evidence of intrauterine death and maceration at delivery . t2-weighted pmmr imaging shows that the lung bases contain air ( dark ) on both coronal ( a ) and axial images ( b ) and gas within the hepatobiliary system ( c ) . we suggest that these findings may be related to initial resuscitation rather than spontaneous breathing two false negative diagnoses on pmmr were made , where neonates had documented spontaneous breaths , but no gas was seen in the lung parenchyma ( fig . both were neonates who died from perinatal asphyxia and ischaemic brain injury in the first week of life.fig . coronal ( a ) and axial ( b ) t2-weighted pmmr imaging of the lungs in a 2-day - old neonate who died of ischaemic brain injury . the baby had no heart rate at birth and very poor apgar score and was resuscitated and mechanically ventilated but care was withdrawn . despite the ventilation , the lungs did not contain air on pmmr . interestingly , we also did not see gas in the airway ( not shown ) example of false negative pmmr . coronal ( a ) and axial ( b ) t2-weighted pmmr imaging of the lungs in a 2-day - old neonate who died of ischaemic brain injury . the baby had no heart rate at birth and very poor apgar score and was resuscitated and mechanically ventilated but care was withdrawn . despite the ventilation , the lungs did not contain air on pmmr . interestingly , we also did not see gas in the airway ( not shown ) as it was possible that the use of both lung and gi tract aeration or either lung or gi tract aeration together could improve the diagnostic accuracy of pmmr , we repeated the statistical analysis for both of these measures . both of these analyses gave the same results as those for lung aeration only and did not improve the diagnostic accuracy . our results indicate that subjective lung parenchyma aeration on pmmr is a good indicator of spontaneous breathing and can be useful to distinguish live birth from stillbirth in most cases . this method of assessing stillbirth has similar accuracy to traditional lung flotation methods but has the primary advantage of being completely non - invasive and may be acquired along with routine pmmr imaging . with the increasing use , experience and acceptability of non - invasive imaging - based post - mortem examinations in addition to conventional invasive autopsy techniques , pmmr can give reliable information about the body without the need for invasive testing . our results are similar to the most recent formal study of the lung flotation test by groe ostendorf et al . which included 208 cases , in which they report 98.0 % ( 95.199.3 % ) concordance , with sensitivity of 71.4 % ( 45.488.3 % ) , specificity of 100 % ( 98.0100 % ) , ppv 100 % ( 72.3100 % ) and npv 98.0 % ( 94.999.2 ) . they had no false positives ( lungs of a stillborn which float ) and four false negatives ( lungs of a live birth which sink ) . they argue that a negative test result can not be taken as proof for a newborn never to have breathed at all . in keeping with their results , we found an absence of air in the lungs in two live births ( two false negatives ) , with specificity of lung aeration for live birth higher than sensitivity . however , 194 of their 208 cases were born dead , and 125 of these were below 22-week gestation which we excluded in our study due to the potential for confounding due to lung immaturity and the relative lack of clinical relevance in this population . the very large proportion of stillbirths and low gestation of some of their cases may have contributed to their higher specificity . in all studies of this type , uncertainty will remain over the factors such as the accurate identification of successful spontaneous breathing and , in particular , over the role of resuscitation in introducing air into the lungs of stillbirths . in this study , we attempted to control as many variables as possible , with most cases either terminations who had definitely not breathed and not been resuscitated and early infant deaths who had unequivocally breathed spontaneously . in real clinical scenarios , the situation is rarely so controlled , and it remains to be seen how much aeration that a few gasps or brief attempts at initial resuscitation could introduce into the lungs . the main clinical circumstance in which this information is important is to distinguish stillbirth from live birth in cases of suspected infanticide and medico - legal cases in which there is uncertainty regarding optimal management of labour and delivery . resuscitation as a confounder in this context could be tested by attempting to ventilate known stillborn infants in future studies . in adults , post - mortem imaging has been performed during lung ventilation ( ventilated pmct ) in order to differentiate collapsed post - mortem lungs from true pathology , and the introduction of this technique into perinatal pm imaging may allow resolution of this issue . we primarily identified the presence or absence of gas on whole - body 3-d t2-weighted turbo spin echo ( tse ) pmmr sequences , which can give excellent signal to noise at high resolution in these cases . air is typically black or very low signal intensity compared to the surrounding tissues and is usually easy to identify in particular within the lungs . where there was doubt , other sequences can be useful ; for instance , we found the vibe sequence useful , as it is particularly sensitive to the presence of gas due to its chemical shift artefact . we consider a 3-d t2-weighted and 3-d t2-weighted sequence of the body to be an appropriate minimum acquisition standard in foetal and paediatric pmmr body imaging , as they should allow clinical abnormalities to be identified with confidence , and reconstruction in any plane . in a medico - legal context a false positive result ( interpreting a stillbirth as having breathed ) could incorrectly lead to accusations of an innocent caregiver of infanticide / neonaticide , whereas a false negative result ( incorrectly interpreting a live birth as having no lung aeration ) could inaccurately provide a defence to a guilty party . in this context , as consent from the parents is typically not required when they fall under the jurisdiction of those investigating a suspicious death , formal autopsy tests are likely to remain the gold standard . however , with the recent decline in parental acceptance of invasive autopsy , there has been increasing emphasis on the accurate interpretation of perinatal and paediatric pm imaging as part of a more minimally invasive approach . part of the challenge of pm imaging is the correct interpretation of imaging findings , which this study will help to educate and elucidate further . despite this being the only study to have addressed this issue using pmmr , and our study population being larger than other studies using pm ct [ 8 , 9 ] we were unable to further evaluate the role of resuscitation in potentially generating false positive pmmr results . furthermore , our data is based on the accuracy of clinical data regarding witnessed spontaneous breathing or definite stillbirth ( although for the vast majority of cases , this was clearly documented ) . a further limitation is that we also excluded those with congenital lung abnormalities or lung disease such as pneumonia , in which the presence of additional pathology could affect these results . the diagnosis of pneumonia on pmmr is particularly challenging , and future studies will be needed to clarify how various pathological states ( pneumonia , haemorrhage etc . ) and physiological changes ( post - mortem fluid accumulation ) could affect these imaging investigations . in summary , these findings demonstrate a high overall accuracy for determination of lung aeration by pmmr as an indicator for spontaneous breathing in foetal and early infant deaths . this approach has a similar accuracy to traditional invasive tests , but is non - invasive , and therefore likely to become more acceptable in future autopsies which involve pmmr as a standard investigation .

objectiveaim of this study was to investigate whether lung assessment on post - mortem magnetic resonance imaging ( pmmr ) can reliably differentiate between live birth and stillbirth.materials and methodswe retrospectively assessed pmmr imaging features of a group of late foetal terminations following fetocide and stillbirths ( without witnessed breathing ) and early infant deaths ( breathed spontaneously before death ) . pmmr images were reviewed for evidence of lung aeration and other features , blinded to the clinical and autopsy data.resultsnineteen infant deaths ( mean age 3.0 6.5 post - natal weeks ) and 23 foetal terminations or stillbirths ( mean age 32.6 10.2-week gestation ) were compared . subjective appearances of lung aeration on pmmr were the best indicator of live birth , with a sensitivity of 89.5 % ( 95 % confidence interval 68.6 , 97.1 % ) and specificity of 95.6 % ( 79.0 , 99.2 % ) and positive and negative predictive values of 94.4 % and 91.7 % , respectively.conclusionslung aeration on pmmr appears to have high overall accuracy for confirmation of live birth versus intrauterine foetal death but now requires validating in a larger cohort of perinatal deaths .

Introduction Methods PMMR imaging Autopsy Data and statistical analysis Results Demographics Imaging Discussion Conclusion Ethical standards Conflict of interest

however , there is uncertainty regarding the reliability of this test , with various factors potentially contributing to both false positive and false negative findings [ 2 , 3 ] . post - mortem imaging is becoming widely accepted as an important component of the examination after death [ 5 , 6 ] , with excellent diagnostic agreement between perinatal and paediatric post - mortem magnetic resonance imaging ( pmmr ) and autopsy findings . part of the challenge of post - mortem ( pm ) imaging is the correct interpretation of imaging findings , and two small case series ( 11 cases in total ) have described the use of post - mortem computed tomography ( pmct ) to assess lung aeration in infant deaths [ 8 , 9 ] . in this study , we investigate the accuracy of pmmr imaging characteristics to distinguish between liveborn and stillborn infants to establish a non - invasive alternative or adjunct to the lung flotation test , which could be more acceptable to parents who decline an invasive autopsy , and to more accurately interpret routine pmmr imaging . we retrospectively reviewed our hospital database for cases of late foetal death , termination of pregnancy , and infant deaths in the first 3 months of life , who had undergone pmmr as part of their post - mortem assessment in the last 5 years . this study did not require further specific institutional approval , as all parents had consented to a clinical pre - autopsy pmmr as part of our institution s clinical post - mortem assessment , and use of routinely collected post - mortem data is approved by the local research ethics committee . our inclusion criteria identified two groups : group 1 : live births defined as infants who were born alive and witnessed spontaneously breathing before death , and group 2 : non - live births defined as either late foetal terminations of pregnancy for abnormality following fetocide ( over 24-week gestation ) without documented evidence of breathing and stillbirths who had been born dead . pmmr findings were reviewed by two radiologists with 3- ( jb ) and 7-year ( oa ) clinical radiology experience ( 0- and 2-year pmmr reporting experience , respectively ) , using the osirix platform ( osirix foundation , geneva , switzerland ) , blinded to the clinical history and autopsy findings . the presence of post - mortem pleural fluid collections was also recorded but predicted to be consistent across the two groups ( a control measure ) , as this was assumed to be unrelated to breathing . conventional autopsies were performed by one of several experienced specialized perinatal / paediatric pathologists in accordance with national guidelines and were reported blinded to the pmmr findings . autopsy findings were reviewed for this study to exclude any cases which had abnormal lung pathology . primary outcomes were sensitivity , specificity , positive predictive value ( ppv ) and negative predictive value ( npv ) where pmmr ( the index test ) was compared with clinical history ( the gold standard ) , with 95 % confidence intervals ( cis ) . pmmr findings were reviewed by two radiologists with 3- ( jb ) and 7-year ( oa ) clinical radiology experience ( 0- and 2-year pmmr reporting experience , respectively ) , using the osirix platform ( osirix foundation , geneva , switzerland ) , blinded to the clinical history and autopsy findings . the presence of post - mortem pleural fluid collections was also recorded but predicted to be consistent across the two groups ( a control measure ) , as this was assumed to be unrelated to breathing . conventional autopsies were performed by one of several experienced specialized perinatal / paediatric pathologists in accordance with national guidelines and were reported blinded to the pmmr findings . autopsy findings were reviewed for this study to exclude any cases which had abnormal lung pathology . primary outcomes were sensitivity , specificity , positive predictive value ( ppv ) and negative predictive value ( npv ) where pmmr ( the index test ) was compared with clinical history ( the gold standard ) , with 95 % confidence intervals ( cis ) . group 1 live births comprised 19 infant deaths ( mean post - natal age at death 3.0 6.6 weeks ) . group 2 non - live births comprised 23 foetal terminations or stillbirths ( mean age 32.6 10.2-week gestation ) of which 17 were late foetal terminations ( known to have not breathed and not undergone resuscitation ) and six stillbirths without spontaneous breathing . gas in almost all locations , including airways , lungs , or gi tract , was identified more commonly in live births than stillbirths ( table 1).table 1diagnostic accuracy of imaging indices for live birthindexfp / tpfn / tnsensitivity ( % ) specificity ( % ) ppv ( % ) npv ( % ) concordance ( % ) gas in airway7/163/1684.2 % ( 62.4 , 94.5)*69.6 % ( 49.1 , 84.4)69.6 % ( 49.1 , 84.4)84.2 % ( 62.4 , 94.5)76.2 % ( 61.5 , 86.5)lung aeration1/172/2289.5 % ( 68.6 , 97.1)95.6 % ( 79.0 , 99.2)94.4 % ( 74.2 , 99.0)91.7 % ( 74.2 , 97.7)92.9 % ( 81.0 , 97.5)gas in gi tract5/190/18100 % ( 83.2 , 100)78.3 % ( 58.1 , 90.3)79.2 % ( 59.5 , 90.8)100 % ( 82.4 , 100)88.1 % ( 75.0 , 94.8)absence of r ht gas9/145/1473.7 % ( 51.2 , 88.2)60.9 % ( 40.8 , 77.8)60.9 % ( 40.8 , 77.8)73.7 % ( 51.2 , 88.2)66.7 % ( 51.6 , 79.0)absence of l ht gas8/172/1589.5 % ( 68.6 , 97.1)65.2 % ( 44.9 , 81.2)68.0 % ( 48.4 , 82.8)88.2 % ( 65.7 , 96.7)76.2 % ( 61.5 , 86.5)absence of hepatobiliary gas12/163/1184.2 % ( 62.4 , 94.5)47.8 % ( 29.2 , 67.0)57.1 % ( 39.1 , 73.5)78.6 % ( 52.4 , 92.4)64.3 % ( 49.2 , 77.0)bilateral effusions11/109/1252.6 % ( 31.7 , 72.7)52.2 % ( 33.0 , 70.8)47.6 % ( 28.3 , 67.6)57.1 % ( 36.6 , 75.5)52.4 % ( 37.7 , 66.6)a positive test result was defined as the presence of any aeration in the given location fp false positive , tp true positive , fn false negative , tn true negative , r ht right heart ( atrium or ventricle ) , l ht left heart ( atrium or ventricle)*all percentages are given 95 % confidence interval diagnostic accuracy of imaging indices for live birth a positive test result was defined as the presence of any aeration in the given location fp false positive , tp true positive , fn false negative , tn true negative , r ht right heart ( atrium or ventricle ) , l ht left heart ( atrium or ventricle ) * all percentages are given 95 % confidence interval lung aeration on pmmr was the best indicator of live birth , with highest concordance between pmmr and autopsy of 92.9 % ( 95 % ci 81.0 , 97.5 ) . lung aeration was the most specific marker of live birth , with specificity of 95.6 % ( 79.0 , 99.2 ) and ppv of 94.4 % ( 74.2 , 99.0 ) , whereas gas in the gi tract was the most sensitive at 100 % ( 86.2 , 100 ) and highest npv 100 % ( 82.4 , 100 ) ( fig . it demonstrates air within the airways and lungs ( dark ) and gas within the stomach ( dark ) , as would be expected in an infant who had breathed before death ( true positive in this study ) . there were no signs of life at delivery , no resuscitation implemented , and pmmr demonstrated no gas in the lungs ( grey lungs ) , nor gi tract ( not shown ) , as would be expected ( true negative in this study ) example of agreement between pmmr and autopsy . it demonstrates air within the airways and lungs ( dark ) and gas within the stomach ( dark ) , as would be expected in an infant who had breathed before death ( true positive in this study ) . there were no signs of life at delivery , no resuscitation implemented , and pmmr demonstrated no gas in the lungs ( grey lungs ) , nor gi tract ( not shown ) , as would be expected ( true negative in this study ) intracardiac gas and hepatobiliary gas were more commonly seen in stillbirths than in live births , but , together with the presence of pleural effusions , were not reliable indicators of live birth versus stillbirth status . for lung aeration , a single false positive diagnosis was made where apparently well - aerated lungs were seen in a stillborn infant ( fig . we suggest that these findings may be related to initial resuscitation rather than spontaneous breathing two false negative diagnoses on pmmr were made , where neonates had documented spontaneous breaths , but no gas was seen in the lung parenchyma ( fig . coronal ( a ) and axial ( b ) t2-weighted pmmr imaging of the lungs in a 2-day - old neonate who died of ischaemic brain injury . coronal ( a ) and axial ( b ) t2-weighted pmmr imaging of the lungs in a 2-day - old neonate who died of ischaemic brain injury . group 1 live births comprised 19 infant deaths ( mean post - natal age at death 3.0 6.6 weeks ) . sixteen breathed spontaneously at birth and had lived for up to 11 weeks prior to death , which was attributed to a variety of causes including trauma and sudden unexpected death in infancy ( sudi ) . group 2 non - live births comprised 23 foetal terminations or stillbirths ( mean age 32.6 10.2-week gestation ) of which 17 were late foetal terminations ( known to have not breathed and not undergone resuscitation ) and six stillbirths without spontaneous breathing . gas in almost all locations , including airways , lungs , or gi tract , was identified more commonly in live births than stillbirths ( table 1).table 1diagnostic accuracy of imaging indices for live birthindexfp / tpfn / tnsensitivity ( % ) specificity ( % ) ppv ( % ) npv ( % ) concordance ( % ) gas in airway7/163/1684.2 % ( 62.4 , 94.5)*69.6 % ( 49.1 , 84.4)69.6 % ( 49.1 , 84.4)84.2 % ( 62.4 , 94.5)76.2 % ( 61.5 , 86.5)lung aeration1/172/2289.5 % ( 68.6 , 97.1)95.6 % ( 79.0 , 99.2)94.4 % ( 74.2 , 99.0)91.7 % ( 74.2 , 97.7)92.9 % ( 81.0 , 97.5)gas in gi tract5/190/18100 % ( 83.2 , 100)78.3 % ( 58.1 , 90.3)79.2 % ( 59.5 , 90.8)100 % ( 82.4 , 100)88.1 % ( 75.0 , 94.8)absence of r ht gas9/145/1473.7 % ( 51.2 , 88.2)60.9 % ( 40.8 , 77.8)60.9 % ( 40.8 , 77.8)73.7 % ( 51.2 , 88.2)66.7 % ( 51.6 , 79.0)absence of l ht gas8/172/1589.5 % ( 68.6 , 97.1)65.2 % ( 44.9 , 81.2)68.0 % ( 48.4 , 82.8)88.2 % ( 65.7 , 96.7)76.2 % ( 61.5 , 86.5)absence of hepatobiliary gas12/163/1184.2 % ( 62.4 , 94.5)47.8 % ( 29.2 , 67.0)57.1 % ( 39.1 , 73.5)78.6 % ( 52.4 , 92.4)64.3 % ( 49.2 , 77.0)bilateral effusions11/109/1252.6 % ( 31.7 , 72.7)52.2 % ( 33.0 , 70.8)47.6 % ( 28.3 , 67.6)57.1 % ( 36.6 , 75.5)52.4 % ( 37.7 , 66.6)a positive test result was defined as the presence of any aeration in the given location fp false positive , tp true positive , fn false negative , tn true negative , r ht right heart ( atrium or ventricle ) , l ht left heart ( atrium or ventricle)*all percentages are given 95 % confidence interval diagnostic accuracy of imaging indices for live birth a positive test result was defined as the presence of any aeration in the given location fp false positive , tp true positive , fn false negative , tn true negative , r ht right heart ( atrium or ventricle ) , l ht left heart ( atrium or ventricle ) * all percentages are given 95 % confidence interval lung aeration on pmmr was the best indicator of live birth , with highest concordance between pmmr and autopsy of 92.9 % ( 95 % ci 81.0 , 97.5 ) . lung aeration was the most specific marker of live birth , with specificity of 95.6 % ( 79.0 , 99.2 ) and ppv of 94.4 % ( 74.2 , 99.0 ) , whereas gas in the gi tract was the most sensitive at 100 % ( 86.2 , 100 ) and highest npv 100 % ( 82.4 , 100 ) ( fig . it demonstrates air within the airways and lungs ( dark ) and gas within the stomach ( dark ) , as would be expected in an infant who had breathed before death ( true positive in this study ) . it demonstrates air within the airways and lungs ( dark ) and gas within the stomach ( dark ) , as would be expected in an infant who had breathed before death ( true positive in this study ) . there were no signs of life at delivery , no resuscitation implemented , and pmmr demonstrated no gas in the lungs ( grey lungs ) , nor gi tract ( not shown ) , as would be expected ( true negative in this study ) intracardiac gas and hepatobiliary gas were more commonly seen in stillbirths than in live births , but , together with the presence of pleural effusions , were not reliable indicators of live birth versus stillbirth status . t2-weighted pmmr imaging shows that the lung bases contain air ( dark ) on both coronal ( a ) and axial images ( b ) and gas within the hepatobiliary system ( c ) . we suggest that these findings may be related to initial resuscitation rather than spontaneous breathing two false negative diagnoses on pmmr were made , where neonates had documented spontaneous breaths , but no gas was seen in the lung parenchyma ( fig . coronal ( a ) and axial ( b ) t2-weighted pmmr imaging of the lungs in a 2-day - old neonate who died of ischaemic brain injury . coronal ( a ) and axial ( b ) t2-weighted pmmr imaging of the lungs in a 2-day - old neonate who died of ischaemic brain injury . our results indicate that subjective lung parenchyma aeration on pmmr is a good indicator of spontaneous breathing and can be useful to distinguish live birth from stillbirth in most cases . which included 208 cases , in which they report 98.0 % ( 95.199.3 % ) concordance , with sensitivity of 71.4 % ( 45.488.3 % ) , specificity of 100 % ( 98.0100 % ) , ppv 100 % ( 72.3100 % ) and npv 98.0 % ( 94.999.2 ) . they had no false positives ( lungs of a stillborn which float ) and four false negatives ( lungs of a live birth which sink ) . in keeping with their results , we found an absence of air in the lungs in two live births ( two false negatives ) , with specificity of lung aeration for live birth higher than sensitivity . in this study , we attempted to control as many variables as possible , with most cases either terminations who had definitely not breathed and not been resuscitated and early infant deaths who had unequivocally breathed spontaneously . in adults , post - mortem imaging has been performed during lung ventilation ( ventilated pmct ) in order to differentiate collapsed post - mortem lungs from true pathology , and the introduction of this technique into perinatal pm imaging may allow resolution of this issue . in a medico - legal context a false positive result ( interpreting a stillbirth as having breathed ) could incorrectly lead to accusations of an innocent caregiver of infanticide / neonaticide , whereas a false negative result ( incorrectly interpreting a live birth as having no lung aeration ) could inaccurately provide a defence to a guilty party . however , with the recent decline in parental acceptance of invasive autopsy , there has been increasing emphasis on the accurate interpretation of perinatal and paediatric pm imaging as part of a more minimally invasive approach . and physiological changes ( post - mortem fluid accumulation ) could affect these imaging investigations . in summary , these findings demonstrate a high overall accuracy for determination of lung aeration by pmmr as an indicator for spontaneous breathing in foetal and early infant deaths .

rice ( oryza sativa l. ) is the staple food for half of the world s population . the eating quality of rice is increasingly important to meet the market demand . therefore , one of the major goals in a breeding program is to develop rice varieties of better eating quality to satisfy the requirements of both the food industry and consumers . even though indica rice varieties are popular worldwide , consumers in northeastern asian countries such as korea , japan , northern china , and taiwan prefer japonica rice , mainly due to its moderate elasticity and stickiness . the eating quality of rice is a complex trait involving many physicochemical properties , and thus it has been challenging to accurately evaluate eating quality for selection in rice - breeding programs . some key physicochemical properties affecting the eating quality are amylose content ( ac ) ( 1 ) , pasting properties ( pp ) ( 2 ) , gel consistency ( gc ) , gelatinization temperature ( gt ) ( 3 ) , and protein content ( pc ) ( 4 ) . good eating quality is also associated with stickiness , sweet flavor , glossiness of the cooked rice , and palatability . palatability , the trait directly related to rice eating quality , is determined by aroma , appearance , taste , and texture ( 4 ) . in addition to genetic determinants , such as genes involved in the synthesis of starch and protein , rice eating quality is also largely affected by environmental factors , cultural practices , and postharvest practices such as air temperature during ripening , the amount of fertilizer , irrigation management , grain - drying after harvest , and cooking methods ( 5 ) . in breeding programs , however , because this method both requires a large amount of rice per sample and allows the evaluation of only a few samples per day , the sensory test is more efficient when performed at a later stage when selected lines are homozygous ( 6 ) . moreover , the results of sensory evaluation are sometimes not consistent even for the same sample , presumably due to the physical and emotional condition of members of the panel or subtle differences in sample preparation . recently , an instrument for evaluating the palatability value of rice has been developed and used for line selection in breeding programs ( 7 , 8) . however , it also requires a large amount of rice per sample , and thus the palatability test using this instrument is usually performed only for advanced breeding generations . these have revealed that some rice physicochemical properties such as ac , gt , gc , and pasting viscosity are controlled by one to three major genes with one or more modifiers . the enzymes involved in starch biosynthesis , such as starch branching enzyme ( sbe ) , starch synthase ( ss ) , and granule bound starch synthase ( gbss ) contribute greatly to the variation of starch physicochemical properties and thus eating quality ( 2 ) . major genes and/or quantitative trait loci ( qtls ) associated with eating quality ( 9 ) , pc ( 10 ) , and palatability ( 9 , 11 ) such as wx ( waxy gene ) and alk ( starch synthase ii ) ( 3 ) have been reported . in addition , interaction among these genes along with others may govern rice grain physicochemical properties , which in turn determine the eating quality of cooked rice . collectively , the genetic complexity of eating quality , as well as the difficulty in accurate evaluation of eating quality at early breeding generations , has constrained the development of rice varieties with high eating quality . to complement the physicochemical analyses and sensory tests available to evaluate eating quality , dna marker - based approaches have been developed . these methods offer the additional advantages of screening at early breeding generations as well as simplicity and accuracy . markers based on the polymerase chain reaction ( pcr ) have been tested for quality evaluation of rice varieties ( 12 ) . recently , sequence - tagged site ( sts ) primers developed from random amplified polymorphic dna ( rapd ) analysis were able to differentiate rice varieties according to their palatability ( 12 , 13 ) . several functional markers have also been developed to distinguish the physicochemical properties of rice , especially the effect of the waxy locus on pp ( 14 ) , that of sbe on starch viscosity ( 15 ) , and those of ac ( 2 ) and starch synthase iia ( ssiia ) on gt ( 3 ) . additional gene - tagged markers have also been developed from starch - synthesizing genes ( 2 , 14 , 15 ) . despite the recent progress in developing markers and identification of qtls associated with eating quality , a marker - assisted breeding ( mab ) system for better eating quality has not been established . in this study , our aim was to develop dna markers associated with eating quality and to formulate a marker - based evaluation and prediction method of eating quality of cooked rice in japonica varieties . a total of 22 japonica rice varieties , mostly bred in korea , were evaluated for palatability . these consisted of 2 varieties from japan ( koshihikari and hitomebore ) , 1 variety from china ( hexi41 ) , and 19 varieties from korea ( gopum , ilpum , samgwang , chucheong , dongjin , sinkeumo , hwaseong , hwacheong , dobong , samnam , palkong , baekjinju1 , seonong4 , onnuri , manmi , giho , geuman , nakdong , and samdeok ) . these varieties were chosen because they represent diverse palatability scores among japonica rice ( national institute of crop science ( nics ) , personal communication ) . varieties were grown using conventional cultural practices at the experimental farm of seoul national university , suwon , in 2006 . in addition , 32 japonica breeding lines along with three check varieties grown in a regional yield trial plot in 2007 at nics were also used for validation of the marker set developed in this study . for physicochemical analysis , rice grains all rice varieties and lines were grown in a light- and temperature - controlled greenhouse until the tillering stage , at which point tissue was collected for dna extraction . genomic dna was extracted using the modified cetyltrimethylammonium bromide ( ctab ) method based on the protocol of murray and thompson ( 16 ) . twenty previously reported sts primer pairs developed from rapd ( 12 , 13 , 17 ) were tested in this study . to identify the amplicon sequences , the amplified bands were excised , purified , ta cloned , and sequenced . in a homology search , each cloned sequence was compared against all sequences in the nonredundant databases using the blastx and blastn programs ( http://www.ncbi.nlm.nih.gov/blast and www.gramene.org ) . ( 3 ) , was polymorphic among the 22 rice varieties and was actually used in this study . the sequences and banding patterns of markers previously reported are presented in table 1 and figure 1 , respectively . ( a ) polymorphic indel in tre ( m , 100 bp dna marker ; 1 , koshihikari ; 2 , samgwang ; 3 , ilpum ; 4 , sinkeumo ; 5 , dobong ; 6 , samnam ; 7 , palkong ; 8 , hitomebore ; 9 , hexi41 ; 10 , samdeok ) ; a , insertion of cttt . ( b ) polymorphic snp in s3 ( 1 , koshihikari ; 2 , samgwang ; 3 , ilpum ; 4 , sinkeumo ; 5 , dobong ; 6 , samnam ; 7 , hexi41 ) ; b , point mutation t to g allele . ( c ) variation in ( ctt)n repeats in cbg ( 1 , koshihikari ; 2 , gopum ; 3 , samgwang ; 4 , ilpum ; 5 , chucheong ; 6 , dongjin ; 7 , sinkeumo ; 8 , hwaseong ; 9 , hwacheong ; 10 , dobong ; 11 , samnam ; 12 , palkong ; 13 , hitomebore ; 14 , baekjinju1 ; 15 , seonong 4 ; 16 , manmi ) ; c , ( ctt)8 alleles , others are ( ctt)19 alleles . ( d ) dna banding patterns produced from 11 sts markers developed by ohtsubo et al . ( 13 , 17 ) and ohtsubo and nakamura ( 12 ) observed on koshihikari ( marker ; 1 , b7 ; 2 , a7 ; 3 , g81 ; 4 , b43 ; 5 , e30 ; 6 , f6 ; 7 , g4 ; 8 , g22 ; 9 , g28 ; 10 , j6 ; 11 , m11 ) . on the basis of selected regions either close to or within the genes linked to interesting qtls ( 11 , 18 , 19 ) for rice eating quality traits ( table 2 ) , analysis of nucleotide polymorphism of the sequences among japonica varieties was performed to develop primers for eating quality . to design the purified pcr products from various japonica varieties were ta - cloned into pgem - t easy vectors and transformed into eschericia colidh5-competent cells prepared according to the protocol of sambrook and russell ( 21 ) . plasmids were isolated using the dna - spin plasmid dna purification kit ( intron biotechnology , korea ) and sequenced with an abi-3700 dna sequencer following the manufacturer s instructions ( applied biosystems , inc . ) . to identify snps , insertions and deletions ( indels ) , and/or microsatellite repeats in the rice varieties , sequence results were aligned using the clustal w program ( 22 ) from embl - european bioinformatics institute ( http://www.ebi.ac.uk/tools ) , with assistance from codoncode aligner 2.0.6 ( codoncode corporation , dedham , ma ) as well as bioedit ( http://www.mbio.ncsu.edu/bioedit/bioedit.html ) . to detect a 1 bp substitution in a specific fragment , a dcaps ( derived cleaved amplified polymorphic sequences ) primer was designed , facilitated by dcaps finder 2.0 ( 23 ) ( http://helix.wustl.edu/dcaps ) . finally , nine molecular markers were successfully developed on the basis of indels , snps , and microsatellite repeats identified in this study ( table 3 ) . http://www.ncbi.nlm.nih.gov/ , http://rgp.dna.affrc.go.jp/e/irgsp/index.html , http://rice.plantbiology.msu.edu/ , and http://www.gramene.org/. pcr amplification of markers was carried out in a ptc-200 peltier thermal cycler ( mj research , inc . ) in a total volume of 20 l with the following genotyping pcr reagents : 2 l of dna at 20 ng/l , 2 l of 10 buffer containing 25 mm mgcl2 , 1 l of 2.5 mm dntps , 1 unit of taq polymerase ( intron biotechnology , korea ) , and 1 l each of forward and reverse primers ( 10 m ) . the pcr reaction for further sequencing analysis consisted of 1 unit of extaq polymerase ( takara ) in a total reaction of 50 l . all amplifications were performed for a total of 35 cycles of 1 min at 95 c , 30 s at 55 c , and 1 min at 72 c . for sts markers developed by ohtsubo et al . ( 13 , 17 ) and ohtsubo and nakamura ( 12 ) , the initial denaturation was at 95 c for 5 min , followed by 40 cycles of 96 c for 1 min , 62 c for 1 min , and 72 c for 2 min . ( 2 , 3 ) were performed under the following conditions : 5 min at 94 c followed by 35 cycles of 45 s at 94 c , 1 min at 55 c , and 1 min at 72 c , with a final extension of 7 min at 72 c . amplified pcr products were analyzed by electrophoresis on 3% agarose gels stained with ethidium bromide and/or by nondenaturing electrophoresis on 8% polyacrylamide gels stained with ethidium bromide ( model mgv , cbs scientific co. ) . for palatability and physicochemical analyses , the rice grains were hulled and milled to 91% yield . palatability was measured using a rice taste measuring system ( toyo taste meter , model ma-90 ) in accordance with the operation manual ( trcm co. ) ( toyo rice polishing machine factory , japan ) . pc was calculated using total nitrogen multiplied by 5.95 after determination of the nitrogen content of rice material by the micro - kjeldahl method ( 24 ) . the ac of milled rice was determined by the relative absorbency of starchiodine color in a digested solution of 100-mesh rice flour according to the method of perez and juliano ( 25 ) . rva pasting properties were determined on a rapid visco analyzer ( rva ) in accordance with the operation manual ( newport sci . rice starch paste profile characteristics were described by six parameters : peak viscosity ( pv ) , hot paste viscosity ( hpv ) , cool paste viscosity ( cpv ) , breakdown viscosity ( bdv = pv hpv ) , setback viscosity ( sbv = cpv pv ) , and consistency viscosity ( ctv = cpv hpv ) in accordance with the procedure of bao and xia ( 26 ) . the milled rice was cooked according to the protocol of nics , the rural development administration ( rda ) , korea . dry - milled head rice ( 300 g ) was rinsed four times and soaked for 30 min with distilled water , and the water was then strained for 10 min . rice was cooked using an electric rice cooker with the ratio of rice / water = 1:1.25 w / w . after completion of the automatic cooking cycle , samples were transferred to plates and kept at room temperature for about 10 min until cooled to 3537 c . sensory evaluation of cooked rice samples by 11 well - trained panel members was performed with five replications . the overall palatability was assessed according to appearance ( glossiness ) , fragrance , taste , stickiness , texture , and palatability score ( overall score ; overall eating quality ) and scored from + 3 to 3 compared to a cooked rice reference sample , chucheong ( score = 0 ) . the palatability score of each variety by the sensory test was the average value scored by 11 panel members . the collected data were analyzed and subjected to analysis of variance using sas software version 8.2 ( 27 ) . the least significant difference ( duncan ) method was used to evaluate differences between trait means . regression and correlation analyses were also performed to determine the relationships between rice eating quality traits . multiple regression analysis was also conducted to determine the relationship between palatability scored both by taste analyzer and by sensory test and also palatability evaluated by molecular markers . sts marker data were scored as 1 ( present ) and 0 ( absent ) . similarly , the two different alleles resulting from each ssr and snp marker were also converted into binary values of 1 or 0 . by using the palatability scores as dependent variables and the binary data from molecular markers as independent variables or regressors , the best model equation to predict rice palatability was obtained . the most accurate prediction gave the lowest standard error and significantly highest coefficient of determination ( r ) , which consisted of the highly significant regression coefficient for each marker in the model . using the binary matrix , a cluster analysis was performed with the unweighted pair group method ( upgma ) in ntsys ( exeter software , setauket , ny ) ( 28 ) . dna sequences of pcr amplicons produced by 20 previously reported sts primer pairs ( 12 , 13 , 17 ) were compared against all sequences in the nonredundant databases using the blastx and blastn programs . however , no candidate gene of any known function possibly related to quality traits was found among the sequences derived from sts primers . therefore , a total of 21 markers , including ssiia ( 3 ) , were used directly in this study ( table 1 ) as 5 of the markers listed in table 1 from bao et al . on the basis of qtl analyses for rice eating quality ( 11 , 18 , 19 ) , we were able to select seven candidate genes underlying the qtl regions : sucrose synthase 3 ( s3 , clone ap004988 ) , trehalose phosphatase ( tre , clone ap004341 ) , granule bound starch synthase 1 ( gbss1/waxy gene , clone ap002542 ) , udp - n - acetylglucosamine pyrophosphorylase ( acph , clone ap003875 ) , glucosamine - fructose-6-phosphate aminotransferase ( gpa , clone ac138454 ) , aspartate aminotransferase ( am , clone ap003991 ) , and noncyanogenic -glucosidase ( cbg , clone ac074354 ) ( table 2 ) . in addition , by searching the genomics date base ( db ) , adp - glucose pyrophosphorylase ( shrunken gene , sh51 , ap004317 ) , a gene involved in starch biosynthesis and located on chromosome 1 , was also chosen for marker development . by comparing the sequence of each gene ( table 2 ) among japonica varieties , we developed a total of nine dna markers ( table 3 ) . examples of pcr amplicons of different marker types among japonica rice varieties are shown in figure 1 . the insertion of cttt alleles in the tre locus ( figure 1a ) and the g allele in the s3 locus ( figure 1b ) were rarely found in japonica varieties . in the cbg locus were distinguished two ( ctt)n repeats of ( ctt)8 and ( ctt)19 , but only a rice of low palatability ( palkong ) possessed the ( ctt)8 allele ( figure 1c ) . moreover , the dna banding pattern from the sts primers for palatability on koshihikari was similar to those of previous studies ( 12 , 13 , 17 ) ( figure 1d ; table 4 ) . tt : ggtttc ( 0 ) and gc : gggctc ( 1 ) at nt 43294330 ( exon ) . insertion of cttt ( 1 ) and no insertion ( 0 ) at nt 7982 of consensus region ( intron ) . point mutation from t ( 1 ) to g allele ( 0 ) at nt 1454 of consensus region ( intron ) . no deletion ( 1 ) and deletion ( ctc ) ( 0 ) at nt 12551257 of consensus region ( intron ) . point mutation from t ( 1 ) to g allele ( 0 ) at nt 397 of consensus region ( intron ) . point mutation from a ( 0 ) to t allele ( 1 ) at nt 51 ( intron ) . . we genotyped 22 japonica rice varieties with diverse palatability values using a total of 30 markers comprising 21 markers developed previously and 9 markers developed in this study ( table 4 ) . cluster analysis was performed on similarity coefficient matrices calculated from molecular markers to generate a dendrogram ( figure 2 ) . the varieties chucheong and hwacheong showed maximum genetic similarity ( 0.96 ) , whereas koshihikari and seonong4 showed the least genetic similarity ( 0.41 ) . when a cutoff value of 0.64 was used for genetic similarity among all varieties , three clusters were formed , i , ii , and iii ( figure 2 ) , which contained 12 , 7 , and 3 varieties , respectively . the two japanese cultivars , koshihikari and hitomebore , formed an independent subcluster , supporting the fact that hitomebore was bred using koshihikari as a parent . similarly , other varieties sharing parentage grouped in the same subclusters . dendrogram of 22 japonica varieties constructed on the basis of similarity coefficients in upgma analysis . the palatability value according to the toyo taste meter ( p ) , the palatability score according to the sensory test ( st ) , and the physicochemical properties of 22 japonica rice varieties are summarized in tables 5 and 6 . all traits except sbv showed significant differences among varieties . four varieties ( koshihikari , ilpum , samgwang , and geuman ) showed good palatability in both p and st . eight varieties exhibited better st than chucheong , which was used as a check variety in the sensory test . ac was similar among varieties except for two , baekjinju1 and seonong4 , which were developed as extremely low ac varieties . p , palatability value ; st , palatability score from sensory test ; ac , amylose content ; pc , protein content ; cpv , cold paste viscosity ; bdv , breakdown viscosity ; pv , peak viscosity ; hpv , hot paste viscosity ; sbv , setback viscosity ; ctv , consistency viscosity ; m , palatability value estimated from the equation based on marker data ; rvu , rapid visco unit . ns , nonsignificant at 5% level ; * * and * , significant at 1 and 5% level , respectively . correlation analysis among quality traits revealed that p and st were significantly correlated ( r = 0.85 * * ) as expected ( table 7 ) . however , p and st were not significantly correlated with other traits , indicating that palatability is a complex trait in which a number of factors are involved . ns , nonsignificant at 5% level ; * and * * , significant at 5 and 1% level , respectively . association analyses between marker genotypes based on 30 primer sets ( table 4 ) and palatability values according to the toyo taste meter ( p ) ( table 6 ) and sensory test ( st ) were performed ( table 8) . general linear model regression analysis indicated that individual molecular markers could not differentiate the eating qualities . thus , multiple regression analysis of the entire set of markers was performed . by using only significant markers as independent variables , model equations were generated that could predict the eating quality with highly significant resolution ( r = 0.99 ) ( table 8) . this indicates that palatability values of 22 varieties estimated by the toyo taste meter and/or by the sensory test can be estimated with high resolution by these regression equations based on the marker genotypes . the marker sets for the equations consisted of 13 markers for palatability by the toyo taste meter and 14 markers for palatability by the sensory test , with 9 markers shared for both equations . three newly developed markers ( gpa , s3ci , and cbg ) were included among these 9 markers . the other 3 markers ( ams , ssiia , and acph ) were included among either 13 markers for palatability by the toyo taste meter or 14 markers for palatability by the sensory test . * * and * , significant at 1% and 5% level , respectively . to evaluate the validity of the regression equation , 32 breeding lines along with 3 japonica check varieties were genotyped with the set of 13 markers mentioned above and their palatability values there was a highly significant correlation ( r = 0.715 * * ) between palatability estimates by the regression equation and palatability values according to the toyo taste meter ( figure 3 ) , indicating that the model equation we developed reliably predicts the palatability of random breeding lines of japonica rice . three check varieties . correlation between palatability values of 32 breeding lines and 3 check varieties measured by the toyo taste meter and estimated by the regression equation . the highly significant correlation between palatability values according to the toyo taste meter and palatability values according to the sensory test is in good agreement with previous studies ( 9 ) . japanese researchers have also reported that palatability values from the toyo taste meter and palatability scores from the sensory test exhibit a high positive correlation ( 7 , 8) . this indicates that the palatability value according to the toyo taste meter can be used as a good measure of the eating quality of rice . for breeding better eating quality rice , the toyo taste meter is the measurement method of choice because of its relative simplicity and reproducibility compared to the sensory test ( 79 ) . this is why the toyo taste meter has been widely used in breeding programs for eating quality in korea , japan , and northeastern asia . no correlation between palatability and any pasting property was found in this study , suggesting that pasting properties could not be a good indicator for palatability value due to its complex nature . pc was the only trait to affect the palatability value as reported previously ( 29 ) . as nucleotide differences among genotypes are a major source of heritable variation , molecular markers derived from them should provide an effective measure of genotypic variation and hence phenotypic differences among varieties . when 22 varieties were classified on the basis of the cluster analysis of 30 markers , they formed three major clusters at a genetic similarity level of 0.640.67 . however , the clusters did not appear to be related to eating quality , but to the parental origin of the strains , although the 30 markers were derived from quality - associated traits or qtls for eating quality . this may be explained by the fact that the cluster diagram was constructed on the basis of genetic similarities simply calculated on the basis of the assumption that each of the markers had the same effect on the genotype determination . this demonstrates that simple genotyping and similarity analysis might not be a good measure to evaluate the eating quality of rice , and thus there should be differential weighting of markers . multiple regression equations developed in this study ( table 8) proved to be effective in predicting the palatability of rice varieties and breeding lines ( figure 3 ) . ( 13 , 17 ) and ohtsubo and nakamura ( 12 ) were able to estimate rice palatability ( sensory test of japanese people ) by using a combination of sts markers and estimation equations with significant multiple regression coefficients . similarly , other marker sets derived from starch synthesizing genes ( gbss , ss , sbe , and sdbe ) ( 2 , 3 ) and storage protein genes ( glutelin and prolamin ) were also tested to evaluate ac , pc , and the adhesiveness of cooked rice ( 30 ) . of the significant markers in the equations ( table 8) , nine were important for palatability as measured by both the toyo taste meter ( p ) and the sensory test ( st ) , and the r values of the nine markers explained most of the variation in both equations . this indicates that palatability by the toyo taste meter and palatability by the sensory test are in good accordance as revealed by the significant correlation ( r = 0.85 * * ) ( table 7 ) . of the nine significant markers , the sts marker , p5 , showed the highest partial r in both equations , implying that it might represent a major qtl for palatability . because we used most of the reported markers developed for taste evaluation in japonica rice , the regression equations ( table 8) are of sufficiently high resolution to estimate the palatability of japonica rice in korea . however , because palatability varies among countries and varietal groups even within japonica rice , the equations may need to be modified to be applicable in other countries . particularly in countries we expect that the set of 13 markers used in this study will be useful for the selection of early breeding or even individual f2 plants to accelerate breeding for improvement of rice eating quality in korea or other japonica rice - consuming countries .

evaluation of eating quality in early breeding generations of rice is critical to developing varieties with better palatability . this paper reports dna markers associated with eating quality of temperate japonica rice and an evaluation method aided by multiple regression analysis . a total of 30 markers comprising stss , snps , and ssrs were tested for their association with palatability using 22 temperate japonica varieties with different palatability values . eating quality - related traits of the 22 varieties were also measured . of the 30 markers , 18 were found to be significantly associated with palatability and , consequently , a model regression equation with an r2 value of 0.99 was formulated to estimate the palatability by the marker data set . validation of the model equation using selected breeding lines indicated that the marker set and the equation are highly applicable to evaluation of the palatability of cooked rice in temperate japonica varieties .

Introduction Materials and Methods Results Discussion

the eating quality of rice is increasingly important to meet the market demand . therefore , one of the major goals in a breeding program is to develop rice varieties of better eating quality to satisfy the requirements of both the food industry and consumers . even though indica rice varieties are popular worldwide , consumers in northeastern asian countries such as korea , japan , northern china , and taiwan prefer japonica rice , mainly due to its moderate elasticity and stickiness . the eating quality of rice is a complex trait involving many physicochemical properties , and thus it has been challenging to accurately evaluate eating quality for selection in rice - breeding programs . some key physicochemical properties affecting the eating quality are amylose content ( ac ) ( 1 ) , pasting properties ( pp ) ( 2 ) , gel consistency ( gc ) , gelatinization temperature ( gt ) ( 3 ) , and protein content ( pc ) ( 4 ) . good eating quality is also associated with stickiness , sweet flavor , glossiness of the cooked rice , and palatability . palatability , the trait directly related to rice eating quality , is determined by aroma , appearance , taste , and texture ( 4 ) . in addition to genetic determinants , such as genes involved in the synthesis of starch and protein , rice eating quality is also largely affected by environmental factors , cultural practices , and postharvest practices such as air temperature during ripening , the amount of fertilizer , irrigation management , grain - drying after harvest , and cooking methods ( 5 ) . in breeding programs , however , because this method both requires a large amount of rice per sample and allows the evaluation of only a few samples per day , the sensory test is more efficient when performed at a later stage when selected lines are homozygous ( 6 ) . moreover , the results of sensory evaluation are sometimes not consistent even for the same sample , presumably due to the physical and emotional condition of members of the panel or subtle differences in sample preparation . recently , an instrument for evaluating the palatability value of rice has been developed and used for line selection in breeding programs ( 7 , 8) . however , it also requires a large amount of rice per sample , and thus the palatability test using this instrument is usually performed only for advanced breeding generations . these have revealed that some rice physicochemical properties such as ac , gt , gc , and pasting viscosity are controlled by one to three major genes with one or more modifiers . the enzymes involved in starch biosynthesis , such as starch branching enzyme ( sbe ) , starch synthase ( ss ) , and granule bound starch synthase ( gbss ) contribute greatly to the variation of starch physicochemical properties and thus eating quality ( 2 ) . major genes and/or quantitative trait loci ( qtls ) associated with eating quality ( 9 ) , pc ( 10 ) , and palatability ( 9 , 11 ) such as wx ( waxy gene ) and alk ( starch synthase ii ) ( 3 ) have been reported . in addition , interaction among these genes along with others may govern rice grain physicochemical properties , which in turn determine the eating quality of cooked rice . collectively , the genetic complexity of eating quality , as well as the difficulty in accurate evaluation of eating quality at early breeding generations , has constrained the development of rice varieties with high eating quality . to complement the physicochemical analyses and sensory tests available to evaluate eating quality , dna marker - based approaches have been developed . these methods offer the additional advantages of screening at early breeding generations as well as simplicity and accuracy . markers based on the polymerase chain reaction ( pcr ) have been tested for quality evaluation of rice varieties ( 12 ) . several functional markers have also been developed to distinguish the physicochemical properties of rice , especially the effect of the waxy locus on pp ( 14 ) , that of sbe on starch viscosity ( 15 ) , and those of ac ( 2 ) and starch synthase iia ( ssiia ) on gt ( 3 ) . despite the recent progress in developing markers and identification of qtls associated with eating quality , a marker - assisted breeding ( mab ) system for better eating quality has not been established . in this study , our aim was to develop dna markers associated with eating quality and to formulate a marker - based evaluation and prediction method of eating quality of cooked rice in japonica varieties . a total of 22 japonica rice varieties , mostly bred in korea , were evaluated for palatability . these consisted of 2 varieties from japan ( koshihikari and hitomebore ) , 1 variety from china ( hexi41 ) , and 19 varieties from korea ( gopum , ilpum , samgwang , chucheong , dongjin , sinkeumo , hwaseong , hwacheong , dobong , samnam , palkong , baekjinju1 , seonong4 , onnuri , manmi , giho , geuman , nakdong , and samdeok ) . these varieties were chosen because they represent diverse palatability scores among japonica rice ( national institute of crop science ( nics ) , personal communication ) . in addition , 32 japonica breeding lines along with three check varieties grown in a regional yield trial plot in 2007 at nics were also used for validation of the marker set developed in this study . twenty previously reported sts primer pairs developed from rapd ( 12 , 13 , 17 ) were tested in this study . to identify the amplicon sequences , the amplified bands were excised , purified , ta cloned , and sequenced . ( 3 ) , was polymorphic among the 22 rice varieties and was actually used in this study . on the basis of selected regions either close to or within the genes linked to interesting qtls ( 11 , 18 , 19 ) for rice eating quality traits ( table 2 ) , analysis of nucleotide polymorphism of the sequences among japonica varieties was performed to develop primers for eating quality . to design the purified pcr products from various japonica varieties were ta - cloned into pgem - t easy vectors and transformed into eschericia colidh5-competent cells prepared according to the protocol of sambrook and russell ( 21 ) . to identify snps , insertions and deletions ( indels ) , and/or microsatellite repeats in the rice varieties , sequence results were aligned using the clustal w program ( 22 ) from embl - european bioinformatics institute ( http://www.ebi.ac.uk/tools ) , with assistance from codoncode aligner 2.0.6 ( codoncode corporation , dedham , ma ) as well as bioedit ( http://www.mbio.ncsu.edu/bioedit/bioedit.html ) . finally , nine molecular markers were successfully developed on the basis of indels , snps , and microsatellite repeats identified in this study ( table 3 ) . http://www.ncbi.nlm.nih.gov/ , http://rgp.dna.affrc.go.jp/e/irgsp/index.html , http://rice.plantbiology.msu.edu/ , and http://www.gramene.org/. in a total volume of 20 l with the following genotyping pcr reagents : 2 l of dna at 20 ng/l , 2 l of 10 buffer containing 25 mm mgcl2 , 1 l of 2.5 mm dntps , 1 unit of taq polymerase ( intron biotechnology , korea ) , and 1 l each of forward and reverse primers ( 10 m ) . the pcr reaction for further sequencing analysis consisted of 1 unit of extaq polymerase ( takara ) in a total reaction of 50 l . all amplifications were performed for a total of 35 cycles of 1 min at 95 c , 30 s at 55 c , and 1 min at 72 c . ( 13 , 17 ) and ohtsubo and nakamura ( 12 ) , the initial denaturation was at 95 c for 5 min , followed by 40 cycles of 96 c for 1 min , 62 c for 1 min , and 72 c for 2 min . ( 2 , 3 ) were performed under the following conditions : 5 min at 94 c followed by 35 cycles of 45 s at 94 c , 1 min at 55 c , and 1 min at 72 c , with a final extension of 7 min at 72 c . for palatability and physicochemical analyses , the rice grains were hulled and milled to 91% yield . pc was calculated using total nitrogen multiplied by 5.95 after determination of the nitrogen content of rice material by the micro - kjeldahl method ( 24 ) . the ac of milled rice was determined by the relative absorbency of starchiodine color in a digested solution of 100-mesh rice flour according to the method of perez and juliano ( 25 ) . rice starch paste profile characteristics were described by six parameters : peak viscosity ( pv ) , hot paste viscosity ( hpv ) , cool paste viscosity ( cpv ) , breakdown viscosity ( bdv = pv hpv ) , setback viscosity ( sbv = cpv pv ) , and consistency viscosity ( ctv = cpv hpv ) in accordance with the procedure of bao and xia ( 26 ) . dry - milled head rice ( 300 g ) was rinsed four times and soaked for 30 min with distilled water , and the water was then strained for 10 min . rice was cooked using an electric rice cooker with the ratio of rice / water = 1:1.25 w / w . after completion of the automatic cooking cycle , samples were transferred to plates and kept at room temperature for about 10 min until cooled to 3537 c . sensory evaluation of cooked rice samples by 11 well - trained panel members was performed with five replications . the overall palatability was assessed according to appearance ( glossiness ) , fragrance , taste , stickiness , texture , and palatability score ( overall score ; overall eating quality ) and scored from + 3 to 3 compared to a cooked rice reference sample , chucheong ( score = 0 ) . the palatability score of each variety by the sensory test was the average value scored by 11 panel members . regression and correlation analyses were also performed to determine the relationships between rice eating quality traits . multiple regression analysis was also conducted to determine the relationship between palatability scored both by taste analyzer and by sensory test and also palatability evaluated by molecular markers . sts marker data were scored as 1 ( present ) and 0 ( absent ) . by using the palatability scores as dependent variables and the binary data from molecular markers as independent variables or regressors , the best model equation to predict rice palatability was obtained . the most accurate prediction gave the lowest standard error and significantly highest coefficient of determination ( r ) , which consisted of the highly significant regression coefficient for each marker in the model . therefore , a total of 21 markers , including ssiia ( 3 ) , were used directly in this study ( table 1 ) as 5 of the markers listed in table 1 from bao et al . on the basis of qtl analyses for rice eating quality ( 11 , 18 , 19 ) , we were able to select seven candidate genes underlying the qtl regions : sucrose synthase 3 ( s3 , clone ap004988 ) , trehalose phosphatase ( tre , clone ap004341 ) , granule bound starch synthase 1 ( gbss1/waxy gene , clone ap002542 ) , udp - n - acetylglucosamine pyrophosphorylase ( acph , clone ap003875 ) , glucosamine - fructose-6-phosphate aminotransferase ( gpa , clone ac138454 ) , aspartate aminotransferase ( am , clone ap003991 ) , and noncyanogenic -glucosidase ( cbg , clone ac074354 ) ( table 2 ) . in addition , by searching the genomics date base ( db ) , adp - glucose pyrophosphorylase ( shrunken gene , sh51 , ap004317 ) , a gene involved in starch biosynthesis and located on chromosome 1 , was also chosen for marker development . by comparing the sequence of each gene ( table 2 ) among japonica varieties , we developed a total of nine dna markers ( table 3 ) . examples of pcr amplicons of different marker types among japonica rice varieties are shown in figure 1 . the insertion of cttt alleles in the tre locus ( figure 1a ) and the g allele in the s3 locus ( figure 1b ) were rarely found in japonica varieties . we genotyped 22 japonica rice varieties with diverse palatability values using a total of 30 markers comprising 21 markers developed previously and 9 markers developed in this study ( table 4 ) . when a cutoff value of 0.64 was used for genetic similarity among all varieties , three clusters were formed , i , ii , and iii ( figure 2 ) , which contained 12 , 7 , and 3 varieties , respectively . dendrogram of 22 japonica varieties constructed on the basis of similarity coefficients in upgma analysis . the palatability value according to the toyo taste meter ( p ) , the palatability score according to the sensory test ( st ) , and the physicochemical properties of 22 japonica rice varieties are summarized in tables 5 and 6 . four varieties ( koshihikari , ilpum , samgwang , and geuman ) showed good palatability in both p and st . p , palatability value ; st , palatability score from sensory test ; ac , amylose content ; pc , protein content ; cpv , cold paste viscosity ; bdv , breakdown viscosity ; pv , peak viscosity ; hpv , hot paste viscosity ; sbv , setback viscosity ; ctv , consistency viscosity ; m , palatability value estimated from the equation based on marker data ; rvu , rapid visco unit . association analyses between marker genotypes based on 30 primer sets ( table 4 ) and palatability values according to the toyo taste meter ( p ) ( table 6 ) and sensory test ( st ) were performed ( table 8) . general linear model regression analysis indicated that individual molecular markers could not differentiate the eating qualities . thus , multiple regression analysis of the entire set of markers was performed . by using only significant markers as independent variables , model equations were generated that could predict the eating quality with highly significant resolution ( r = 0.99 ) ( table 8) . this indicates that palatability values of 22 varieties estimated by the toyo taste meter and/or by the sensory test can be estimated with high resolution by these regression equations based on the marker genotypes . the marker sets for the equations consisted of 13 markers for palatability by the toyo taste meter and 14 markers for palatability by the sensory test , with 9 markers shared for both equations . three newly developed markers ( gpa , s3ci , and cbg ) were included among these 9 markers . the other 3 markers ( ams , ssiia , and acph ) were included among either 13 markers for palatability by the toyo taste meter or 14 markers for palatability by the sensory test . to evaluate the validity of the regression equation , 32 breeding lines along with 3 japonica check varieties were genotyped with the set of 13 markers mentioned above and their palatability values there was a highly significant correlation ( r = 0.715 * * ) between palatability estimates by the regression equation and palatability values according to the toyo taste meter ( figure 3 ) , indicating that the model equation we developed reliably predicts the palatability of random breeding lines of japonica rice . correlation between palatability values of 32 breeding lines and 3 check varieties measured by the toyo taste meter and estimated by the regression equation . the highly significant correlation between palatability values according to the toyo taste meter and palatability values according to the sensory test is in good agreement with previous studies ( 9 ) . japanese researchers have also reported that palatability values from the toyo taste meter and palatability scores from the sensory test exhibit a high positive correlation ( 7 , 8) . this indicates that the palatability value according to the toyo taste meter can be used as a good measure of the eating quality of rice . for breeding better eating quality rice , the toyo taste meter is the measurement method of choice because of its relative simplicity and reproducibility compared to the sensory test ( 79 ) . this is why the toyo taste meter has been widely used in breeding programs for eating quality in korea , japan , and northeastern asia . no correlation between palatability and any pasting property was found in this study , suggesting that pasting properties could not be a good indicator for palatability value due to its complex nature . pc was the only trait to affect the palatability value as reported previously ( 29 ) . when 22 varieties were classified on the basis of the cluster analysis of 30 markers , they formed three major clusters at a genetic similarity level of 0.640.67 . however , the clusters did not appear to be related to eating quality , but to the parental origin of the strains , although the 30 markers were derived from quality - associated traits or qtls for eating quality . this may be explained by the fact that the cluster diagram was constructed on the basis of genetic similarities simply calculated on the basis of the assumption that each of the markers had the same effect on the genotype determination . this demonstrates that simple genotyping and similarity analysis might not be a good measure to evaluate the eating quality of rice , and thus there should be differential weighting of markers . multiple regression equations developed in this study ( table 8) proved to be effective in predicting the palatability of rice varieties and breeding lines ( figure 3 ) . ( 13 , 17 ) and ohtsubo and nakamura ( 12 ) were able to estimate rice palatability ( sensory test of japanese people ) by using a combination of sts markers and estimation equations with significant multiple regression coefficients . similarly , other marker sets derived from starch synthesizing genes ( gbss , ss , sbe , and sdbe ) ( 2 , 3 ) and storage protein genes ( glutelin and prolamin ) were also tested to evaluate ac , pc , and the adhesiveness of cooked rice ( 30 ) . of the significant markers in the equations ( table 8) , nine were important for palatability as measured by both the toyo taste meter ( p ) and the sensory test ( st ) , and the r values of the nine markers explained most of the variation in both equations . this indicates that palatability by the toyo taste meter and palatability by the sensory test are in good accordance as revealed by the significant correlation ( r = 0.85 * * ) ( table 7 ) . of the nine significant markers , the sts marker , p5 , showed the highest partial r in both equations , implying that it might represent a major qtl for palatability . because we used most of the reported markers developed for taste evaluation in japonica rice , the regression equations ( table 8) are of sufficiently high resolution to estimate the palatability of japonica rice in korea . however , because palatability varies among countries and varietal groups even within japonica rice , the equations may need to be modified to be applicable in other countries . particularly in countries we expect that the set of 13 markers used in this study will be useful for the selection of early breeding or even individual f2 plants to accelerate breeding for improvement of rice eating quality in korea or other japonica rice - consuming countries .

the original birth cohort consists of 8,760 people who were born as singletons at the helsinki university central hospital during 1934 and 1944 and who attended child welfare clinics and were residents in finland in 1971 . details of the cohort have been described previously ( 16,17 ) . from the original study cohort , 2,003 men and women were selected at random to attend a clinical examination in 2003 . no significant difference in diabetes - related outcomes between individuals selected and not selected was observed . the examination included the measurement of weight , length , and waist circumference and a standard 2-h 75-g oral glucose tolerance test ( ogtt ) , with plasma glucose and insulin concentrations measured at 0 and 120 min . the study was approved by the local ethical committee , and informed written consent was obtained from the participants . serum total and hdl cholesterol and triglyceride concentrations were measured using standard enzymatic methods and apolipoprotein b using an immunoturbinometric assay . from the ogtt the diagnosis of diabetes was based on an ogtt and the world health organization 1999 criteria for glucose intolerance . the pop analyses were performed in the national institute for health and welfare , chemical exposure unit , which is accredited for measurement of pops in serum samples ( according to the international standard iso / iec 17025 ) . the compounds analyzed were oxychlordane , trans - nonachlor , 1,1-dichloro-2,2-bis-(p - chlorophenyl)-ethylene ( p , p-dde ) , 2,2,4,4,5,5-hexachlorobiphenyl ( pcb 153 ) , 2,2,4,4-tetrabromodiphenyl ether ( bde 47 ) , and 2,2,4,4,5,5-hexabromodiphenyl ether ( bde 153 ) . analytical grade n - hexane , diethyl ether , ethanol , sodium chloride , n - butyl acetate , dichloromethane , silica gel 60 , silver nitrate , and heptanoic acid were used . for each compound , serum samples , 2 ml each ( the internal standard in a toluene solution ) , and 3.0 ml ethanol were pipetted into glass test tubes . the samples were shaken mechanically and sonicated for 5 min to precipitate the proteins and equilibrate the internal standards . to extract the analytes , 1.0 ml solid sodium chloride , followed by 3.0 ml diethyl ether and 3.0 ml hexane , were added . the samples were extracted in a carousel extractor for 15 min at 35 rpm and centrifuged for 5 min at 3,500 rpm . during centrifugation , a solid plug was formed in the interface between the organic and water phase , and the organic extract was poured into another glass tube . the extract was evaporated to dryness in a warm water bath under a gentle stream of nitrogen gas with heptanoic acid as a keeper . a 6-ml solid phase extraction column was packed with a filter paper , 1.0 ml 44% sulfuric acid impregnated silica , 1.0 ml activated silica , 1.0 ml 10% silver nitrate impregnated silica , and a wad of glass wool on top to prevent dusting . the column was precleaned and equilibrated by elution with 3.0 ml 20% dichloromethane : hexane three times , followed by 3.0 ml hexane . the extract reconstituted in hexane was poured into the column , and the analytes were eluted with 1218 ml 20% dichloromethane : hexane . the eluate was evaporated into 0.5 ml , and n - butyl acetate was added as keeper . the determination of the analytes was performed with a hewlett - packard 6890 gas chromatograph with a combi pal autosampler , connected to an autospec ultima high - resolution mass spectrometer ( with a resolution of 8,000 ) . the gas chromatograph was equipped with a split - splitless injector and a db-5ms capillary column ( 30 m , 0.25-mm internal diameter , 0.25-m film ) . the linearity of the gas chromatograph high resolution mass spectrometer analysis was checked with calibration solutions that covered the concentration range expected in real samples . two laboratory reagent and equipment blank samples and two in - house control samples ( human plasma and serum ) were analyzed with each batch of 36 actual samples . the accuracy and precision of the method was verified by analyzing standard reference material 1589a ( srm 1589a ) from the national institute of standards and technology ( gaithersburg , md ) , which has certified concentrations or reference concentrations for all compounds analyzed . furthermore , the laboratory participates regularly in the arctic monitoring and assessment program ( amap ) interlaboratory exercises ( ring test for persistent organic pollutants in human serum , the national institute of public health , quebec , canada ) , offering assigned values for all pops analyzed . the concentration of total cholesterol as millimoles per liter was converted into milligram per milliliter by multiplying by the molecular mass of 386.7 g / mol . the concentration of triglycerides as millimoles per liter was converted into milligram per milliliter by multiplying by the molecular mass of triolein ( 885.5 g / mol ) . the limits of quantification were calculated individually for each analyte in each run of samples and varied between 0.0122.3 , 0.00230.57 , 0.09647 , 0.05024 , 0.03314 , and 0.00407.5 ng / g lipid for oxychlordane , trans - nonachlor , p , p-dde , pcb 153 , bde 47 , and bde 153 , respectively . complete data from the clinical examination and the serum pop analyses were available for 1,988 subjects . pop concentrations below the limits of quantification were treated as one - half of the respective limits of quantification ( middle bound ) . pearson s correlation coefficients were calculated between the six pops using log - transformed data ( nontransformed data were used for other analyses ) . the concentrations of each pop were categorized into four groups , on the basis of percentile intervals < 10th , 10th to < 50th , 50th to < 90th , and 90th . logistic regression was performed to obtain the odds ratio ( or ) for prevalent type 2 diabetes across the categories of each pop , adjusting for sex , age , waist circumference , and mean arterial pressure using the lowest category ( < 10th ) as the reference group . the adjusting variables were selected to obtain the best - fitting model possible using most relevant risk factors for type 2 diabetes . mean arterial pressure was used instead of systolic and diastolic pressure to avoid multicollinearity effects . the analysis was performed also without adjusting for mean arterial pressure , which did not considerably change the results ( data not shown ) . furthermore , we were not able to simultaneously adjust for both waist circumference and bmi because of multicollinearity effects . therefore , logistic regression was performed stratified by bmi , with the bmi categories being < 25 , 25 to < 30 , and 30 kg / m . the original birth cohort consists of 8,760 people who were born as singletons at the helsinki university central hospital during 1934 and 1944 and who attended child welfare clinics and were residents in finland in 1971 . details of the cohort have been described previously ( 16,17 ) . from the original study cohort , 2,003 men and women were selected at random to attend a clinical examination in 2003 . no significant difference in diabetes - related outcomes between individuals selected and not selected was observed . the examination included the measurement of weight , length , and waist circumference and a standard 2-h 75-g oral glucose tolerance test ( ogtt ) , with plasma glucose and insulin concentrations measured at 0 and 120 min . the study was approved by the local ethical committee , and informed written consent was obtained from the participants . serum total and hdl cholesterol and triglyceride concentrations were measured using standard enzymatic methods and apolipoprotein b using an immunoturbinometric assay . from the ogtt , the diagnosis of diabetes was based on an ogtt and the world health organization 1999 criteria for glucose intolerance . the pop analyses were performed in the national institute for health and welfare , chemical exposure unit , which is accredited for measurement of pops in serum samples ( according to the international standard iso / iec 17025 ) . the compounds analyzed were oxychlordane , trans - nonachlor , 1,1-dichloro-2,2-bis-(p - chlorophenyl)-ethylene ( p , p-dde ) , 2,2,4,4,5,5-hexachlorobiphenyl ( pcb 153 ) , 2,2,4,4-tetrabromodiphenyl ether ( bde 47 ) , and 2,2,4,4,5,5-hexabromodiphenyl ether ( bde 153 ) . analytical grade n - hexane , diethyl ether , ethanol , sodium chloride , n - butyl acetate , dichloromethane , silica gel 60 , silver nitrate , and heptanoic acid were used . for each compound , corresponding c - labeled compounds were used as the internal standard . a toluene solution of c serum samples , 2 ml each ( the internal standard in a toluene solution ) , and 3.0 ml ethanol were pipetted into glass test tubes . the samples were shaken mechanically and sonicated for 5 min to precipitate the proteins and equilibrate the internal standards . to extract the analytes , 1.0 ml solid sodium chloride , followed by 3.0 ml diethyl ether and 3.0 ml hexane , were added . the samples were extracted in a carousel extractor for 15 min at 35 rpm and centrifuged for 5 min at 3,500 rpm . during centrifugation , a solid plug was formed in the interface between the organic and water phase , and the organic extract was poured into another glass tube . the extract was evaporated to dryness in a warm water bath under a gentle stream of nitrogen gas with heptanoic acid as a keeper . a 6-ml solid phase extraction column was packed with a filter paper , 1.0 ml 44% sulfuric acid impregnated silica , 1.0 ml activated silica , 1.0 ml 10% silver nitrate impregnated silica , and a wad of glass wool on top to prevent dusting . the column was precleaned and equilibrated by elution with 3.0 ml 20% dichloromethane : hexane three times , followed by 3.0 ml hexane . the extract reconstituted in hexane was poured into the column , and the analytes were eluted with 1218 ml 20% dichloromethane : hexane . the eluate was evaporated into 0.5 ml , and n - butyl acetate was added as keeper . the determination of the analytes was performed with a hewlett - packard 6890 gas chromatograph with a combi pal autosampler , connected to an autospec ultima high - resolution mass spectrometer ( with a resolution of 8,000 ) . the gas chromatograph was equipped with a split - splitless injector and a db-5ms capillary column ( 30 m , 0.25-mm internal diameter , 0.25-m film ) . the linearity of the gas chromatograph high resolution mass spectrometer analysis was checked with calibration solutions that covered the concentration range expected in real samples . two laboratory reagent and equipment blank samples and two in - house control samples ( human plasma and serum ) were analyzed with each batch of 36 actual samples . the accuracy and precision of the method was verified by analyzing standard reference material 1589a ( srm 1589a ) from the national institute of standards and technology ( gaithersburg , md ) , which has certified concentrations or reference concentrations for all compounds analyzed . furthermore , the laboratory participates regularly in the arctic monitoring and assessment program ( amap ) interlaboratory exercises ( ring test for persistent organic pollutants in human serum , the national institute of public health , quebec , canada ) , offering assigned values for all pops analyzed . the concentration of total cholesterol as millimoles per liter was converted into milligram per milliliter by multiplying by the molecular mass of 386.7 g / mol . the concentration of triglycerides as millimoles per liter was converted into milligram per milliliter by multiplying by the molecular mass of triolein ( 885.5 g / mol ) . the limits of quantification were calculated individually for each analyte in each run of samples and varied between 0.0122.3 , 0.00230.57 , 0.09647 , 0.05024 , 0.03314 , and 0.00407.5 ng / g lipid for oxychlordane , trans - nonachlor , p , p-dde , pcb 153 , bde 47 , and bde 153 , respectively . complete data from the clinical examination and the serum pop analyses were available for 1,988 subjects . pop concentrations below the limits of quantification were treated as one - half of the respective limits of quantification ( middle bound ) . pearson s correlation coefficients were calculated between the six pops using log - transformed data ( nontransformed data were used for other analyses ) . the concentrations of each pop were categorized into four groups , on the basis of percentile intervals < 10th , 10th to < 50th , 50th to < 90th , and 90th . logistic regression was performed to obtain the odds ratio ( or ) for prevalent type 2 diabetes across the categories of each pop , adjusting for sex , age , waist circumference , and mean arterial pressure using the lowest category ( < 10th ) as the reference group . the adjusting variables were selected to obtain the best - fitting model possible using most relevant risk factors for type 2 diabetes . mean arterial pressure was used instead of systolic and diastolic pressure to avoid multicollinearity effects . the analysis was performed also without adjusting for mean arterial pressure , which did not considerably change the results ( data not shown ) . furthermore , we were not able to simultaneously adjust for both waist circumference and bmi because of multicollinearity effects . therefore , logistic regression was performed stratified by bmi , with the bmi categories being < 25 , 25 to < 30 , and 30 kg / m . descriptive statistics of the study population are provided in table 1 . in total , 308 participants were diagnosed with type 2 diabetes , and the total prevalence in the study population was 15.5% . the prevalence of type 2 diabetes was higher among men than among women ( 19.4 and 12.1% , respectively ) . descriptive statistics of the study population data are means se ( median , range ) , unless otherwise indicated . * unpaired t test ( two - tailed ) for age , bmi , waist circumference , and serum lipids ( normally distributed ) ; mann - whitney u test ( two - tailed ) for serum pops ( not normally distributed ) . the age distribution of the population was narrow ( average age 62 years , ranging between 57 and 70 years ) . the average bmi and waist circumference for men and women were 27 and 28 kg / m and 100 and 91 cm , respectively , and both were higher among the participants diagnosed with type 2 diabetes than among individuals not diagnosed ( p < 0.001 ) . the average concentration of serum lipids was 360 mg / dl , of which the majority was accounted for by cholesterol ( 64% ) . men had a slightly lower concentration of serum cholesterol than women ( 220 and 240 mg / dl , p < 0.001 ) . among the participants with diabetes , the total concentration of serum lipids was higher than among the participants without diabetes ( 400 and 360 mg / dl , p < 0.001 ) . the median concentrations of oxychlordane , trans - nonachlor , p , p-dde , pcb 153 , bde 47 , and bde 153 were 11 , 28 , 470 , 290 , 2.9 , and 1.6 ng / g lipid , respectively . the concentrations of oxychlordane , trans - nonachlor , and pcb 153 were higher among men than among women ( p < 0.001 ) . on the contrary , the concentrations of the bdes were lower among men than women ( p < 0.001 ) . among the participants with diabetes , the concentrations of trans - nonachlor and p , p-dde were higher ( p = 0.067 and p < 0.001 , respectively ) than among individuals without diabetes , but the concentration of bde 153 was lower ( p = 0.005 ) . significant associations were observed between all of the analytes , when log - transformed data were studied for bivariate correlation . the associations between oxychlordane , trans - nonachlor , p , p-dde , and pcb 153 ( pearson s correlation coefficient r = 0.490.93 , p < 0.001 ) were strong , but the association of these four compounds with the bdes was weaker ( r = 0.170.37 , p < 0.001 ) . however , the bdes were strongly associated with each other ( r = 0.51 , p < 0.001 ) . the associations among the analytes and age , bmi , and waist circumference were weak ( r = 0.0530.145 , 0.200.17 , and 0.140.13 , respectively , p was 0.001 in most cases ) . there was strong correlation between bmi and waist circumference ( r = 0.84 , p < 0.001 ) . in the logistic regression analyses , oxychlordane ( p for linear trend across categories = 0.003 ) , trans - nonachlor ( plin = 0.003 , where plin is the p value for linear trend across pop categories ) , p , p-dde ( plin = 0.020 ) , and pcb 153 ( plin = 0.050 ) had a statistically significant positive association with prevalent type 2 diabetes ( table 2 ) . the individual ors in the highest exposure categories were 2.08 ( 95% ci 1.183.69 , p = 0.012 ) , 2.24 ( 1.254.03 , p = 0.007 ) , 1.75 ( 0.963.19 , p = 0.069 ) , and 1.64 ( 0.922.93 , p = 0.097 ) , respectively , compared with the group with the lowest exposure category . for the bdes , no association was observed ( plin = 0.57 and 0.20 , respectively ) . serum concentration of pops and adjusted ors for type 2 diabetes across categories of each pop data are or ( 95% ci ) , unless otherwise indicated . ors are adjusted for sex , age , waist circumference , and mean arterial pressure . in the stratified adjusted logistic regression , none of the pops showed association with type 2 diabetes among the group with bmi < 25 kg / m ( normal weight ) . however , oxychlordane and trans - nonachlor were significantly associated with type 2 diabetes among the groups with bmi between 25 and 30 kg / m ( overweight , plin = 0.011 and = 0.030 , respectively ) and those with bmi higher than 30 kg / m ( obese , plin = 0.020 and = 0.034 ) . for p , p-dde and pcb 153 , borderline significant associations were observed , only among those with bmi > 30 kg / m ( plin = 0.087 and = 0.062 , respectively ) . for all of these four pops , the associations were strongest among the group with bmi between 25 and 30 kg / m . in the current study , high exposure to ocps and pcb 153 was associated with an approximately double risk of prevalent type 2 diabetes . among the participants with the highest levels of the organochlorine pesticide metabolites oxychlordane , trans - nonachlor , and p , p-dde , as well as pcb 153 , the prevalence of type 2 diabetes was 1.642.24 times higher than among participants with the lowest exposure . furthermore , the prevalence of type 2 diabetes increased in an exposure - response manner across the increasing categories of these pops . in contrast , exposure to pbdes did not seem to be associated with type 2 diabetes . it is generally assumed a single measurement of pops in the body reflects lifetime exposure , although there is variation in the elimination rate between individuals , caused by , for example , age , lactation , and changes in body weight . these results are in accordance with previous findings among populations with relatively low exposure ( 38 ) . in particular , among nhanes , lee et al . ( 5 ) found that type 2 diabetes had a strong association with concentrations of oxychlordane , trans - nonachlor , p , p-dde , and pcb 153 , with the individual ors among the highest exposure category being 6.5 , 11.8 , 4.3 , and 6.8 , respectively . ( 7 ) reported much weaker associations between several pbde congeners and prevalent type 2 diabetes among nhanes : among the highest exposure group , ors ranged between 0.8 and 1.7 and were statistically nonsignificant . ( 8) , who reported increased risk of type 2 diabetes among great lakes sport fish consumers highly exposed to p , p-dde , but not among individuals highly exposed to pbdes . in the stratified analysis , we observed that among the normal - weight participants ( bmi < 25 kg / m ) , even high pop exposure did not increase the risk of type 2 diabetes . additionally , we observed that , among individuals with low pop exposure , overweight and obesity did not seem to increase the prevalence of diabetes as much as among individuals with high pop exposure . this is in line with nhanes ( 5 ) , where a significant association was observed between type 2 diabetes and a sum of six pops among normal - weight participants . together , these studies suggest that overweight and exposure to pops may have a synergistic effect on the risk of type 2 diabetes . additionally , we observed that exposure to the pbdes was weakly related to the exposure to the other pops , suggesting different or additional routes of exposure . it is generally accepted that diet ( animal foods , in particular ) is the most important source of pcdd / fs and pcbs for humans . for pbdes , other sources such as indoor air or dust have been identified , although their contribution to total pbde intake appears to be higher in north america than in europe ( 18 ) . in finland , the most important dietary source of pcdd / fs and pcbs is fish , contributing 95 and 80% of total dietary intake , respectively , and for pbdes , the contribution of fish is much lower ( 55% ) and other sources are more diverse ( 12 ) . however , the different dietary intake patterns between pcdd / fs , pcbs , and pbdes do not explain the weak association observed between the pbdes and the other pops . our findings suggest that , in finland , exposure to pbdes through inhalation of indoor air or inhalation or ingestion of dust may be more significant than previously believed . the associations between pops and type 2 diabetes observed in the current study were not as striking as those observed in nhanes ( 5 ) . in their study ( 5 ) , the ors for type 2 diabetes in the highest exposure categories of oxychlordane , trans - nonachlor , p , p-dde , and pcb 153 were 6.5 , 11.8 , 4.3 , and 6.8 , respectively , whereas the respective ors in our study were 2.08 , 2.24 , 1.75 , and 1.64 . however , in nhanes , participants represented all age - groups 20 years of age , and the age of the participants in our study was 5770 years . among the subgroup of participants 60 years of age , lee et al . ( 5 ) observed that the risk of type 2 diabetes associated with high exposure to the sum of pops ( calculated by adding ranks of the individual pops ) was lower than among younger age - groups , which is more consistent with our findings . one important factor that may explain why the association of pops and type 2 diabetes was lower among our elderly study group , and among the nhanes subgroup aged 60 years , is the lack of exposure during the developmental period . participants in our study population , who were born in the 1930s and 1940s , probably experienced little exposure in utero and during childhood , which is often thought to be critical when considering the health effects of pops . in fact , most of the study population s exposure probably occurred during their adult life . even so , a statistically significant association between pop exposure and type 2 diabetes was observed here . this , together with an increasing number of studies , suggesting that a causal relationship exists ( 18 ) , might encourage public health professionals concerned with the diabetes epidemic : limiting adult exposure might help to decrease the risk of developing this disorder . another significant factor explaining why the associations observed in the current study were not as strong as in nhanes is the clear difference in the level of exposure . among nhanes , the same pop compounds as in the current study were reported , and with the exception ofpcb 153 , their concentrations were systematically higher . in the highest exposure categories ( 90th ) , in particular , the concentrations of oxychlordane , trans - nonachlor , and p , p-dde were 75190% higher in nhanes than in the current study , whereas the concentration of pcb 153 was 72% lower ( 5 ) . the difference in the concentrations of the pbdes was even more pronounced , with the concentrations of the congeners 47 and 153 in the highest exposure category ( 75th ) being 590650% higher than in the current study ( 7 ) . this difference is striking , especially when it is considered that , in nhanes , participants from all age - groups are represented , whereas in the current study , participants are all aged 5770 years , with longer overall exposure history . no studies previously reported serum concentrations of pops among the general finnish population , but pcb 153 concentrations of 116 ng / g ( range 16.8958 ) of lipid were observed in adipose tissue of a general southern finnish population aged 1381 years , which is slightly lower than that in the current study ( 19 ) . because of the cross - sectional study design , we were not able to assess the causal relationship between pops and type 2 diabetes . within human populations , studies addressing this question are scarce . in a follow - up study by rignell - hydbom et al . ( 20 ) within a general female population , the risk of type 2 diabetes among individuals with high baseline exposure to p , p-dde compared with individuals with low exposure was more pronounced when the lag time between baseline and diagnosis was longer , suggesting that high exposure may predispose individuals to developing type 2 diabetes later in life . in a case - control study nested in a prospective study , lee et al . ( 21 ) observed indications of nonmonotone associations between pops and type 2 diabetes among a general population , although the cases in this study had elevated baseline concentrations of triglycerides , hdl cholesterol , and fasting glucose . recently , strong evidence of causality was obtained in in vitro and in vivo studies . ( 11 ) showed that low - level chronic pop exposure induced significant impairment of whole - body insulin action in rats . in addition , they showed that , in differentiated adipocytes , pop exposure induced a significant inhibition of insulin - dependent glucose uptake . it is interesting that there were no threshold doses in the inhibition of glucose uptake , suggesting that the risk assessments on the basis of tolerable intakes may not be applicable to insulin resistance . the biological mechanisms by which pops are thought to promote diabetogenesis remain obscure . in general , pops are thought to exert their toxic effects through direct binding and activation of the aryl hydrocarbon receptor ( ahr ) pathway ( 22 ) . based on this assumption , a toxic equivalency concept was developed to assess the risks associated with exposure to dioxins and dioxin - like compounds . however , the diabetogenic effects of pops might also be mediated through ahr - independent oxidative stress and mitochondrial dysfunction , which is known to play an important role in the etiology of diabetes ( 23 ) . ( 24 ) demonstrated that an intracellular ca increase and insulin release from rinm5f cells is stimulated by pcb 47 and pcb 153 , which have low affinity for the ahr but that such an effect is not produced by the coplanar pcb 77 , a congener with moderate affinity for the ahr . in addition , biswas et al . ( 25 ) observed that 2,3,7,8-tetrachlorodibenzo - p - dioxin , a high - affinity ahr ligand , induces mitochondrial dysfunction in mouse skeletal muscle c2c12 myoblasts , independent of ahr activation . we were unable to include some important risk factors for type 2 diabetes in this study , e.g. , physical inactivity , dietary composition , and genetic susceptibility . therefore , we were unable to assess the role of these factors as possible confounders . in conclusion , the current study confirms the association between adult exposure to ocps and type 2 diabetes in a general finnish population . in addition , our results suggest that regarding exposure to pbdes , a significant contribution from nondietary sources is probable . furthermore , this is the first study to report serum concentrations of the selected pops in a population representing the general urban finnish population .

objectivethe prevalence of type 2 diabetes is increasing alarmingly in both developed and developing countries . recently , exposure to persistent organic pollutants ( pops ) has been associated with the prevalence of type 2 diabetes . the purpose of this cross - sectional study is to examine the association between type 2 diabetes and pop exposure in the helsinki birth cohort study.research design and methodsthe cohort consists of 8,760 people born in helsinki during 19341944 , before the global pop emission peak . in 2003 , a clinical examination was performed , including blood sampling for laboratory analyses of serum lipids and pops . complete data from the examination were available for 1,988 participants . the concentrations of each pop were categorized into four groups on the basis of percentile intervals , and logistic regression was performed to examine diabetes prevalence across the pop categories , adjusting for sex , age , waist circumference , and mean arterial pressure and using the lowest category as the reference group.resultsamong the participants with the highest exposure to oxychlordane , trans - nonachlor , 1,1-dichloro-2,2-bis-(p - chlorophenyl)-ethylene ( p , p-dde , and polychlorinated biphenyl 153 , the risk of type 2 diabetes was 1.642.24 times higher than that among individuals with the lowest exposure ( plin = 0.0030.050 , where plin is the p value for linear trend across pop categories ) . in the stratified analysis , the associations between type 2 diabetes and oxychlordane and trans - nonachlor remained significant and were strongest among the overweight participants . exposure to 2,2,4,4-tetrabromodiphenyl ether ( bde 47 ) and 2,2,4,4,5,5-hexabromodiphenyl ether ( bde 153 ) was not associated with type 2 diabetes.conclusionsthis study confirms the association between type 2 diabetes and adult - only exposure to organochlorine pesticides in a general urban population .

RESEARCH DESIGN AND METHODS Study population and data collection Laboratory analyses of diabetes-related markers Laboratory analyses of POPs Statistical analysis RESULTS CONCLUSIONS

the original birth cohort consists of 8,760 people who were born as singletons at the helsinki university central hospital during 1934 and 1944 and who attended child welfare clinics and were residents in finland in 1971 . from the original study cohort , 2,003 men and women were selected at random to attend a clinical examination in 2003 . the examination included the measurement of weight , length , and waist circumference and a standard 2-h 75-g oral glucose tolerance test ( ogtt ) , with plasma glucose and insulin concentrations measured at 0 and 120 min . the study was approved by the local ethical committee , and informed written consent was obtained from the participants . from the ogtt the diagnosis of diabetes was based on an ogtt and the world health organization 1999 criteria for glucose intolerance . the pop analyses were performed in the national institute for health and welfare , chemical exposure unit , which is accredited for measurement of pops in serum samples ( according to the international standard iso / iec 17025 ) . the compounds analyzed were oxychlordane , trans - nonachlor , 1,1-dichloro-2,2-bis-(p - chlorophenyl)-ethylene ( p , p-dde ) , 2,2,4,4,5,5-hexachlorobiphenyl ( pcb 153 ) , 2,2,4,4-tetrabromodiphenyl ether ( bde 47 ) , and 2,2,4,4,5,5-hexabromodiphenyl ether ( bde 153 ) . for each compound , serum samples , 2 ml each ( the internal standard in a toluene solution ) , and 3.0 ml ethanol were pipetted into glass test tubes . during centrifugation , a solid plug was formed in the interface between the organic and water phase , and the organic extract was poured into another glass tube . furthermore , the laboratory participates regularly in the arctic monitoring and assessment program ( amap ) interlaboratory exercises ( ring test for persistent organic pollutants in human serum , the national institute of public health , quebec , canada ) , offering assigned values for all pops analyzed . the limits of quantification were calculated individually for each analyte in each run of samples and varied between 0.0122.3 , 0.00230.57 , 0.09647 , 0.05024 , 0.03314 , and 0.00407.5 ng / g lipid for oxychlordane , trans - nonachlor , p , p-dde , pcb 153 , bde 47 , and bde 153 , respectively . complete data from the clinical examination and the serum pop analyses were available for 1,988 subjects . the concentrations of each pop were categorized into four groups , on the basis of percentile intervals < 10th , 10th to < 50th , 50th to < 90th , and 90th . logistic regression was performed to obtain the odds ratio ( or ) for prevalent type 2 diabetes across the categories of each pop , adjusting for sex , age , waist circumference , and mean arterial pressure using the lowest category ( < 10th ) as the reference group . mean arterial pressure was used instead of systolic and diastolic pressure to avoid multicollinearity effects . the analysis was performed also without adjusting for mean arterial pressure , which did not considerably change the results ( data not shown ) . therefore , logistic regression was performed stratified by bmi , with the bmi categories being < 25 , 25 to < 30 , and 30 kg / m . the original birth cohort consists of 8,760 people who were born as singletons at the helsinki university central hospital during 1934 and 1944 and who attended child welfare clinics and were residents in finland in 1971 . from the original study cohort , 2,003 men and women were selected at random to attend a clinical examination in 2003 . the examination included the measurement of weight , length , and waist circumference and a standard 2-h 75-g oral glucose tolerance test ( ogtt ) , with plasma glucose and insulin concentrations measured at 0 and 120 min . the study was approved by the local ethical committee , and informed written consent was obtained from the participants . from the ogtt , the diagnosis of diabetes was based on an ogtt and the world health organization 1999 criteria for glucose intolerance . the pop analyses were performed in the national institute for health and welfare , chemical exposure unit , which is accredited for measurement of pops in serum samples ( according to the international standard iso / iec 17025 ) . the compounds analyzed were oxychlordane , trans - nonachlor , 1,1-dichloro-2,2-bis-(p - chlorophenyl)-ethylene ( p , p-dde ) , 2,2,4,4,5,5-hexachlorobiphenyl ( pcb 153 ) , 2,2,4,4-tetrabromodiphenyl ether ( bde 47 ) , and 2,2,4,4,5,5-hexabromodiphenyl ether ( bde 153 ) . during centrifugation , a solid plug was formed in the interface between the organic and water phase , and the organic extract was poured into another glass tube . furthermore , the laboratory participates regularly in the arctic monitoring and assessment program ( amap ) interlaboratory exercises ( ring test for persistent organic pollutants in human serum , the national institute of public health , quebec , canada ) , offering assigned values for all pops analyzed . the limits of quantification were calculated individually for each analyte in each run of samples and varied between 0.0122.3 , 0.00230.57 , 0.09647 , 0.05024 , 0.03314 , and 0.00407.5 ng / g lipid for oxychlordane , trans - nonachlor , p , p-dde , pcb 153 , bde 47 , and bde 153 , respectively . complete data from the clinical examination and the serum pop analyses were available for 1,988 subjects . the concentrations of each pop were categorized into four groups , on the basis of percentile intervals < 10th , 10th to < 50th , 50th to < 90th , and 90th . logistic regression was performed to obtain the odds ratio ( or ) for prevalent type 2 diabetes across the categories of each pop , adjusting for sex , age , waist circumference , and mean arterial pressure using the lowest category ( < 10th ) as the reference group . the adjusting variables were selected to obtain the best - fitting model possible using most relevant risk factors for type 2 diabetes . the analysis was performed also without adjusting for mean arterial pressure , which did not considerably change the results ( data not shown ) . therefore , logistic regression was performed stratified by bmi , with the bmi categories being < 25 , 25 to < 30 , and 30 kg / m . in total , 308 participants were diagnosed with type 2 diabetes , and the total prevalence in the study population was 15.5% . the prevalence of type 2 diabetes was higher among men than among women ( 19.4 and 12.1% , respectively ) . * unpaired t test ( two - tailed ) for age , bmi , waist circumference , and serum lipids ( normally distributed ) ; mann - whitney u test ( two - tailed ) for serum pops ( not normally distributed ) . the average bmi and waist circumference for men and women were 27 and 28 kg / m and 100 and 91 cm , respectively , and both were higher among the participants diagnosed with type 2 diabetes than among individuals not diagnosed ( p < 0.001 ) . the average concentration of serum lipids was 360 mg / dl , of which the majority was accounted for by cholesterol ( 64% ) . among the participants with diabetes , the total concentration of serum lipids was higher than among the participants without diabetes ( 400 and 360 mg / dl , p < 0.001 ) . the median concentrations of oxychlordane , trans - nonachlor , p , p-dde , pcb 153 , bde 47 , and bde 153 were 11 , 28 , 470 , 290 , 2.9 , and 1.6 ng / g lipid , respectively . the concentrations of oxychlordane , trans - nonachlor , and pcb 153 were higher among men than among women ( p < 0.001 ) . on the contrary , the concentrations of the bdes were lower among men than women ( p < 0.001 ) . among the participants with diabetes , the concentrations of trans - nonachlor and p , p-dde were higher ( p = 0.067 and p < 0.001 , respectively ) than among individuals without diabetes , but the concentration of bde 153 was lower ( p = 0.005 ) . the associations between oxychlordane , trans - nonachlor , p , p-dde , and pcb 153 ( pearson s correlation coefficient r = 0.490.93 , p < 0.001 ) were strong , but the association of these four compounds with the bdes was weaker ( r = 0.170.37 , p < 0.001 ) . however , the bdes were strongly associated with each other ( r = 0.51 , p < 0.001 ) . the associations among the analytes and age , bmi , and waist circumference were weak ( r = 0.0530.145 , 0.200.17 , and 0.140.13 , respectively , p was 0.001 in most cases ) . there was strong correlation between bmi and waist circumference ( r = 0.84 , p < 0.001 ) . in the logistic regression analyses , oxychlordane ( p for linear trend across categories = 0.003 ) , trans - nonachlor ( plin = 0.003 , where plin is the p value for linear trend across pop categories ) , p , p-dde ( plin = 0.020 ) , and pcb 153 ( plin = 0.050 ) had a statistically significant positive association with prevalent type 2 diabetes ( table 2 ) . the individual ors in the highest exposure categories were 2.08 ( 95% ci 1.183.69 , p = 0.012 ) , 2.24 ( 1.254.03 , p = 0.007 ) , 1.75 ( 0.963.19 , p = 0.069 ) , and 1.64 ( 0.922.93 , p = 0.097 ) , respectively , compared with the group with the lowest exposure category . for the bdes , no association was observed ( plin = 0.57 and 0.20 , respectively ) . serum concentration of pops and adjusted ors for type 2 diabetes across categories of each pop data are or ( 95% ci ) , unless otherwise indicated . ors are adjusted for sex , age , waist circumference , and mean arterial pressure . in the stratified adjusted logistic regression , none of the pops showed association with type 2 diabetes among the group with bmi < 25 kg / m ( normal weight ) . however , oxychlordane and trans - nonachlor were significantly associated with type 2 diabetes among the groups with bmi between 25 and 30 kg / m ( overweight , plin = 0.011 and = 0.030 , respectively ) and those with bmi higher than 30 kg / m ( obese , plin = 0.020 and = 0.034 ) . for p , p-dde and pcb 153 , borderline significant associations were observed , only among those with bmi > 30 kg / m ( plin = 0.087 and = 0.062 , respectively ) . for all of these four pops , the associations were strongest among the group with bmi between 25 and 30 kg / m . in the current study , high exposure to ocps and pcb 153 was associated with an approximately double risk of prevalent type 2 diabetes . among the participants with the highest levels of the organochlorine pesticide metabolites oxychlordane , trans - nonachlor , and p , p-dde , as well as pcb 153 , the prevalence of type 2 diabetes was 1.642.24 times higher than among participants with the lowest exposure . furthermore , the prevalence of type 2 diabetes increased in an exposure - response manner across the increasing categories of these pops . in contrast , exposure to pbdes did not seem to be associated with type 2 diabetes . it is generally assumed a single measurement of pops in the body reflects lifetime exposure , although there is variation in the elimination rate between individuals , caused by , for example , age , lactation , and changes in body weight . ( 5 ) found that type 2 diabetes had a strong association with concentrations of oxychlordane , trans - nonachlor , p , p-dde , and pcb 153 , with the individual ors among the highest exposure category being 6.5 , 11.8 , 4.3 , and 6.8 , respectively . ( 7 ) reported much weaker associations between several pbde congeners and prevalent type 2 diabetes among nhanes : among the highest exposure group , ors ranged between 0.8 and 1.7 and were statistically nonsignificant . ( 8) , who reported increased risk of type 2 diabetes among great lakes sport fish consumers highly exposed to p , p-dde , but not among individuals highly exposed to pbdes . in the stratified analysis , we observed that among the normal - weight participants ( bmi < 25 kg / m ) , even high pop exposure did not increase the risk of type 2 diabetes . additionally , we observed that , among individuals with low pop exposure , overweight and obesity did not seem to increase the prevalence of diabetes as much as among individuals with high pop exposure . this is in line with nhanes ( 5 ) , where a significant association was observed between type 2 diabetes and a sum of six pops among normal - weight participants . together , these studies suggest that overweight and exposure to pops may have a synergistic effect on the risk of type 2 diabetes . in finland , the most important dietary source of pcdd / fs and pcbs is fish , contributing 95 and 80% of total dietary intake , respectively , and for pbdes , the contribution of fish is much lower ( 55% ) and other sources are more diverse ( 12 ) . however , the different dietary intake patterns between pcdd / fs , pcbs , and pbdes do not explain the weak association observed between the pbdes and the other pops . our findings suggest that , in finland , exposure to pbdes through inhalation of indoor air or inhalation or ingestion of dust may be more significant than previously believed . the associations between pops and type 2 diabetes observed in the current study were not as striking as those observed in nhanes ( 5 ) . in their study ( 5 ) , the ors for type 2 diabetes in the highest exposure categories of oxychlordane , trans - nonachlor , p , p-dde , and pcb 153 were 6.5 , 11.8 , 4.3 , and 6.8 , respectively , whereas the respective ors in our study were 2.08 , 2.24 , 1.75 , and 1.64 . however , in nhanes , participants represented all age - groups 20 years of age , and the age of the participants in our study was 5770 years . ( 5 ) observed that the risk of type 2 diabetes associated with high exposure to the sum of pops ( calculated by adding ranks of the individual pops ) was lower than among younger age - groups , which is more consistent with our findings . one important factor that may explain why the association of pops and type 2 diabetes was lower among our elderly study group , and among the nhanes subgroup aged 60 years , is the lack of exposure during the developmental period . participants in our study population , who were born in the 1930s and 1940s , probably experienced little exposure in utero and during childhood , which is often thought to be critical when considering the health effects of pops . even so , a statistically significant association between pop exposure and type 2 diabetes was observed here . this , together with an increasing number of studies , suggesting that a causal relationship exists ( 18 ) , might encourage public health professionals concerned with the diabetes epidemic : limiting adult exposure might help to decrease the risk of developing this disorder . another significant factor explaining why the associations observed in the current study were not as strong as in nhanes is the clear difference in the level of exposure . among nhanes , the same pop compounds as in the current study were reported , and with the exception ofpcb 153 , their concentrations were systematically higher . in the highest exposure categories ( 90th ) , in particular , the concentrations of oxychlordane , trans - nonachlor , and p , p-dde were 75190% higher in nhanes than in the current study , whereas the concentration of pcb 153 was 72% lower ( 5 ) . the difference in the concentrations of the pbdes was even more pronounced , with the concentrations of the congeners 47 and 153 in the highest exposure category ( 75th ) being 590650% higher than in the current study ( 7 ) . no studies previously reported serum concentrations of pops among the general finnish population , but pcb 153 concentrations of 116 ng / g ( range 16.8958 ) of lipid were observed in adipose tissue of a general southern finnish population aged 1381 years , which is slightly lower than that in the current study ( 19 ) . because of the cross - sectional study design , we were not able to assess the causal relationship between pops and type 2 diabetes . ( 20 ) within a general female population , the risk of type 2 diabetes among individuals with high baseline exposure to p , p-dde compared with individuals with low exposure was more pronounced when the lag time between baseline and diagnosis was longer , suggesting that high exposure may predispose individuals to developing type 2 diabetes later in life . ( 21 ) observed indications of nonmonotone associations between pops and type 2 diabetes among a general population , although the cases in this study had elevated baseline concentrations of triglycerides , hdl cholesterol , and fasting glucose . it is interesting that there were no threshold doses in the inhibition of glucose uptake , suggesting that the risk assessments on the basis of tolerable intakes may not be applicable to insulin resistance . based on this assumption , a toxic equivalency concept was developed to assess the risks associated with exposure to dioxins and dioxin - like compounds . however , the diabetogenic effects of pops might also be mediated through ahr - independent oxidative stress and mitochondrial dysfunction , which is known to play an important role in the etiology of diabetes ( 23 ) . ( 24 ) demonstrated that an intracellular ca increase and insulin release from rinm5f cells is stimulated by pcb 47 and pcb 153 , which have low affinity for the ahr but that such an effect is not produced by the coplanar pcb 77 , a congener with moderate affinity for the ahr . we were unable to include some important risk factors for type 2 diabetes in this study , e.g. in conclusion , the current study confirms the association between adult exposure to ocps and type 2 diabetes in a general finnish population . in addition , our results suggest that regarding exposure to pbdes , a significant contribution from nondietary sources is probable . furthermore , this is the first study to report serum concentrations of the selected pops in a population representing the general urban finnish population .

there has been a progressive increase in the incidence of type 1 dm , and several advances in its treatment have been achieved . as a result , an increasing number of patients , who are older and have longer disease durations , are more severely affected by chronic complications [ 13 ] . chronic subclinical inflammation , impaired fibrinolytic system activity , and elevated procoagulant factor levels form the basis of atherosclerotic diseases . consequently , dm has been regarded as a major risk factor for cardiovascular diseases [ 4 , 5 ] . insulin resistance is characterized by limited stimulation of glucose metabolism in muscle and the liver and has been described in patients with poorly controlled type 1 dm [ 68 ] . cytokines such as leptin , resistin , and adiponectin released from these tissues critically impact nutritional status , body fat distribution , metabolic parameters , inflammatory status , atherosclerotic alterations , and insulin resistance . the levels of adiponectin , which is regarded as an antidiabetic , anti - inflammatory , and antiatherogenic cytokine , are reported to be depressed in patients with type 2 dm [ 10 , 11 ] . however , in some studies , increased adiponectin levels have been reported in patients with type 1 dm . resistin , on the other hand , impairs cellular glucose intake , because it is stimulated by insulin ; as a result , hepatic glucose production is increased , leading to impaired glucose tolerance and eventual development of insulin resistance . owing to its augmenting effect on the production of adhesion molecules , resistin is considered to be having proinflammatory effects in the vascular endothelium [ 13 , 14 ] . leptin has been shown to have important effects on both body energy balance and fat distribution [ 15 , 16 ] . glucose uptake rates by peripheral tissues , which can be stimulated by insulin in skeletal muscles , decrease over time in patients with type 1 dm and poor glycemic control . these patients have significant hepatic insulin resistance , and the effects of insulin are impaired because of plasma free fatty acids ( ffas ) [ 1719 ] . despite recent advances in the management of dm , it has been suggested that cardiovascular disease - related mortality rates increase as more intensive therapies are required to ensure tight blood glucose control . therefore , combination therapies are required to ensure tight glycemic control , minimize the risk of macrovascular disease , and reduce other cardiovascular risk factors . thiazolidinediones ( tzds ) act by binding to nuclear peroxisome proliferator activated receptor - gamma ( ppar- ) , which is chiefly expressed in fatty tissue , and mediate their effects by activating the transcription of the genes that influence adipocyte differentiation as well as glucose and lipid metabolism [ 2123 ] . tzds , apart from their direct effect on fatty tissue , might influence the release of adipocyte - derived signal factors that determine the insulin sensitivity of muscles , such as ffas , adiponectin , leptin , and tumor necrosis factor - alpha ( tnf- ) . in addition to their favorable effects on glycemic control , tzds directly influence vessel walls , decrease vasoconstriction , and inhibit inflammation . therefore , they inhibit insulin resistance and slow down the atherosclerotic process [ 2427 ] . in addition , it has been shown that tzds could inhibit hyperglycemia - induced reactive oxygen species production from mitochondria ( mtros ) by activating the ppar- coactivator-1 alpha ( pgc-1 ) pathway which could contribute to the prevention of diabetic vascular complications [ 28 , 29 ] . agonists of ppar - gamma and ppar - alpha have been shown to upregulate the heme - oxygenase- ( ho- ) system which has been shown to increase insulin sensitivity , improve glucose / lipid metabolism , suppress inflammation / oxidative stress , decrease immune response , and modulate cell - growth / differentiation . it has been also shown that there were beneficial effects of the ho - system in the pathogenesis of type 1 diabetes and related cardiometabolic complications . although , euglycemic - hyperinsulinemic clamp study is accepted standard for measurement of insulin sensitivity in patients with type 1 dm , it is not practical for use and is labor - intensive . estimated glucose disposal rate ( egdr ) is a derived measure of insulin resistance and can be calculated using routine clinical measures such as waist circumferences or waist - to - hip ratio , presence of hypertension , and hba1c levels . as an insulin sensitivity index , it is well correlated with results obtained from clamp studies and it should be emphasized that lower egdr levels indicate greater insulin resistance [ 32 , 33 ] . the purpose of the present study was to investigate the efficacy of combined therapy of insulin and the oral antihyperglycemic agent rosiglitazone , a ppar- agonist , on blood glucose regulation , total administered daily insulin dose , metabolic parameters , egdr , ffas , inflammatory indicators , and adipocytokine levels in patients with type 1 dm and poor glycemic control despite intensive insulin therapy . the study was conducted between march 2007 and january 2008 at the clinic of endocrinology and metabolism diseases , following the approval of the uluda university medical school ethics committee . after providing written consent , the patients were considered eligible based on the following criteria : age 1865 years , diagnosis of type 1 dm , and a glycosylated hemoglobin ( hba1c ) level > 6.5% despite 40-unit ( u ) intensive insulin therapy , on average , for 6 months . exclusion criteria were renal failure ( glomerular filtration rate < 75 mg / min or creatinine > 1.5 mg / dl ) , chronic hepatic disease or aspartate aminotransferase ( ast ) and alanine aminotransferase ( alt ) levels 2.5 times the normal values , current antidiabetic therapy other than insulin therapy , known history of rosiglitazone allergy , stage ii iv heart failure according to the new york heart association ( nyha ) classification , inability of the patient to enforce strict lifestyle changes , medical nutrition therapy or self - monitoring of blood glucose levels , ongoing or planned pregnancy , and current lactation . this prospective , open - label , randomized trial investigated the effectiveness of 4 mg / day rosiglitazone for 18 weeks in patients undergoing insulin therapy and without acute metabolic complications in combination with strict lifestyle changes and effective medical nutrition therapy . the patients attended a screening visit ( visit 1 ) two weeks before randomization , and they were classified into two open - label groups , such that clinical and demographical characteristics were similar in the two groups . the patients were evaluated for lifestyle changes , diet and exercise compliance , and insulin requirements . their therapies were modified , and the randomization visit ( visit 2 ) occurred two weeks later . the patients in both groups underwent insulin titration at visit 2 , and the patients in one group had 4 mg / day rosiglitazone added to their ongoing therapy ( group 1 ) , while the patients in the other group were monitored from that point with their most recent insulin titration ( group 2 ) . to reduce the risk of side effects , 4 mg rosiglitazone then , the patients attended a control visit ( visit 3 ) four weeks after randomization and a final visit ( visit 4 ) after 16 weeks . during each of the four visits , variables within six different categories were monitored , including a detailed physical examination ; self - monitoring of bg levels ; glycemic control ; therapy alterations and insulin requirements ; adverse events ; and biochemical , hematological , and inflammatory parameters , including adipocytokine levels . in order to measure the direct effect of rosiglitazone recurrent hypoglycemia , presence of symptomatic hyperglycemia , diabetic ketosis ( dk ) , diabetic ketoacidosis ( dka ) , and nonketotic hyperosmolar syndrome ( nkhs ) were considered metabolic complications . while patients with dk , dka , nkhs , or major hypoglycemia were planned to be excluded from the study and hospitalized if necessary , patients with symptomatic hyperglycemia and recurrent minor hypoglycemia underwent insulin titration . patients with symptoms of polydipsia , polyuria , weight loss , and nocturia were considered to be having symptomatic hyperglycemia if they also had mean bg levels > 276 mg / dl ; minor hypoglycemia if they were aware enough of their condition to administer self - therapy , were symptomatic , and had bg levels < 56 mg / dl ; or major hypoglycemia if they were unconscious , were unable to treat themselves , and recovered after therapy administered by others at home or at hospital . blood samples were analyzed for fasting plasma glucose ( fpg ) , urea , creatinine ( cr ) , ast , alt , total cholesterol ( total - c ) , high - density lipoprotein cholesterol ( hdl - c ) , low - density lipoprotein cholesterol ( ldl - c ) , and triglycerides ( tg ) using an autoanalyzer ( aeroset system operations manual ; abbot laboratories , abbott park , il , usa ) . ldl - c levels in patients with tg values < 400 mg / dl were calculated using the friedewald formula as follows : ldl - c = total - c [ ( tg/5 ) + hdl - c ] . hba1c levels were measured using high performance liquid chromatography ( hplc ; bio rad diagnostic group , hercules , ca , usa ) . spot urine albumin excretion was measured by chemiluminescence immunoassay ( immulite 2500 analyzer ; siemens , ca , usa ) , and creatinine levels were measured spectrophotometrically ( aeroset system operations manual ; abbot laboratories , abbott park , il , usa ) ; in addition , the albumin / creatinine ratio ( acr ) was calculated . the egdr was calculated as follows : 21.158 + ( 0.009 wc ) + ( 3.407 htn ) + ( 0.51 hba1c ) , where the wc indicates the waist circumferences and htn indicates blood pressure and is expressed as 0 : no , 1 : yes . the range of clamp - measured glucose disposal rates in the egdr validation study was 3.8 to 13.4 , with a range of ~9 to 11 in those with normal insulin resistance . ffa measurements were conducted using wako nefa - hr ( wako chemicals , gmbh , neuss , germany ) and an in vitro enzymatic calorimetric assay method . white blood cell ( wbc ) , hemoglobin ( hgb ) , hematocrit ( hct ) , and platelet ( plt ) measurements were conducted using cell - dyn 3700 ( mapss laser differential ; abbott laboratories , abbott park , il , usa ) . blood samples were collected as indicated and placed in westergren tubes to establish erythrocyte sedimentation rates ( esrs ) in a 1-hour period . fibrinogen was measured using the coagulation system , dade behring bnii ( dade behring inc . , the solid phase enzyme linked immunosorbent assay ( elisa ) method was used with a high sensitivity crp enzyme immunoassay ( drg international inc . , mountainside , nj , usa ) for the measurement of high - sensitivity c - reactive protein ( hs - crp ) . 0.110 mg / l , and intra- and interassay coefficients were 2.5% and 2.3% , respectively . the solid phase elisa method and a drg leptin enzyme immunoassay kit ( drg gmbh , marburg , germany ) were used to measure leptin . the expected results were 3.84 1.79 ng / ml for men and 7.36 3.73 ng / ml for women . the intra- and interassay coefficients were 5.95% and 8.66% , respectively . resistin was measured using the elisa method with human resistin elisa ( biovendor , brno , czech republic ) . adiponectin was measured using the elisa method with a high sensitivity human adiponectin elisa kit ( biovendor , brno , czech republic ) . the normal range was dependent upon bmi and was 9.5 3.9 g / ml for men and 13.2 6.1 g / ml for women . the intra- and interassay coefficients were 4.1% and 4.0% , respectively . according to pilot study , a sample size of 30 patients per group was calculated to give 80% power to detect a difference of 6 in change from baseline in mean adiponectin between groups at the 5% 2-sided significance level , assuming 8 as the common sd . statistical analyses were conducted using spss for windows ( version 22.0 ; spss inc . , chicago , il , usa ) . continuous variables are expressed as the mean standard deviation or median ( minimum - maximum ) , as appropriate ; categorical variables are expressed as frequencies ( n , % ) . a one - way anova was used to compare mean values for normally distributed variables when there were more than two independent groups , and the kruskal - wallis test was used when the assumptions for parametric tests were not met . paired data were analyzed using paired t - test and the wilcoxon signed rank test when data were not normally distributed . there were no significant differences between the groups in sex , mean age , disease duration , bw , bmi , waist circumferences , waist - to - hip ratios , fpg , hba1c values , and egdr in the baseline and mean p value was higher than 0.05 for all parameters mentioned above . egdr was 8.53 2.70 mg / kg / min in group 1 and 9.25 3.18 mg / kg / min in group 2 . patients in group 1 had higher fpg and hba1c and slightly low egdr , in other words more insulin resistant than group 2 . the comparisons between the final and baseline values for each group are shown in table 2 . the total number of patients in each group that completed the study was 28 , as two patients in each group were excluded owing to acute metabolic complications and one patient in group 2 was excluded owing to noncompliance with the visit schedule . during the follow - up period , the changes in bw and bmi were statistically significant in both groups ( p < 0.05 for group 1 and p < 0.01 for group 2 ) , whereas the changes in wc and waist - to - hip ratio ( whr ) were not significant . systolic blood pressure ( sbp ) significantly decreased in group 2 ; the changes in diastolic blood pressure ( dbp ) were similar in both groups . although the final fpg and hba1c values were lower than the baseline values in both groups , the changes were statistically significant only in group 2 . no statistically significant differences were found in egdr during the study period in both groups . egdr was 8.53 2.70 mg / kg / min at baseline and 8.36 2.45 mg / kg / min at final visits in group 1 . it was 9.25 3.18 mg / kg / min at baseline and 9.22 3.20 at final visits in group 2 . p value was 0.185 for group 1 and 0.235 for group 2 in terms of egdr changes between two visits . urea , cr , spot urine acr , ast , and alt levels did not significantly change in either group . of the total - c , hdl - c , ldl - c , tg , and ffa levels , only the hdl - c levels in group 2 significantly changed ( p = 0.038 ) . while hgb and hct values did not change significantly in group 2 , a significant decrease in hgb levels was observed in group 1 . of the esr , fibrinogen , and hs - crp levels , only fibrinogen levels significantly decreased in group 1 , and the changes in the other parameters were not significant for either group . although the resistin and leptin levels decreased significantly and changes were statistically significant in both groups , adiponectin levels increased but the change was not significant . the total daily insulin doses administered during the follow - up period are also shown in table 2 . the changes observed both between and within the groups in total daily insulin doses per kilogram of bw were not significant . comparisons between the groups for the final and baseline values of inflammatory markers , adipocytokine , and ffa levels are shown in table 3 . the differences between the groups in esr and hs - crp levels were not significant at both baseline and final visits . however , while the difference in fibrinogen levels was significant at the baseline visit , the values at the final visit were not different owing to the decrease in fibrinogen levels in group 1 . also , the differences between the groups in resistin , leptin , and adiponectin levels were not significant at both baseline and final visits . diabetes - related complications experienced by the patients over the 18-week follow - up period are shown in table 4 . the mean hypoglycemia frequency was calculated by dividing the number of total hypoglycemia episodes for all of the patients over the 18-week period by the number of patients , and the mean change in bw for the same period was calculated similarly . the number of patients with acute complications or major hypoglycemic events was expressed as the total frequency for the group . two patients in group 1 experienced major hypoglycemic events , while two patients in group 2 had to be hospitalized due to dka ( table 4 ) . while 5.35 minor hypoglycemia episodes were experienced per patient over the 18 weeks in group 1 , the mean weight gain was 2.58 3.10 kg during the same period . the patients in group 2 , on the other hand , experienced 4.61 minor hypoglycemic episodes per patient , and the mean weight gain was 1.47 1.53 kg during the same period . the p value for minor hypoglycemia was 0.437 , and it was 0.142 for weight gain . therefore , the difference between the groups with respect to either parameter was not statistically significant . because of the low number of major hypoglycemia events and acute metabolic complications , statistical comparisons were not possible . the combined therapy approaches used in type 2 diabetes management have also become increasingly common in type 1 diabetes management . furthermore , studies have demonstrated that insulin resistance is not exclusively observed in patients with type 2 diabetes and that it is also a critical factor for patients with type 1 diabetes because it can be overlooked in patients without adequate bg regulation [ 32 , 3538 ] . the primary goals when introducing insulin - sensitizing agents in combined therapy are ensuring good glycemic control , decreasing insulin demand , achieving favorable effects on cardiovascular risk factors , and minimizing alterations in bw . strowig and raskin reported improved glycemic control without an increase in insulin demand in 25 overweight adult patients with type 1 diabetes following therapy with 4 mg rosiglitazone twice a day for 8 months . although patients in both groups in the present study had decreased fpg and hba1c levels , the changes were not significant . baseline glucose and hba1c values were higher in the patients receiving rosiglitazone ; however , the differences in the changes between the groups were not significant . in a study conducted by stone et al . , 36 patients with type 1 diabetes whose daily insulin requirement was > 1.1 u / kg were administered 8 mg / day rosiglitazone . the investigators reported that the change in hba1c levels was not significant when compared with the placebo group . for decades , type 1 diabetes has been traditionally known as insulin - dependent , while type 2 has been known as noninsulin - dependent diabetes . however , it is becoming increasingly clear that insulin deficiency and insulin resistance are manifested in both forms of diabetes at different stages . unlike other studies [ 39 , 40 ] which investigate the effects of tzds on insulin resistance and consist of obese or overweight patients with type 1 dm , almost all of the patients were lean in present study . egdr is a validated clinical tool for estimating insulin sensitivity in patients with type 1 dm . it was near normal at baseline and did not change significantly during our study period in both groups . this result was important to show effects of rosiglitazone , except insulin - sensitizing characteristics . thus , it can be thought that the effects of rosiglitazone observed in present study were not associated with insulin sensitivity . one of the known major effects of these drugs is on oxidative stress and mitochondrial ros production . it has been shown that tzds could inhibit hyperglycemia - induced mtros and contribute to the prevention of diabetic vascular complications . although there were some studies investigating these effects in patients with type 2 dm , it is unclear in patients with type 1 dm . it is obvious that there is a need to investigate this effect in prospective cohort studies composed of patients with type 1 dm . in addition , agonists of ppar - gamma might increase insulin sensitivity via upregulating heme - oxygenase - system . the ho - system and related products have been shown to decrease inflammation and enhance insulin sensitivity . more importantly , in experimental models of type 1 dm , upregulating ho - system caused increase in pancreatic beta cell insulin production . beneficial effects of tzds on insulin resistance and inflammation resulting from ho - system have been studied in patients with both type 2 and type 1 dm [ 30 , 31 , 42 ] . these developments may offer new options , either prevention of disease or development of the complications . we observed significant bw and bmi changes in all patients at the final visit compared with baseline values . the most significant side effect of tzds , particularly when combined with sulfonylurea and insulin , is weight gain . tzds increase overall fat tissue , and the most affected area is subcutaneous fat tissue . another major cause of tzd - related weight gain is increased water and salt retention , which leads to increased plasma volume . edema is caused by depressed renal sodium excretion and free water retention [ 4345 ] . reported a 10% increase in left ventricular mass without significant alterations in cardiac structure and function in patients undergoing rosiglitazone therapy . although we observed weight gain in our patient groups in this present study , no patient had heart failure . moreover , peripheral edema or dyspnea was not among the adverse events reported by our patients . this could be attributed to the relatively young mean age in our sample or to the fact that heart failure at baseline was among the exclusion criteria . furthermore , no patient in this cohort had diabetic nephropathy that could lead to edema , which might have decreased the risk for edema . the hypoglycemic effects of tzds include increased insulin sensitivity that is mediated through tg and ffa metabolism and is associated with the agonistic effects of ppar-. increased ffa levels lead to insulin resistance and fasting plasma ffa levels in patients with type 2 diabetes administered tzd might decrease by 2030% . ppar- activation impairs tg and fatty acid synthesis , leading to decreased very - ldl - c and hdl - c synthesis as well as increased ldl - c and total - c levels [ 47 , 48 ] . the patients in the present study with bmi and wc levels in the normal range had baseline tg and hdl - c levels within the desired range , and the ldl - c levels in all patients were close to 100 mg / dl . while slight decreases in total - c , ldl - c , tg , and ffa levels were noted in the patients receiving rosiglitazone at the end of the present study , hdl - c levels had increased slightly . patients receiving only insulin had similar values to those in the combined therapy group except for slightly higher tg levels , although this was not statistically significant . we suggest that the strict enforcement of lifestyle changes during the follow - up period might have contributed to these results . tzds have been demonstrated to have significant anti - inflammatory characteristics in studies conducted on patients with type 2 diabetes . reported that rosiglitazone reduces diabetes - related atherosclerosis and that this effect is possibly associated with oxidative stress and inflammation , independent of metabolic effects , unrelated to the insulin dose . in particular , the diabetes control and complications trial , as well as a number of other studies , revealed that hs - crp levels increased in patients undergoing intensive therapy . furthermore , hs - crp and increased fibrinogen levels are independent risk factors for coronary heart disease , and a number of studies have reported elevated fibrinogen levels in patients with diabetes [ 50 , 51 ] . the esr levels in the present study were within the normal range at baseline , and they continued to be so until the end of study . while hs - crp levels decreased with respect to baseline values in the patients receiving rosiglitazone in the present study , they increased slightly in the patients receiving insulin alone . to the best of our knowledge , this is the first study to measure fibrinogen levels in patients with type 1 diabetes after rosiglitazone administration . the levels decreased significantly in these patients , while the patients undergoing therapy with insulin alone showed minimal decreases in fibrinogen levels . independent of the improvement in bg regulation , levels of hs - crp and fibrinogen , which are conventional inflammatory markers , decreased significantly following rosiglitazone therapy in the present study . plasma leptin levels are positively correlated with female sex , bmi , and age but not with diabetes duration , hba1c , or total insulin dose per kilogram . most patients with type 1 diabetes are either underweight or in the normal bw range . data concerning leptin levels in this group of patients vary , and exogenous insulin therapy leads to elevated leptin levels in patients with type 1 diabetes [ 53 , 54 ] . resistin , which is released from adipose tissue , is directly associated with insulin resistance factors such as wc and whr . reported increased serum resistin levels in patients with type 1 diabetes and reported that levels returned to the normal range after pancreas transplant [ 55 , 56 ] . while no significant differences were observed between the groups at the baseline or final visit or in the change between the visits , the within - group changes during the follow - up period were significant , despite the weight gain . resistin levels were low in both groups , and the change in the patients receiving rosiglitazone was significant , despite weight gain , and was associated with the favorable effects of rosiglitazone . adiponectin has been shown to have positive effects on cardiometabolic risk , and adiponectin levels are negatively correlated with insulin resistance and weight gain . moreover , good glycemic control increases adiponectin levels , whereas poor glycemic control decreases adiponectin levels [ 57 , 58 ] . in the cacti trial , maahs et al . reported negative correlations between adiponectin levels and male sex , central adiposity , sbp , dbp , daily insulin dose , hba1c , fibrinogen , albumin excretion rate , and tg levels ; positive correlations were noted with type 1 diabetes , hdl - c , and homocysteine . in the present study , adiponectin levels increased parallel to glycemic improvement both in patients undergoing combined therapy and in those receiving insulin therapies alone . the diverse favorable effects of tzds previously reported in patients with type 2 diabetes were not fully experienced in patients with type 1 diabetes in the present study . the addition of 4 mg / day rosiglitazone to intensive insulin therapy did not significantly improve glycemic parameter , lipid parameter , ffa , esr , hs - crp , leptin , or resistin levels . patients receiving rosiglitazone showed weight gain , a major side effect of tzds , whereas insulin sensitivity was not significantly different and the incidence of hypoglycemia was not lower . hba1c , fpg , and total daily insulin doses decreased significantly after combination therapy with tzd and insulin in patients with type 2 diabetes mellitus , suggesting that the insulin - sensitizing characteristics of tzds are likely more pronounced in patients who are not totally devoid of endogenous insulin secretion . we acknowledge the limitations of the current study including single centre experience and small sample size . patients treated in our series did not appear to have driven any meaningful benefit from combination therapy . due to small number of patients included in study , prospective clinical trials are however required to define optimal treatment regimens . clinical prognostic factors evaluated in our series may be useful for stratification and eligibility considerations in future clinical trials .

aim . to investigate the efficacy of combined therapy of insulin and rosiglitazone on metabolic and inflammatory parameters , insulin sensitivity , and adipocytokine levels in patients with type 1 diabetes mellitus ( type 1 dm ) . material and methods . a total of 61 adults with type 1 dm were randomly and prospectively assigned in open - label fashion to take insulin and rosiglitazone 4 mg / day ( n = 30 ) or insulin alone ( n = 31 ) for a period of 18 weeks while undergoing insulin therapy without acute metabolic complications . results . combination therapy did not significantly improve metabolic and inflammatory parameters , insulin sensitivity , and adiponectin levels . while leptin and resistin levels decreased in both groups ( group 1 : resistin 6.96 3.06 to 4.99 2.64 , p = 0.006 ; leptin 25.8 17.6 to 20.1 12.55 , p = 0.006 ; group 2 : resistin 7.16 2.30 to 5.57 2.48 , p = 0.031 ; leptin 16.72 16.1 to 14.0 13.4 , p = 0.007 ) hgb and fibrinogen levels decreased only in group 1 ( hgb 13.72 1.98 to 13.16 1.98 , p = 0.015 , and fibrinogen 4.00 1.08 to 3.46 0.90 , p = 0.002 ) . patients in both groups showed weight gain and the incidence of hypoglycemia was not lower . discussion . the diverse favorable effects of tzds were not fully experienced in patients with type 1 dm . these results are suggesting that insulin sensitizing and anti - inflammatory characteristics of tzds were likely to be more pronounced in patients who were not totally devoid of endogenous insulin secretion .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

there has been a progressive increase in the incidence of type 1 dm , and several advances in its treatment have been achieved . chronic subclinical inflammation , impaired fibrinolytic system activity , and elevated procoagulant factor levels form the basis of atherosclerotic diseases . insulin resistance is characterized by limited stimulation of glucose metabolism in muscle and the liver and has been described in patients with poorly controlled type 1 dm [ 68 ] . cytokines such as leptin , resistin , and adiponectin released from these tissues critically impact nutritional status , body fat distribution , metabolic parameters , inflammatory status , atherosclerotic alterations , and insulin resistance . the levels of adiponectin , which is regarded as an antidiabetic , anti - inflammatory , and antiatherogenic cytokine , are reported to be depressed in patients with type 2 dm [ 10 , 11 ] . however , in some studies , increased adiponectin levels have been reported in patients with type 1 dm . glucose uptake rates by peripheral tissues , which can be stimulated by insulin in skeletal muscles , decrease over time in patients with type 1 dm and poor glycemic control . these patients have significant hepatic insulin resistance , and the effects of insulin are impaired because of plasma free fatty acids ( ffas ) [ 1719 ] . tzds , apart from their direct effect on fatty tissue , might influence the release of adipocyte - derived signal factors that determine the insulin sensitivity of muscles , such as ffas , adiponectin , leptin , and tumor necrosis factor - alpha ( tnf- ) . in addition to their favorable effects on glycemic control , tzds directly influence vessel walls , decrease vasoconstriction , and inhibit inflammation . agonists of ppar - gamma and ppar - alpha have been shown to upregulate the heme - oxygenase- ( ho- ) system which has been shown to increase insulin sensitivity , improve glucose / lipid metabolism , suppress inflammation / oxidative stress , decrease immune response , and modulate cell - growth / differentiation . it has been also shown that there were beneficial effects of the ho - system in the pathogenesis of type 1 diabetes and related cardiometabolic complications . although , euglycemic - hyperinsulinemic clamp study is accepted standard for measurement of insulin sensitivity in patients with type 1 dm , it is not practical for use and is labor - intensive . estimated glucose disposal rate ( egdr ) is a derived measure of insulin resistance and can be calculated using routine clinical measures such as waist circumferences or waist - to - hip ratio , presence of hypertension , and hba1c levels . the purpose of the present study was to investigate the efficacy of combined therapy of insulin and the oral antihyperglycemic agent rosiglitazone , a ppar- agonist , on blood glucose regulation , total administered daily insulin dose , metabolic parameters , egdr , ffas , inflammatory indicators , and adipocytokine levels in patients with type 1 dm and poor glycemic control despite intensive insulin therapy . after providing written consent , the patients were considered eligible based on the following criteria : age 1865 years , diagnosis of type 1 dm , and a glycosylated hemoglobin ( hba1c ) level > 6.5% despite 40-unit ( u ) intensive insulin therapy , on average , for 6 months . exclusion criteria were renal failure ( glomerular filtration rate < 75 mg / min or creatinine > 1.5 mg / dl ) , chronic hepatic disease or aspartate aminotransferase ( ast ) and alanine aminotransferase ( alt ) levels 2.5 times the normal values , current antidiabetic therapy other than insulin therapy , known history of rosiglitazone allergy , stage ii iv heart failure according to the new york heart association ( nyha ) classification , inability of the patient to enforce strict lifestyle changes , medical nutrition therapy or self - monitoring of blood glucose levels , ongoing or planned pregnancy , and current lactation . this prospective , open - label , randomized trial investigated the effectiveness of 4 mg / day rosiglitazone for 18 weeks in patients undergoing insulin therapy and without acute metabolic complications in combination with strict lifestyle changes and effective medical nutrition therapy . the patients attended a screening visit ( visit 1 ) two weeks before randomization , and they were classified into two open - label groups , such that clinical and demographical characteristics were similar in the two groups . their therapies were modified , and the randomization visit ( visit 2 ) occurred two weeks later . the patients in both groups underwent insulin titration at visit 2 , and the patients in one group had 4 mg / day rosiglitazone added to their ongoing therapy ( group 1 ) , while the patients in the other group were monitored from that point with their most recent insulin titration ( group 2 ) . during each of the four visits , variables within six different categories were monitored , including a detailed physical examination ; self - monitoring of bg levels ; glycemic control ; therapy alterations and insulin requirements ; adverse events ; and biochemical , hematological , and inflammatory parameters , including adipocytokine levels . in order to measure the direct effect of rosiglitazone recurrent hypoglycemia , presence of symptomatic hyperglycemia , diabetic ketosis ( dk ) , diabetic ketoacidosis ( dka ) , and nonketotic hyperosmolar syndrome ( nkhs ) were considered metabolic complications . while patients with dk , dka , nkhs , or major hypoglycemia were planned to be excluded from the study and hospitalized if necessary , patients with symptomatic hyperglycemia and recurrent minor hypoglycemia underwent insulin titration . patients with symptoms of polydipsia , polyuria , weight loss , and nocturia were considered to be having symptomatic hyperglycemia if they also had mean bg levels > 276 mg / dl ; minor hypoglycemia if they were aware enough of their condition to administer self - therapy , were symptomatic , and had bg levels < 56 mg / dl ; or major hypoglycemia if they were unconscious , were unable to treat themselves , and recovered after therapy administered by others at home or at hospital . ldl - c levels in patients with tg values < 400 mg / dl were calculated using the friedewald formula as follows : ldl - c = total - c [ ( tg/5 ) + hdl - c ] . spot urine albumin excretion was measured by chemiluminescence immunoassay ( immulite 2500 analyzer ; siemens , ca , usa ) , and creatinine levels were measured spectrophotometrically ( aeroset system operations manual ; abbot laboratories , abbott park , il , usa ) ; in addition , the albumin / creatinine ratio ( acr ) was calculated . the egdr was calculated as follows : 21.158 + ( 0.009 wc ) + ( 3.407 htn ) + ( 0.51 hba1c ) , where the wc indicates the waist circumferences and htn indicates blood pressure and is expressed as 0 : no , 1 : yes . the range of clamp - measured glucose disposal rates in the egdr validation study was 3.8 to 13.4 , with a range of ~9 to 11 in those with normal insulin resistance . white blood cell ( wbc ) , hemoglobin ( hgb ) , hematocrit ( hct ) , and platelet ( plt ) measurements were conducted using cell - dyn 3700 ( mapss laser differential ; abbott laboratories , abbott park , il , usa ) . 0.110 mg / l , and intra- and interassay coefficients were 2.5% and 2.3% , respectively . a one - way anova was used to compare mean values for normally distributed variables when there were more than two independent groups , and the kruskal - wallis test was used when the assumptions for parametric tests were not met . paired data were analyzed using paired t - test and the wilcoxon signed rank test when data were not normally distributed . egdr was 8.53 2.70 mg / kg / min in group 1 and 9.25 3.18 mg / kg / min in group 2 . patients in group 1 had higher fpg and hba1c and slightly low egdr , in other words more insulin resistant than group 2 . the total number of patients in each group that completed the study was 28 , as two patients in each group were excluded owing to acute metabolic complications and one patient in group 2 was excluded owing to noncompliance with the visit schedule . during the follow - up period , the changes in bw and bmi were statistically significant in both groups ( p < 0.05 for group 1 and p < 0.01 for group 2 ) , whereas the changes in wc and waist - to - hip ratio ( whr ) were not significant . systolic blood pressure ( sbp ) significantly decreased in group 2 ; the changes in diastolic blood pressure ( dbp ) were similar in both groups . although the final fpg and hba1c values were lower than the baseline values in both groups , the changes were statistically significant only in group 2 . no statistically significant differences were found in egdr during the study period in both groups . egdr was 8.53 2.70 mg / kg / min at baseline and 8.36 2.45 mg / kg / min at final visits in group 1 . it was 9.25 3.18 mg / kg / min at baseline and 9.22 3.20 at final visits in group 2 . p value was 0.185 for group 1 and 0.235 for group 2 in terms of egdr changes between two visits . urea , cr , spot urine acr , ast , and alt levels did not significantly change in either group . of the total - c , hdl - c , ldl - c , tg , and ffa levels , only the hdl - c levels in group 2 significantly changed ( p = 0.038 ) . while hgb and hct values did not change significantly in group 2 , a significant decrease in hgb levels was observed in group 1 . of the esr , fibrinogen , and hs - crp levels , only fibrinogen levels significantly decreased in group 1 , and the changes in the other parameters were not significant for either group . although the resistin and leptin levels decreased significantly and changes were statistically significant in both groups , adiponectin levels increased but the change was not significant . comparisons between the groups for the final and baseline values of inflammatory markers , adipocytokine , and ffa levels are shown in table 3 . the differences between the groups in esr and hs - crp levels were not significant at both baseline and final visits . however , while the difference in fibrinogen levels was significant at the baseline visit , the values at the final visit were not different owing to the decrease in fibrinogen levels in group 1 . also , the differences between the groups in resistin , leptin , and adiponectin levels were not significant at both baseline and final visits . the mean hypoglycemia frequency was calculated by dividing the number of total hypoglycemia episodes for all of the patients over the 18-week period by the number of patients , and the mean change in bw for the same period was calculated similarly . two patients in group 1 experienced major hypoglycemic events , while two patients in group 2 had to be hospitalized due to dka ( table 4 ) . while 5.35 minor hypoglycemia episodes were experienced per patient over the 18 weeks in group 1 , the mean weight gain was 2.58 3.10 kg during the same period . the patients in group 2 , on the other hand , experienced 4.61 minor hypoglycemic episodes per patient , and the mean weight gain was 1.47 1.53 kg during the same period . the p value for minor hypoglycemia was 0.437 , and it was 0.142 for weight gain . therefore , the difference between the groups with respect to either parameter was not statistically significant . because of the low number of major hypoglycemia events and acute metabolic complications , statistical comparisons were not possible . the combined therapy approaches used in type 2 diabetes management have also become increasingly common in type 1 diabetes management . furthermore , studies have demonstrated that insulin resistance is not exclusively observed in patients with type 2 diabetes and that it is also a critical factor for patients with type 1 diabetes because it can be overlooked in patients without adequate bg regulation [ 32 , 3538 ] . the primary goals when introducing insulin - sensitizing agents in combined therapy are ensuring good glycemic control , decreasing insulin demand , achieving favorable effects on cardiovascular risk factors , and minimizing alterations in bw . strowig and raskin reported improved glycemic control without an increase in insulin demand in 25 overweight adult patients with type 1 diabetes following therapy with 4 mg rosiglitazone twice a day for 8 months . although patients in both groups in the present study had decreased fpg and hba1c levels , the changes were not significant . baseline glucose and hba1c values were higher in the patients receiving rosiglitazone ; however , the differences in the changes between the groups were not significant . , 36 patients with type 1 diabetes whose daily insulin requirement was > 1.1 u / kg were administered 8 mg / day rosiglitazone . for decades , type 1 diabetes has been traditionally known as insulin - dependent , while type 2 has been known as noninsulin - dependent diabetes . however , it is becoming increasingly clear that insulin deficiency and insulin resistance are manifested in both forms of diabetes at different stages . unlike other studies [ 39 , 40 ] which investigate the effects of tzds on insulin resistance and consist of obese or overweight patients with type 1 dm , almost all of the patients were lean in present study . egdr is a validated clinical tool for estimating insulin sensitivity in patients with type 1 dm . it was near normal at baseline and did not change significantly during our study period in both groups . thus , it can be thought that the effects of rosiglitazone observed in present study were not associated with insulin sensitivity . although there were some studies investigating these effects in patients with type 2 dm , it is unclear in patients with type 1 dm . it is obvious that there is a need to investigate this effect in prospective cohort studies composed of patients with type 1 dm . more importantly , in experimental models of type 1 dm , upregulating ho - system caused increase in pancreatic beta cell insulin production . beneficial effects of tzds on insulin resistance and inflammation resulting from ho - system have been studied in patients with both type 2 and type 1 dm [ 30 , 31 , 42 ] . the most significant side effect of tzds , particularly when combined with sulfonylurea and insulin , is weight gain . tzds increase overall fat tissue , and the most affected area is subcutaneous fat tissue . reported a 10% increase in left ventricular mass without significant alterations in cardiac structure and function in patients undergoing rosiglitazone therapy . the hypoglycemic effects of tzds include increased insulin sensitivity that is mediated through tg and ffa metabolism and is associated with the agonistic effects of ppar-. increased ffa levels lead to insulin resistance and fasting plasma ffa levels in patients with type 2 diabetes administered tzd might decrease by 2030% . the patients in the present study with bmi and wc levels in the normal range had baseline tg and hdl - c levels within the desired range , and the ldl - c levels in all patients were close to 100 mg / dl . patients receiving only insulin had similar values to those in the combined therapy group except for slightly higher tg levels , although this was not statistically significant . tzds have been demonstrated to have significant anti - inflammatory characteristics in studies conducted on patients with type 2 diabetes . furthermore , hs - crp and increased fibrinogen levels are independent risk factors for coronary heart disease , and a number of studies have reported elevated fibrinogen levels in patients with diabetes [ 50 , 51 ] . the esr levels in the present study were within the normal range at baseline , and they continued to be so until the end of study . while hs - crp levels decreased with respect to baseline values in the patients receiving rosiglitazone in the present study , they increased slightly in the patients receiving insulin alone . to the best of our knowledge , this is the first study to measure fibrinogen levels in patients with type 1 diabetes after rosiglitazone administration . the levels decreased significantly in these patients , while the patients undergoing therapy with insulin alone showed minimal decreases in fibrinogen levels . independent of the improvement in bg regulation , levels of hs - crp and fibrinogen , which are conventional inflammatory markers , decreased significantly following rosiglitazone therapy in the present study . most patients with type 1 diabetes are either underweight or in the normal bw range . data concerning leptin levels in this group of patients vary , and exogenous insulin therapy leads to elevated leptin levels in patients with type 1 diabetes [ 53 , 54 ] . reported increased serum resistin levels in patients with type 1 diabetes and reported that levels returned to the normal range after pancreas transplant [ 55 , 56 ] . while no significant differences were observed between the groups at the baseline or final visit or in the change between the visits , the within - group changes during the follow - up period were significant , despite the weight gain . resistin levels were low in both groups , and the change in the patients receiving rosiglitazone was significant , despite weight gain , and was associated with the favorable effects of rosiglitazone . adiponectin has been shown to have positive effects on cardiometabolic risk , and adiponectin levels are negatively correlated with insulin resistance and weight gain . reported negative correlations between adiponectin levels and male sex , central adiposity , sbp , dbp , daily insulin dose , hba1c , fibrinogen , albumin excretion rate , and tg levels ; positive correlations were noted with type 1 diabetes , hdl - c , and homocysteine . in the present study , adiponectin levels increased parallel to glycemic improvement both in patients undergoing combined therapy and in those receiving insulin therapies alone . the diverse favorable effects of tzds previously reported in patients with type 2 diabetes were not fully experienced in patients with type 1 diabetes in the present study . the addition of 4 mg / day rosiglitazone to intensive insulin therapy did not significantly improve glycemic parameter , lipid parameter , ffa , esr , hs - crp , leptin , or resistin levels . patients receiving rosiglitazone showed weight gain , a major side effect of tzds , whereas insulin sensitivity was not significantly different and the incidence of hypoglycemia was not lower . hba1c , fpg , and total daily insulin doses decreased significantly after combination therapy with tzd and insulin in patients with type 2 diabetes mellitus , suggesting that the insulin - sensitizing characteristics of tzds are likely more pronounced in patients who are not totally devoid of endogenous insulin secretion . patients treated in our series did not appear to have driven any meaningful benefit from combination therapy .

chronic fatigue syndrome ( cfs ) , as defined by the centers for disease control and prevention ( cdcp ) , is a complex illness characterized by prolonged debilitating fatigue and multiple non - specific symptoms including headaches , recurrent sore throats , fever , muscle and joint pain , and neurocognitive complaints [ 1 , 2 ] . in addition to the chronic fatigue , widespread and persistent pain is common in individuals with cfs [ 35 ] . a population - based study revealed that 94% of the persons diagnosed with cfs report muscle aches and pain and 84% report joint pain . seventy - four patients ( 64.9% ) complained of arthralgia . in another study , 24 of 44 patients suffered from chronic widespread pain . chronic fatigue accompanied by chronic musculoskeletal impairments such as myalgias and arthralgias could be considered an important subclass of cfs . evidence supportive of the clinical importance of widespread pain in cfs has been provided : chronic pain accounts for up to 34% of the cfs patients self - reported activity limitations and participation restrictions . chronic pain is more disabling than chronic fatigue . given these facts , it may be surprising that the etiology of these pain complaints has not been studied extensively in patients with cfs . the systematic literature review by meeus et al . shows that only little progress has been made in understanding chronic widespread pain in patients with cfs . a few hypotheses have been proposed , but they have not been studied in depth or relatively little work has been performed to test these hypotheses . in contrast , a large body of scientific literature regarding the etiology of chronic pain complaints in fibromyalgia ( fm ) is currently available . the diagnosis of fm is based on the 1990 american college of rheumatology criteria . following these criteria , fm patients present with 11 of 18 positive tender points and with widespread pain . validity of both the definition for cfs and fm has been shown [ 12 , 13 ] . especially , investigations focusing on the phenomenon central sensitization are presented in force in fm . in cfs , given the great overlap between cfs and fm and given the dearth of studies focusing on the explanation for the chronic widespread pain in patients with cfs , it would be interesting to propose a theoretical model for the chronic pain in cfs based on the current knowledge of cfs and on the evidence for central sensitization in fm , giving rise to further research on that matter . besides the knowledge on chronic pain in fm , it is necessary to gather knowledge on musculoskeletal pain in cfs . the syndromes may overlap , but despite the similarities between the two syndromes , there are also differences . for example , immunological dysregulations such as the abnormal 25a synthetase / rnase l pathway have been revealed in cfs but have never been detected in fm patients . furthermore , there is not yet any good evidence for similar pain mechanisms in cfs and fm . for example , patterns of functional brain activity in patients with fm are quite different from those in patients with cfs . patients with cfs , relative to controls , showed significantly lower blood perfusion in the brain stem [ 17 , 18 ] . patients with fm exhibited significantly lower rcbf levels , during rest , in the thalamus and the caudate nucleus . furthermore , substance p has been found to be elevated in csf of fm patients and not in patients with cfs . therefore , the knowledge on pain in fm can not be applied on cfs patients without further study . based on the similarities and differences between the two syndromes , further research on pain in cfs is advised to get an image of pain processing in the two diseases . first , the concept of central sensitisation as a cause of chronic pain will be explained . this theoretical background will then be applied to fm and an overview of the evidence for central sensitization in fm will follow . finally , based on the theoretical background and the findings in fm , the hypothesis concerning central sensitization in cfs will be unfolded , supported with the present knowledge on cfs . introduction pain is a complex perception that is influenced by prior experience and by the context within which the noxious stimulus occurs ; nociception is the physiologic response to tissue damage or prior tissue damage . the definition of pain is endorsed by the international association for the study of pain : pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage , or described in terms of such damage . there are a host of physiologic mechanisms by which injuries lead to nociceptive responses and ultimately to pain . however , not all nociceptive signals are perceived as pain and vice versa , not every pain sensation originates from nociception . these include low - threshold nociceptors that are connected to fast conducting a - delta pain fibers , and high - threshold nociceptors that conduct impulses in slow ( unmyelinated ) c fibers . within the dorsal horn of the spinal cord , these pain fibers synapse with spinal neurons via synaptic transmission . many neurotransmitters ( i.e. , glutamate , substance p , etc . ) are able to modulate the postsynaptic responses with further transmission to supraspinal sites ( thalamus , anterior cingulated cortex , insular cortex , and somatosensory cortex ) via the ascending pathways [ 22 , 24 , 25].the simplest form of plasticity in nervous systems is that repeated noxious stimulation may lead to habituation ( decreased response ) or sensitization ( increased response ) . prolonged or strong activity of dorsal horn neurons caused by repeated or sustained noxious stimulation may subsequently lead to increased neuronal responsiveness or central sensitization [ 25 , 27 ] . neuroplasticity and subsequent cns sensitization include altered function of chemical , electrophysiological , and pharmacological systems [ 22 , 28 , 29 ] . these changes cause exaggerated perception of painful stimuli ( hyperalgesia ) , a perception of innocuous stimuli as painful ( allodynia ) and may be involved in the generation of referred pain and hyperalgesia across multiple spinal segments [ 25 , 3033].while the exact mechanism by which the spinal cord becomes sensitized or in hyperexcitable state currently remains somewhat unknown , some contributing factors have been proposed . refers to a central spinal mechanism in which repetitive noxious stimulation results in a slow temporal summation that is experienced in humans as increased pain . in 1965 , animal experiments showed for the first time that repetitive c - fiber stimulation could result in a progressive increase of electrical discharges from the second - order neuron in the spinal cord . this mechanism of pain amplification in the spinal cord is related to temporal summation of second pain or wind - up . second pain , which is more dull and strongly related to chronic pain states , is transmitted through unmyelinated c fibers to dorsal horn nociceptive neurons . during the c - fibres transmitted stimuli , n - methyl - d - aspartate ( nmda ) receptors of second - order neurons become activated.it is well - known that nmda activation induces calcium entry into the dorsal horn neurons . calcium entry into sensory neurons in the dorsal horn induces activation of nitric oxide ( no ) synthase , leading to the synthesis of no . no can affect the nociceptor terminals and enhance the release of sensory neuropeptides ( in particular , substance p ) from presynaptic neurons , therefore contributing to the development of hyperalgesia and maintenance of central sensitization . it lowers the threshold of synaptic excitability , resulting in the unmasking of normally silent interspinal synapses and the sensitization of second - order spinal neurons .furthermore , sp can extend for long distances in the spinal cord and sensitize dorsal horn neurons at a distance from the initial input locus . this results in an expansion of receptive fields and the activation of wide dynamic neurons by non - nociceptive afferent impulses .wind - up can be elicited in human patients if identical nociceptive stimuli are applied to the skin or muscles more often than once every 3 s. the resulting progressive increase of pain sensations represents wind - up and has been demonstrated to result from a central rather than a peripheral nervous system mechanism , because the input from c nociceptors has been shown to decline or stay the same with stimulus repetition . endogenous pain modulatory systems the presence of several pain inhibitory and facilitatory centers in the brainstem is well recognized . experimental evidence for the existence of descending inhibitory pathways , and their connection with central sensitization , include the observations that bilateral lesions of the dorsolateral funiculus in the rat led to a significant decrease in latency for paw withdrawal to noxious stimulus . similarly , temporary spinal cord block ( lidocaine ) caused dorsal horn nociceptive neurons to expand their receptive fields and their responsiveness to afferent input . hypersensitivity .the foregoing investigations suggest that disruption of one or more of the elements of the inhibitory system can result in , among other things , the equivalent of central sensitization . one function of the descending inhibitory pathway is to focus the excitation of the dorsal horn neurons . the effect is to generate a more urgent , localized , and rapid pain signal by suppressing surrounding neuronal activity . , descending pathways effectively enhance the biologically valuable pain signal by reducing the level of irrelevant noise in the system.facilitatory pathways leading from the brainstem have also been identified . there is now behavioral evidence that forebrain centers are capable of exerting powerful clinically significant influences on various nuclei of the brainstem , including the nuclei identified as the origin of the descending facilitatory pathway . the activity in descending pathways is not constant but can be modulated , for example , by the level of vigilance or attention and by stress . forebrain products such as cognitions , emotions , attention , and motivation have influence on the clinical pain experience .dubner and ren rewarded subjects for responding to a randomly delivered transient tissue threatening peripheral stimulus . it was found that sensitization of second - order pain pathway neurons was directly related to the strength of attention . the evidence suggests that selective attention to relevant stimuli activated descending pain modulatory systems , turning the balance in favor of facilitation . behavioral variables such as attention to a potentially threatening stimulus result in sensitization of dorsal horns spinal cord neurons . moreover , behavioral modulation associated with selective attention to a perceived threat utilizes the same forebrain and brainstem structures and mechanisms as are involved in the development , amplification , and maintenance of persistent pain after actual tissue damage and inflammation .certain cognitive styles and personality traits have been associated with amplification of pain and its extension in the absence of tissue damage . thus , via descending pathways behavioral and cognitive therapies might also effect synaptic transmission in the spinal cord and thereby have the capacity to prevent or reverse long - term changes of synaptic strength in pain pathways . exaggerated pain is common in whiplash and fm patients . theoretically , peripheral mechanisms can account for the pain hypersensitivity . in fm , however , there is no evidence for peripheral sensitization as the cause of hyperalgesia , given the absence of real tissue damage . peripheral sensitization is defined as a reduction in the threshold of nociceptive afferent receptors caused by a local change in the sensitivity of sensory fibers initiated by tissue damage . peripheral sensitization almost always depends on local inflammation , which may lead to decreased nociceptor thresholds . despite extensive investigations , no tissue pathology , structural abnormalities , or evidence for a source of chronic stimulation of pain afferents this has led to the hypothesis that the central nervous system is hyperexcitable in these patients . central hypersensitivity could explain exaggerated pain in the presence of minimal and undetectable tissue damage , in that the nociceptive signal is amplified by the hyperexcitable neurons . pain measurements studies in patients with chronic pain after whiplash injury and with fm have demonstrated exaggerated pain responses after sensory stimulation of healthy tissues [ 55 , 5863 ] . for example , at same level of thermal stimulation , fm patients perceived pain as 49 and 52% more intense than healthy controls and patients with low - back pain . after - sensations at 15 s after heat taps were regarded as painful on 83% of fm patients , compared to 37% pain reports of healthy controls . the late after - sensations ( 2 min after heat stimulation ) were rated as painful in 55 and 5% of fm and control subjects , respectively . it was not mentioned in the results if all fm patients were subject to increased sensitivity , but there were always significant differences between the fm patients and the healthy controls.although most comparisons were made to pain - free subjects , some researchers , like julien et al . , even compared the pain responses of fm patients to other pain patients , such as patients with chronic low - back pain . they could also report significant differences . despite the lack of research into the contribution of psychological factors in wind - up , these results suggest that input to central nociceptive pathways is abnormally processed in patients with fm . intramuscular electrical stimulation has been used to assess the efficacy of temporal summation of painful muscle stimuli . temporal stimulation was found to be more pronounced and to cause stronger pain and larger referred areas in fm patients compared to controls .the increased efficacy of temporal summation in fm has even so been reproduced with cutaneous heat stimulation and with cold and heat taps . facilitated temporal summation in patients with pain suggests that the efficacy of central processing is increased ( central sensitization ) in these patients . in addition , after - sensation was greater in magnitude , lasted longer , and was more frequently painful in patients with fm . the prolonged decay together with the augmentation provides evidence for the presence of central sensitization [ 24 , 25 ] . immersion of the arm in circulating noxious cold water resulted in a 49% more intense pain in fm patients compared to healthy controls . . found that fibromyalgic patients experienced stronger pain and larger referred areas after intramuscular injection of hypertonic saline.moreover , spatial summation effect during increase of the stimulation area was found only in patients with fm and not in healthy controls or in patients with chronic low - back pain . fm patients perceived pain at the same intensities and unpleasantness during the ascending and the descending sessions ( fingertip to shoulder and shoulder to fingertip immersion ) . in healthy controls and patients with low - back pain , the noxious stimulation of a large surface area results in an activation of a large population of nociceptive afferents that induce endogenous inhibitory responses , resulting in a decreased response in the dorsal horn neurons , and subsequently leads to lower pain intensities afterward.given that fm patients experience similar pain intensities in the descending session after noxious stimulation of the whole arm , this study clearly demonstrated that fm patients present with a lack of activation of endogenous inhibitory systems . secondly , lautenbacher and rollman showed that tonic painful and non - painful thermal stimulation of the foot increased the pain thresholds to electrical stimulation applied to a non - tender point ( inner forearm ) in healthy controls but had no effect on patients with fm . kosek and hansson found that tourniquet ischemic pain in an arm increased the pressure pain threshold in healthy controls but not in fm patients , suggesting once more a deficiency in the latter of a pain - inhibitory phenomenon termed diffuse noxious inhibitory control similarly , aerobic exercise has been shown to decrease wind - up pain in normal subjects but increased it in fm patients , suggesting the possibility of reduced endogenous analgesic mechanisms . also , isometric exercise ( i.e. , hand - grip exercise ) resulted in increased thermal pain ratings and decreased pain thresholds , both ipsilateral and contralateral to the exercised extremity . these investigations support a general hypothesis that fm reflects a disorder affecting modulation of pain sensitivity [ 66 , 67 ] . in one such scenario , tonic dnic is present in the normal situation , and its pathological absence results in the spontaneous pain and evoked pain sensitivity associated with fm . measurements of excitability the results of the above - mentioned studies , however , are based on pain reports of the patients and thus subjective in nature , and it was not clear whether this hypersensitivity was the result of central mechanisms or whether the hypersensitivity was the cause of hypervigilance . could provide objective evidence by quantifying the minimal intensity of transcutaneous electrical stimulation of the sural nerve required to evoke flexion reflex in the biceps femoris . this study clearly demonstrates that spinal cord neurons are sensitized in chronic pain after whiplash and in patients with fm ; because the stimuli were delivered at random time intervals and the latency of emg response was measured , voluntary symptom amplification could be ruled out . cognitive emotional sensitization patients with fm or other pain disorders often receive the message that it is all in their head . one construct that has been hypothesized to explain the pain amplification in fm is that of hypervigilance . also , crombez et al . reported significant correlations between hypervigilance and pain intensity in fm patients . furthermore , exposure to stressful situations , including noise , lights , and weather , is known to exacerbate symptoms of fm , including pain .in addition , fm patients with catastrophic thoughts report increased pain intensities [ 7375 ] . found catastrophizing ( 27% of the variance ) and depression ( 30% of the variance ) to be significant predictors of pain . finally , kinesiophobia and fear of pain are related to pain severity in patients with fm .the foregoing relations between emotions or cognitions and reported pain severity support the hypothesis of cognitive emotional sensitization in fm . central abnormalities in fm fm patients differ from healthy persons in regional cerebral blood flow ( rcbf ) distribution in several brain structures involved in pain processing and pain modulation both at rest and during experimental pain induction . patients with fm exhibited significantly lower rcbf levels , during rest , in the thalamus and the caudate nucleus . dysregulation of thalamic activity and hypoperfusion of the caudate nucleus may contribute to the abnormal pain modulation , given the results of previous investigations [ 7779 ] . in addition to nociceptive transmission , the thalamus also plays an important role in pain modulation . animal studies proved that thalamic stimulation induces analgesia and lesions of the thalamus cause hyperalgesia [ 77 , 78].similarly , stimulating the caudate nucleus decreases pain behavior . during pain induction in patients with fm , the absence of significant thalamic activation and a bilateral activation of the somatosensory cortices and the right anterior cingulated cortex was seen . the patterns of cortical activation may be characteristic of patients with allodynia , and pain - induced activation of the right anterior cingulated cortex is associated with the use of maladaptive coping strategies . patients recruited by bradley et al . indeed reported significantly greater use of emotion - focused coping strategies ( e.g. , praying and hoping ) during pressure stimulation . in addition , increased right frontal brain activity seemed related to increased pain sensitivity .furthermore , fm patients are characterized by relatively high cerebrospinal fluid levels of substance p . this finding indicates that abnormal brain activity in persons with fm is associated with abnormal csf levels of a neuropeptide involved in pain transmission . in addition , it is shown that a subgroup of fm patients present with mycoplasma infections . infection triggers the release of the pro - inflammatory cytokine interleukin-1 , which is known to play a major role in inducing cyclooxygenase-2 ( cox-2 ) and prostaglandin e2 expression in the central nervous system .peripheral nerve terminals can be sensitized by elevated cox-2 amounts and prostaglandin e2 . peripheral infections are even able to activate spinal cord glia , leading to the release of no and proinflammatory cytokines , enhancing the pain response . physiological symptoms , such as pain , can be explained by these mechanisms ( sickness response ) . but the present knowledge concerning cfs is suggestive of a central process similar to that seen in fm , given the great overlap between the two diseases and the observed similarities . first , lower pain thresholds at different sites ( hyperalgesia ) are reported in patients with cfs , compared to controls [ 86 , 87 ] . similar to fm , the lack of peripheral tissue damage and the lack of a distinct localization of the pain complaints are suggestive of a central abnormality responsible for the chronic widespread pain . to our knowledge , there are no investigations that focused on abnormal wind - up , temporal summation , or spatial summation in cfs , to collect evidence for central sensitization in cfs . yet , evidence of a dysfunctional central anti - nociceptive mechanism in cfs has been proved by whiteside et al . . they reported a decrease of pain threshold in patients with cfs after graded exercise , while healthy controls present an increased pain threshold . these findings are similar to those of vierck et al . in fm patients . secondly , the frequently reported opportunistic infections [ 89 , 90 ] may lead to the sickness response and complies with the central sensitization hypothesis , as explained above . as earlier mentioned , no plays an important role in the history of central sensitization and , therefore , central sensitization caused by no would be likely in patients with cfs . the release of excessive amounts of substance p in the csf , however , could not be documented in patients with cfs , in contrast to fm patients . the cfs patients in this study , however , did not experience chronic widespread pain , and it is not clear if they fulfilled the 1994 cdc criteria . given that 70% of the cfs patients do fulfill the acr criteria for widespread pain , it is striking that the patients included in the investigation of evengard et al . further research on that matter should analyze the csf of cfs patients ( fulfilling the fukuda criteria ) suffering from chronic widespread pain . a third important argument in the central sensitization theory for cfs concerns the cognitive , psychological , and behavioral changes in patients diagnosed with cfs . cfs patients often present with depression [ 6 , 92 ] , catastrophizing [ 93 , 94 ] , somatization [ 95 , 96 ] , and kinesiophobia or fear avoidance [ 9799 ] . in cfs , it is known that these psychosocial aspects are important factors in maintaining the complaints of cfs . these cognitions and emotions are able to influence pain perception via modulation of the descending pathways . have earlier been mentioned to be associated with the amplification and the extension of pain [ 4952 ] . finally , brain imaging already provided evidence for altered brain activity in cfs . patterns of functional brain activity in patients with fm are quite different from those in patients with cfs . patients with cfs , relative to controls , showed significantly lower blood perfusion in the brain stem [ 17 , 18 ] . patients with fm exhibited significantly lower rcbf levels , during rest , in the thalamus and the caudate nucleus . however , the areas are different in fm and in cfs ; both the affected areas could be related to central pain processing . low brain stem rcbf levels may contribute to abnormal function of the locus ceruleus in patients with cfs . the locus ceruleus is involved in controlling descending anti - nociceptive pathways from the brain to the spinal dorsal horn . in consequence , pain experiences of patients with cfs may be related to low resting state levels of functional activity in the brain stem . central sensitization is known as an increased central neuronal responsiveness and causes hyperalgesia , allodynia , and referred pain and hyperalgesia across multiple spinal segments , leading to chronic widespread pain . possible triggers for sensitization of the spinal cord have extensively been discussed , such as wind - up or temporal summation , dysregulated descending inhibitory pathways , and upregulated facilitatory modulation . wind - up or temporal summation is the result of repetitive noxious stimuli , leading to an increase in electrical discharges in the dorsal horn . inhibitory modulation can be impaired by abnormalities in the central nervous system and the facilitatory pain pathways can be stimulated by certain behavioral and cognitive factors . this theoretical background can be applied to fm . in fm , studies already provided evidence for central sensitization as the cause of chronic pain . temporal summation was found to be more facilitated , and the inhibitory pain modulation seemed impaired in fm patients . furthermore , some central abnormalities could be examined / objectified in fm : 1 ) hyperexcitability of the spinal cord , 2 ) decreased perfusion of pain - related brain structures , and 3 ) high levels of substance p in csf . in addition , fm patients often present with pain hypervigilance , maladaptive coping strategies , and catastrophic thoughts , leading to cognitive central sensitization . based on the knowledge on central sensitization , on fm and on cfs , it is suggested that chronic widespread pain in cfs is the consequence of central sensitization . also , in other chronic pain populations , central sensitization may play a key role . in fact , there are many similarities between cfs patients and other chronic pain populations such as patients with chronic low - back pain , whiplash , fm , etc . the psychosocial factors , for example , have been proved to contribute to pain perception in these different pain populations . but the specific nature of cfs such as the immunological abnormalities , elevated no amounts , preceding infections etc . , invites further research , in particular , on the possible contributory role of these abnormalities to pain processing in cfs . in fm , many researches have been conducted to prove the theory of central sensitization . in cfs , however , it sticks to supposing . to give a scientific basis to the theory , the protocols applied in fm investigations could be used for patients with cfs . it would , for example , be interesting to test the efficacy of temporal summation in cfs . the influence of exercise on pain tolerance has already been studied in cfs , however , on a relatively small sample . on the contrary , spatial summation has , to our knowledge , never been investigated in cfs . furthermore , the role of depression , hypervigilance , kinesiophobia , catastrophising , etc . on chronic pain in cfs requires further research . to obtain more direct and objective information on central sensitization , the protocol described by banic et al . could be used to test the sensitivity of the central nervous system . clearly , there are many possible research areas to test the hypothesis , but there is still a long way to go to elucidate the nature of the chronic pain in cfs .

in addition to the debilitating fatigue , the majority of patients with chronic fatigue syndrome ( cfs ) experience chronic widespread pain . these pain complaints show the greatest overlap between cfs and fibromyalgia ( fm ) . although the literature provides evidence for central sensitization as cause for the musculoskeletal pain in fm , in cfs this evidence is currently lacking , despite the observed similarities in both diseases . the knowledge concerning the physiological mechanism of central sensitization , the pathophysiology and the pain processing in fm , and the knowledge on the pathophysiology of cfs lead to the hypothesis that central sensitization is also responsible for the sustaining pain complaints in cfs . this hypothesis is based on the hyperalgesia and allodynia reported in cfs , on the elevated concentrations of nitric oxide presented in the blood of cfs patients , on the typical personality styles seen in cfs and on the brain abnormalities shown on brain images . to examine the present hypothesis more research is required . further investigations could use similar protocols to those already used in studies on pain in fm like , for example , studies on temporal summation , spatial summation , the role of psychosocial aspects in chronic pain , etc .

Introduction Central sensitization Evidence in FM Central sensitization in CFS? Conclusion

chronic fatigue syndrome ( cfs ) , as defined by the centers for disease control and prevention ( cdcp ) , is a complex illness characterized by prolonged debilitating fatigue and multiple non - specific symptoms including headaches , recurrent sore throats , fever , muscle and joint pain , and neurocognitive complaints [ 1 , 2 ] . in addition to the chronic fatigue , widespread and persistent pain is common in individuals with cfs [ 35 ] . in another study , 24 of 44 patients suffered from chronic widespread pain . chronic fatigue accompanied by chronic musculoskeletal impairments such as myalgias and arthralgias could be considered an important subclass of cfs . evidence supportive of the clinical importance of widespread pain in cfs has been provided : chronic pain accounts for up to 34% of the cfs patients self - reported activity limitations and participation restrictions . chronic pain is more disabling than chronic fatigue . given these facts , it may be surprising that the etiology of these pain complaints has not been studied extensively in patients with cfs . shows that only little progress has been made in understanding chronic widespread pain in patients with cfs . in contrast , a large body of scientific literature regarding the etiology of chronic pain complaints in fibromyalgia ( fm ) is currently available . the diagnosis of fm is based on the 1990 american college of rheumatology criteria . following these criteria , fm patients present with 11 of 18 positive tender points and with widespread pain . validity of both the definition for cfs and fm has been shown [ 12 , 13 ] . especially , investigations focusing on the phenomenon central sensitization are presented in force in fm . in cfs , given the great overlap between cfs and fm and given the dearth of studies focusing on the explanation for the chronic widespread pain in patients with cfs , it would be interesting to propose a theoretical model for the chronic pain in cfs based on the current knowledge of cfs and on the evidence for central sensitization in fm , giving rise to further research on that matter . besides the knowledge on chronic pain in fm , it is necessary to gather knowledge on musculoskeletal pain in cfs . for example , immunological dysregulations such as the abnormal 25a synthetase / rnase l pathway have been revealed in cfs but have never been detected in fm patients . furthermore , there is not yet any good evidence for similar pain mechanisms in cfs and fm . for example , patterns of functional brain activity in patients with fm are quite different from those in patients with cfs . patients with cfs , relative to controls , showed significantly lower blood perfusion in the brain stem [ 17 , 18 ] . patients with fm exhibited significantly lower rcbf levels , during rest , in the thalamus and the caudate nucleus . therefore , the knowledge on pain in fm can not be applied on cfs patients without further study . based on the similarities and differences between the two syndromes , further research on pain in cfs is advised to get an image of pain processing in the two diseases . first , the concept of central sensitisation as a cause of chronic pain will be explained . this theoretical background will then be applied to fm and an overview of the evidence for central sensitization in fm will follow . finally , based on the theoretical background and the findings in fm , the hypothesis concerning central sensitization in cfs will be unfolded , supported with the present knowledge on cfs . these include low - threshold nociceptors that are connected to fast conducting a - delta pain fibers , and high - threshold nociceptors that conduct impulses in slow ( unmyelinated ) c fibers . are able to modulate the postsynaptic responses with further transmission to supraspinal sites ( thalamus , anterior cingulated cortex , insular cortex , and somatosensory cortex ) via the ascending pathways [ 22 , 24 , 25].the simplest form of plasticity in nervous systems is that repeated noxious stimulation may lead to habituation ( decreased response ) or sensitization ( increased response ) . prolonged or strong activity of dorsal horn neurons caused by repeated or sustained noxious stimulation may subsequently lead to increased neuronal responsiveness or central sensitization [ 25 , 27 ] . in 1965 , animal experiments showed for the first time that repetitive c - fiber stimulation could result in a progressive increase of electrical discharges from the second - order neuron in the spinal cord . this mechanism of pain amplification in the spinal cord is related to temporal summation of second pain or wind - up . second pain , which is more dull and strongly related to chronic pain states , is transmitted through unmyelinated c fibers to dorsal horn nociceptive neurons . calcium entry into sensory neurons in the dorsal horn induces activation of nitric oxide ( no ) synthase , leading to the synthesis of no . no can affect the nociceptor terminals and enhance the release of sensory neuropeptides ( in particular , substance p ) from presynaptic neurons , therefore contributing to the development of hyperalgesia and maintenance of central sensitization . it lowers the threshold of synaptic excitability , resulting in the unmasking of normally silent interspinal synapses and the sensitization of second - order spinal neurons .furthermore , sp can extend for long distances in the spinal cord and sensitize dorsal horn neurons at a distance from the initial input locus . this results in an expansion of receptive fields and the activation of wide dynamic neurons by non - nociceptive afferent impulses .wind - up can be elicited in human patients if identical nociceptive stimuli are applied to the skin or muscles more often than once every 3 s. the resulting progressive increase of pain sensations represents wind - up and has been demonstrated to result from a central rather than a peripheral nervous system mechanism , because the input from c nociceptors has been shown to decline or stay the same with stimulus repetition . endogenous pain modulatory systems the presence of several pain inhibitory and facilitatory centers in the brainstem is well recognized . experimental evidence for the existence of descending inhibitory pathways , and their connection with central sensitization , include the observations that bilateral lesions of the dorsolateral funiculus in the rat led to a significant decrease in latency for paw withdrawal to noxious stimulus . hypersensitivity .the foregoing investigations suggest that disruption of one or more of the elements of the inhibitory system can result in , among other things , the equivalent of central sensitization . the activity in descending pathways is not constant but can be modulated , for example , by the level of vigilance or attention and by stress . forebrain products such as cognitions , emotions , attention , and motivation have influence on the clinical pain experience .dubner and ren rewarded subjects for responding to a randomly delivered transient tissue threatening peripheral stimulus . it was found that sensitization of second - order pain pathway neurons was directly related to the strength of attention . moreover , behavioral modulation associated with selective attention to a perceived threat utilizes the same forebrain and brainstem structures and mechanisms as are involved in the development , amplification , and maintenance of persistent pain after actual tissue damage and inflammation .certain cognitive styles and personality traits have been associated with amplification of pain and its extension in the absence of tissue damage . theoretically , peripheral mechanisms can account for the pain hypersensitivity . in fm , however , there is no evidence for peripheral sensitization as the cause of hyperalgesia , given the absence of real tissue damage . peripheral sensitization is defined as a reduction in the threshold of nociceptive afferent receptors caused by a local change in the sensitivity of sensory fibers initiated by tissue damage . despite extensive investigations , no tissue pathology , structural abnormalities , or evidence for a source of chronic stimulation of pain afferents this has led to the hypothesis that the central nervous system is hyperexcitable in these patients . central hypersensitivity could explain exaggerated pain in the presence of minimal and undetectable tissue damage , in that the nociceptive signal is amplified by the hyperexcitable neurons . pain measurements studies in patients with chronic pain after whiplash injury and with fm have demonstrated exaggerated pain responses after sensory stimulation of healthy tissues [ 55 , 5863 ] . for example , at same level of thermal stimulation , fm patients perceived pain as 49 and 52% more intense than healthy controls and patients with low - back pain . it was not mentioned in the results if all fm patients were subject to increased sensitivity , but there were always significant differences between the fm patients and the healthy controls.although most comparisons were made to pain - free subjects , some researchers , like julien et al . , even compared the pain responses of fm patients to other pain patients , such as patients with chronic low - back pain . despite the lack of research into the contribution of psychological factors in wind - up , these results suggest that input to central nociceptive pathways is abnormally processed in patients with fm . temporal stimulation was found to be more pronounced and to cause stronger pain and larger referred areas in fm patients compared to controls .the increased efficacy of temporal summation in fm has even so been reproduced with cutaneous heat stimulation and with cold and heat taps . facilitated temporal summation in patients with pain suggests that the efficacy of central processing is increased ( central sensitization ) in these patients . in addition , after - sensation was greater in magnitude , lasted longer , and was more frequently painful in patients with fm . the prolonged decay together with the augmentation provides evidence for the presence of central sensitization [ 24 , 25 ] . immersion of the arm in circulating noxious cold water resulted in a 49% more intense pain in fm patients compared to healthy controls . found that fibromyalgic patients experienced stronger pain and larger referred areas after intramuscular injection of hypertonic saline.moreover , spatial summation effect during increase of the stimulation area was found only in patients with fm and not in healthy controls or in patients with chronic low - back pain . in healthy controls and patients with low - back pain , the noxious stimulation of a large surface area results in an activation of a large population of nociceptive afferents that induce endogenous inhibitory responses , resulting in a decreased response in the dorsal horn neurons , and subsequently leads to lower pain intensities afterward.given that fm patients experience similar pain intensities in the descending session after noxious stimulation of the whole arm , this study clearly demonstrated that fm patients present with a lack of activation of endogenous inhibitory systems . secondly , lautenbacher and rollman showed that tonic painful and non - painful thermal stimulation of the foot increased the pain thresholds to electrical stimulation applied to a non - tender point ( inner forearm ) in healthy controls but had no effect on patients with fm . kosek and hansson found that tourniquet ischemic pain in an arm increased the pressure pain threshold in healthy controls but not in fm patients , suggesting once more a deficiency in the latter of a pain - inhibitory phenomenon termed diffuse noxious inhibitory control similarly , aerobic exercise has been shown to decrease wind - up pain in normal subjects but increased it in fm patients , suggesting the possibility of reduced endogenous analgesic mechanisms . , hand - grip exercise ) resulted in increased thermal pain ratings and decreased pain thresholds , both ipsilateral and contralateral to the exercised extremity . in one such scenario , tonic dnic is present in the normal situation , and its pathological absence results in the spontaneous pain and evoked pain sensitivity associated with fm . measurements of excitability the results of the above - mentioned studies , however , are based on pain reports of the patients and thus subjective in nature , and it was not clear whether this hypersensitivity was the result of central mechanisms or whether the hypersensitivity was the cause of hypervigilance . this study clearly demonstrates that spinal cord neurons are sensitized in chronic pain after whiplash and in patients with fm ; because the stimuli were delivered at random time intervals and the latency of emg response was measured , voluntary symptom amplification could be ruled out . one construct that has been hypothesized to explain the pain amplification in fm is that of hypervigilance . furthermore , exposure to stressful situations , including noise , lights , and weather , is known to exacerbate symptoms of fm , including pain .in addition , fm patients with catastrophic thoughts report increased pain intensities [ 7375 ] . finally , kinesiophobia and fear of pain are related to pain severity in patients with fm .the foregoing relations between emotions or cognitions and reported pain severity support the hypothesis of cognitive emotional sensitization in fm . central abnormalities in fm fm patients differ from healthy persons in regional cerebral blood flow ( rcbf ) distribution in several brain structures involved in pain processing and pain modulation both at rest and during experimental pain induction . patients with fm exhibited significantly lower rcbf levels , during rest , in the thalamus and the caudate nucleus . in addition to nociceptive transmission , the thalamus also plays an important role in pain modulation . during pain induction in patients with fm , the absence of significant thalamic activation and a bilateral activation of the somatosensory cortices and the right anterior cingulated cortex was seen . the patterns of cortical activation may be characteristic of patients with allodynia , and pain - induced activation of the right anterior cingulated cortex is associated with the use of maladaptive coping strategies . in addition , it is shown that a subgroup of fm patients present with mycoplasma infections . peripheral infections are even able to activate spinal cord glia , leading to the release of no and proinflammatory cytokines , enhancing the pain response . but the present knowledge concerning cfs is suggestive of a central process similar to that seen in fm , given the great overlap between the two diseases and the observed similarities . first , lower pain thresholds at different sites ( hyperalgesia ) are reported in patients with cfs , compared to controls [ 86 , 87 ] . similar to fm , the lack of peripheral tissue damage and the lack of a distinct localization of the pain complaints are suggestive of a central abnormality responsible for the chronic widespread pain . to our knowledge , there are no investigations that focused on abnormal wind - up , temporal summation , or spatial summation in cfs , to collect evidence for central sensitization in cfs . they reported a decrease of pain threshold in patients with cfs after graded exercise , while healthy controls present an increased pain threshold . secondly , the frequently reported opportunistic infections [ 89 , 90 ] may lead to the sickness response and complies with the central sensitization hypothesis , as explained above . as earlier mentioned , no plays an important role in the history of central sensitization and , therefore , central sensitization caused by no would be likely in patients with cfs . the release of excessive amounts of substance p in the csf , however , could not be documented in patients with cfs , in contrast to fm patients . the cfs patients in this study , however , did not experience chronic widespread pain , and it is not clear if they fulfilled the 1994 cdc criteria . given that 70% of the cfs patients do fulfill the acr criteria for widespread pain , it is striking that the patients included in the investigation of evengard et al . further research on that matter should analyze the csf of cfs patients ( fulfilling the fukuda criteria ) suffering from chronic widespread pain . a third important argument in the central sensitization theory for cfs concerns the cognitive , psychological , and behavioral changes in patients diagnosed with cfs . cfs patients often present with depression [ 6 , 92 ] , catastrophizing [ 93 , 94 ] , somatization [ 95 , 96 ] , and kinesiophobia or fear avoidance [ 9799 ] . in cfs , it is known that these psychosocial aspects are important factors in maintaining the complaints of cfs . finally , brain imaging already provided evidence for altered brain activity in cfs . patterns of functional brain activity in patients with fm are quite different from those in patients with cfs . patients with cfs , relative to controls , showed significantly lower blood perfusion in the brain stem [ 17 , 18 ] . patients with fm exhibited significantly lower rcbf levels , during rest , in the thalamus and the caudate nucleus . however , the areas are different in fm and in cfs ; both the affected areas could be related to central pain processing . the locus ceruleus is involved in controlling descending anti - nociceptive pathways from the brain to the spinal dorsal horn . in consequence , pain experiences of patients with cfs may be related to low resting state levels of functional activity in the brain stem . central sensitization is known as an increased central neuronal responsiveness and causes hyperalgesia , allodynia , and referred pain and hyperalgesia across multiple spinal segments , leading to chronic widespread pain . possible triggers for sensitization of the spinal cord have extensively been discussed , such as wind - up or temporal summation , dysregulated descending inhibitory pathways , and upregulated facilitatory modulation . wind - up or temporal summation is the result of repetitive noxious stimuli , leading to an increase in electrical discharges in the dorsal horn . inhibitory modulation can be impaired by abnormalities in the central nervous system and the facilitatory pain pathways can be stimulated by certain behavioral and cognitive factors . in fm , studies already provided evidence for central sensitization as the cause of chronic pain . temporal summation was found to be more facilitated , and the inhibitory pain modulation seemed impaired in fm patients . furthermore , some central abnormalities could be examined / objectified in fm : 1 ) hyperexcitability of the spinal cord , 2 ) decreased perfusion of pain - related brain structures , and 3 ) high levels of substance p in csf . in addition , fm patients often present with pain hypervigilance , maladaptive coping strategies , and catastrophic thoughts , leading to cognitive central sensitization . based on the knowledge on central sensitization , on fm and on cfs , it is suggested that chronic widespread pain in cfs is the consequence of central sensitization . also , in other chronic pain populations , central sensitization may play a key role . in fact , there are many similarities between cfs patients and other chronic pain populations such as patients with chronic low - back pain , whiplash , fm , etc . the psychosocial factors , for example , have been proved to contribute to pain perception in these different pain populations . , invites further research , in particular , on the possible contributory role of these abnormalities to pain processing in cfs . in fm , many researches have been conducted to prove the theory of central sensitization . in cfs , however , it sticks to supposing . to give a scientific basis to the theory , the protocols applied in fm investigations could be used for patients with cfs . it would , for example , be interesting to test the efficacy of temporal summation in cfs . the influence of exercise on pain tolerance has already been studied in cfs , however , on a relatively small sample . on the contrary , spatial summation has , to our knowledge , never been investigated in cfs . furthermore , the role of depression , hypervigilance , kinesiophobia , catastrophising , etc . on chronic pain in cfs requires further research . to obtain more direct and objective information on central sensitization , the protocol described by banic et al . clearly , there are many possible research areas to test the hypothesis , but there is still a long way to go to elucidate the nature of the chronic pain in cfs .

its significance was reinforced with the launch , in 2003 , of the supporting people programme , which put housing related support at the centre of the government 's strategy to enable vulnerable people to live independently . the supporting people programme is a working partnership between local government and other statutory and non - statutory agencies . this article presents some of the findings from our evaluation of six pilots , which was designed to explore how organisations work across boundaries . we highlight what helps and what hinders joint working across agencies and sectors , at both a strategic and operational level , and emphasise the important role that voluntary sector agencies can play in supporting people with complex needs . housing was recognised as a cornerstone of social care in england in the 1990 , nhs and community care act . attention initially focused on housing adaptation and the development of independent living as a means of helping specific groups of people older people and people with learning disabilities to live independently in the community . over time a more holistic notion of housing support has developed . housing support is provided to a wide range of people including young parents and ex - offenders , and its importance to adult social care was identified in the 2005 green paper . although the relationship between housing and health is well - established , the structural links between housing services and the nhs have historically been poor . recent moves towards a more explicit public health agenda have , however , been mirrored in public policy , with a clear role set out for housing and housing support . for example , easterlow and smith suggest that contemporary public health policy focuses on promoting healthy public policies , inter - sectoral alliances and community care , all of which have housing at their core . the white paper our health , our care , our say notes that , wherever possible , people should have the option to stay in their own homes, and that this may necessitate the provision of intensive support at home allowing more people to continue to live in their own homes for longer . the programme was introduced in england in 2003 as a means of facilitating independent living in the community for groups that require low - intensity support and also for those that are socially excluded , at risk or hard to reach through existing service provision . its broad aim is to provide housing - related support to help people to stay in their own homes or to move towards having their own homes , to increase independence and the capacity for self - care . the programme reflects wider policy aims associated with preventive action , tackling social exclusion , co - ordinating services around the needs of individuals , promoting choice and increasing the role of the voluntary sector . the supporting people programme was launched by the then office of the deputy prime minister in april 2003 and is managed by local authorities . it is designed to be delivered through a working partnership of local government , health services , voluntary sector organisations , probation , housing associations and support agencies . together these organisations commission services that will enable vulnerable people to develop and sustain their capacity to live independently . supporting people services , by their very nature , although the partnership ethos is central to the programme , the relationship between health care and supporting people services has never been considered strong . indeed there has been a perception amongst policy makers that health planners and practitioners were not engaged in local supporting people partnerships , perhaps because they did not understand its relevance to the health agenda . to remedy this the office of the deputy prime minister announced its intentions to establish the supporting people health pilot programme in the summer of 2003 , with the intention of demonstrating : the policy links between supporting people and health and social care , the potential benefits from collaboration and how practitioners and agencies could work together to support the health needs of particularly vulnerable groups . policy reform has focused on : enabling the statutory sector to pool budgets and jointly commission services ; establishing new models of provision as a way of ameliorating some of the difficulties of working across organisational boundaries ( for example the creation of care trusts ) , and encouraging the development of integrated services . recent reforms have encouraged greater contribution from the voluntary sector ensuring that partnerships reach out beyond the statutory sector . there is a rich vein of research focusing on joint working between health and social care services [ 1317 ] , that highlights a number of difficulties associated with organisational , cultural and professional and , contextual issues . although the literature on joint working between housing and health is less well - developed , similar difficulties have been noted [ 3 , 1821 ] . kodner and spreeuwenberg ( 2002 ) defined integration as a set of methods and models of organisational service delivery designed to create connectivity and collaboration within and between different sectors , with the aim of enhancing the quality of life for people with chronic or complex problems . this conceptualisation mirrors the basic aims of the supporting people health pilot programme , which sought to co - ordinate services across housing , health and social care boundaries as a means to support vulnerable people to live independently in the community . it is the experiences of those involved in these endeavours that the article aims to explore . housing was recognised as a cornerstone of social care in england in the 1990 , nhs and community care act . attention initially focused on housing adaptation and the development of independent living as a means of helping specific groups of people older people and people with learning disabilities to live independently in the community . over time a more holistic notion of housing support has developed . housing support is provided to a wide range of people including young parents and ex - offenders , and its importance to adult social care was identified in the 2005 green paper . although the relationship between housing and health is well - established , the structural links between housing services and the nhs have historically been poor . recent moves towards a more explicit public health agenda have , however , been mirrored in public policy , with a clear role set out for housing and housing support . for example , easterlow and smith suggest that contemporary public health policy focuses on promoting healthy public policies , inter - sectoral alliances and community care , all of which have housing at their core . the white paper our health , our care , our say notes that , wherever possible , people should have the option to stay in their own homes, and that this may necessitate the provision of intensive support at home allowing more people to continue to live in their own homes for longer . the programme was introduced in england in 2003 as a means of facilitating independent living in the community for groups that require low - intensity support and also for those that are socially excluded , at risk or hard to reach through existing service provision . its broad aim is to provide housing - related support to help people to stay in their own homes or to move towards having their own homes , to increase independence and the capacity for self - care . the programme reflects wider policy aims associated with preventive action , tackling social exclusion , co - ordinating services around the needs of individuals , promoting choice and increasing the role of the voluntary sector . the supporting people programme was launched by the then office of the deputy prime minister in april 2003 and is managed by local authorities . it is designed to be delivered through a working partnership of local government , health services , voluntary sector organisations , probation , housing associations and support agencies . together these organisations commission services that will enable vulnerable people to develop and sustain their capacity to live independently . supporting people services , by their very nature , although the partnership ethos is central to the programme , the relationship between health care and supporting people services has never been considered strong . indeed there has been a perception amongst policy makers that health planners and practitioners were not engaged in local supporting people partnerships , perhaps because they did not understand its relevance to the health agenda . to remedy this the office of the deputy prime minister announced its intentions to establish the supporting people health pilot programme in the summer of 2003 , with the intention of demonstrating : the policy links between supporting people and health and social care , the potential benefits from collaboration and how practitioners and agencies could work together to support the health needs of particularly vulnerable groups . policy reform has focused on : enabling the statutory sector to pool budgets and jointly commission services ; establishing new models of provision as a way of ameliorating some of the difficulties of working across organisational boundaries ( for example the creation of care trusts ) , and encouraging the development of integrated services . recent reforms have encouraged greater contribution from the voluntary sector ensuring that partnerships reach out beyond the statutory sector . there is a rich vein of research focusing on joint working between health and social care services [ 1317 ] , that highlights a number of difficulties associated with organisational , cultural and professional and , contextual issues . although the literature on joint working between housing and health is less well - developed , similar difficulties have been noted [ 3 , 1821 ] . kodner and spreeuwenberg ( 2002 ) defined integration as a set of methods and models of organisational service delivery designed to create connectivity and collaboration within and between different sectors , with the aim of enhancing the quality of life for people with chronic or complex problems . this conceptualisation mirrors the basic aims of the supporting people health pilot programme , which sought to co - ordinate services across housing , health and social care boundaries as a means to support vulnerable people to live independently in the community . it is the experiences of those involved in these endeavours that the article aims to explore . the office of the deputy prime minister received bids from 122 partnerships in england wishing to be health pilots . the six pilots selected by the office of the deputy prime minister represented a wide range of people who use supporting people services and a range of agencies from the statutory , independent and voluntary sectors . as a precondition of funding each pilot was required to identify aims and objectives , which would demonstrate how the new service could support national health targets . evaluating a diverse range of projects presents a series of challenges . the approach adopted sought to understand both the process and outcome of the intervention as a means to determine what works , for whom and in what circumstances . the evaluation sought to identify common themes and issues as the basis of an overall evaluation of the initiative per se , but took a tailored approach to the evaluation of each individual project . first , that the views of all key stakeholders , including those using services , were important . secondly , that as far as possible relevant data should be collected on as contemporaneous a basis as possible . two main sources of data collection were used : quarterly project evaluation reports from each pilot and semi - structured interviews with key stakeholders , including people who use services . the project reports provided data about process and implementation as well as reporting progress against aims and objectives related to health target(s ) which pilots had been required to identify . each pilot was visited on three occasions : at their inception ; mid way and towards the end of the initiative . during these visits we conducted interviews with between six and eight interviewees across all key professional stakeholder groups , including commissioners , managers of services , representatives of partner agencies as well as project workers . interviewees were selected on the basis that they were involved in the development of the pilot and/or that they were centrally involved in the work of the pilot . interviewees were asked their views on whether or not the pilot was achieving its aims and objectives and to describe the factors that supported or hindered efforts to work across housing , health and social care boundaries . the interviews allowed us to explore the development of the health pilots from a range of perspectives . interviews were transcribed and analysed thematically , emerging themes were discussed within the research team and , as a means to increase the saliency of the analysis , we checked the authenticity of our findings with representatives of the pilots at regular workshops . in this way we were able to identify trends that are generalisable across the pilot sites and beyond . in the following sections we discuss the themes emerging from the data , highlighting the challenges entailed in providing integrated care across housing and health and social care services . previous research has consistently emphasised the importance of agencies understanding the basic aims of their joint effort . they illustrate that joint working was most effective when professionals not only understood the aims and objectives of what they were doing , but appreciated the fundamental role of housing as a means to support the individual people they worked with . in essence , dry exhortation to work in partnership was an insufficient catalyst ; the potential benefits of joint working had to be apparent to individual professionals through the experience of the people they worked with . for example , the idea behind the on - track pilot came from professionals working in local health and voluntary services who identified a gap in services for young people with dual diagnosis ( people with mental health and substance misuse needs ) . falling through the net , being passed between youth to adult services and between drug and mental health services . professionals working in different sectors recognised the need for stable housing and intensive support in order to engage young people with dual diagnosis in relevant health care services . they shared an understanding that only rarely could they achieve their own organisational aims and objectives when working in isolation . the pilot was therefore designed to establish a housing related support service that would help young people to engage with appropriate health services . the aim , as one partner described it , was to provide practical help to get a house and then look at their mental health . if housing needs are n't addressed it is unlikely they will address mental health needs . you need to understand that for people with mental health problems everything is connected . to address mental health you have to address housing . if housing needs are n't addressed it is unlikely they will address mental health needs . you need to understand that for people with mental health problems everything is connected . to address mental health you have to address housing . professionals , including front line staff and senior managers , saw the on - track pilot as a positive response to problems identified by those working directly with young people , rather than a service initiated at a strategic level . consequently , it enjoyed the backing of a wide range of agencies that were willing to invest time , energy and resources to establishing and supporting the new service . as one health partner commented we are working together , we are committed to it , we have common goals , common aims and common wins , it 's not simply about achieving outcomes it is about overcoming cultural barriers . we are working together , we are committed to it , we have common goals , common aims and common wins , it 's not simply about achieving outcomes it is about overcoming cultural barriers . this level of understanding about the central role of housing was not initially evident across all pilots . for example , the place to live pilot was inspired by the valuing people white paper , which calls for people with learning difficulties to be given more control over where they chose to live . although originating from discussions within a multi - disciplinary group , the original proposal was written by social care professionals with little or no involvement from community nursing staff whose participation was crucial to ensuring that individuals moving into supported housing had access to appropriate health care service . as one social worker reflected : things should have been done differently during the planning stage ; we needed to take more people on board , it was important for everyone to own the project . things should have been done differently during the planning stage ; we needed to take more people on board , it was important for everyone to own the project . as well as lacking a shared understanding of what the pilot was trying to achieve , few community nurses appeared to recognise the role of housing to well - being . for example , one health professional described how social workers and community nurses had very different ideas about what constituted a health need and what constituted a social need. as a result few referrals were initially made by community nurses for housing assessments for the people they supported . place to live pilot also illustrated that staff working in allied services , particularly the homelessness unit , neither appreciated the housing rights of people with learning disabilities nor understood the links between housing and well - being . four of the six pilots identified the need for staff working in homelessness units or hostels to have training about the housing and support needs of vulnerable people . for example , housing department staff associated with the on - track pilot did not appreciate the need to house young people with dual diagnosis away from housing estates known to have a drug problem . training resulted in improved working between these agencies and also improved the support they provided to specific individuals . however , in keeping with previous research this raises serious questions about the role of generic housing officers who , within the current policy framework , have to work with a range of individuals with complex lives and health problems , which have a major impact on their ability to live independently . inevitably this requires housing officers to work more closely with other services but without specific knowledge of the needs of different groups , or a specialist service to provide this support , this type of co - ordination may prove difficult to achieve . for example , at a strategic level the housing support outreach and referral pilot evolved from a long history of partnership working in the field of hiv services between two london boroughs . not only had the boroughs previously commissioned hiv services together but they had also worked in partnership with the primary care trust , which commissioned voluntary sector services . this approach was generally regarded as an effective way of addressing a complex problem . as a result the pilot did not have to spend time developing an ethos of joint working from scratch. the two boroughs commissioned the terrence higgins trust / lighthouse to develop the housing support service . although the organisation had no experience of providing housing support services they had an established record of providing a range of services to people living with hiv and a wealth of experience of working with acute health care providers . i know ( the ) terrence higgins trust , i know their roles , that helps , we expect realistic and appropriate things from each other . i know ( the ) terrence higgins trust , i know their roles , that helps , we expect realistic and appropriate things from each other . the pilot was , therefore , developed within a context that valued joint working as a means to support people living with hiv and benefited from a history of collaboration amongst the central partners . the swan nest pilot also profited from a history of joint working , building on the success of the swan programme , a multi agency initiative originally developed to address community safety issues . as well as providing accommodation and floating tenancy support , pilot staff also assisted women to engage with health services as part of a strategy to exit sex work . all of the agencies involved recognised that they could only set up the scheme by working in partnership and that joint working had been made easier because as one partner reflected , they had had a positive experience of joint working in the past , we trust each other , you will deliver because you have in the past . positive experience of joint working in the past , we trust each other , you will deliver because you have in the past . this shared history , however , was based on individuals who had worked together over a long period of time . reorganisation of statutory and non - statutory agencies was a constant threat and partners recognised that changes in personnel could unsteady the partnership . consequently no one took the partnership for granted and partners were willing to invest considerable time and energy into the development and subsequent management of the new service . one of the key themes to emerge from the evaluation was the need for joint working to be based on clear arrangements in respect of governance and managerial responsibility , both at strategic and operational levels . not only does this help establish the democratic accountability of partnerships but transparent arrangements , agreed by all partners , ensure that staff understand to whom they are accountable and enable the work to be managed effectively . however , as previous research indicates , insufficient thought is often given to the complexity of managing complex initiatives . for example , at several pilots the difficulties of sharing information across organisational boundaries were not resolved because it was not clear where responsibility for this lay . the governance arrangements for the swan nest pilot provide a model for how these complex processes can be made straightforward . progress was reported to the existing swan partnership steering group , which met bi - monthly and comprised of senior representatives from the main partner agencies , including the borough council , the primary care trust , the general practice and the specialist housing support agency . additionally , because the primary care trust held the contract for the pilot with the office of the deputy prime minister , the assistant director for public health reported progress to the primary care trust board . these arrangements ensured not only that one individual took overall responsibility for the pilot but also that there was an effective forum in which to tackle problems across the partnership . the pilot also had an operational manager who provided the link between strategic and front line working . the pilots also demonstrated that operational staff working across organisational boundaries need specialist supervision , as well as managerial supervision . this became evident early on , specifically in pilots that were working with people with particularly complex needs and chaotic life styles such as sex workers and homeless people with hiv . staff at these pilots had to work intensively with individuals in order to link them into a variety of general and specialist health services and other agencies such as housing and probation . not only did this require them to have a detailed knowledge of a range of services it also required them to have an understanding of how best to support individual clients . through the provision of specialist supervision , pilots were able to ensure that the practice of individual workers was effective and safe . it also provided them with time to off load and reflect on the difficult nature of the work they were doing . for example the housing support outreach and referral pilot held regular team meetings , sometimes on a daily basis depending on the cases they were covering . as one of the workers described it , we catch up every day , we are always talking cases over , it 's a tight team . we catch up every day , we are always talking cases over , it 's a tight team . the pilot also used professional supervision to ensure that practice met the expectations of terrence higgins trust / lighthouse . these monthly sessions usually lasted for over an hour and gave the workers an opportunity to deal with the demanding nature of their work . both project workers valued these sessions , as one said you need a channel to off - load . a couple of our clients have tried to commit suicide so it is good to have supervision . a couple of our clients have tried to commit suicide so it is good to have supervision . the on - track pilot , for example , did not initially establish formal supervision systems with the result that project workers were anxious about the appropriateness of the support they were providing . these systems play a crucial role in maintaining professional standards and as more integrated services develop agencies will need to provide matrix supervision systems that allow workers to receive professional support for the different elements of their work . after several months the on - track pilot ensured that workers had access to supervision from housing managers as well as from specialist drug agencies . whilst these systems are already common place in many voluntary sector agencies it will be important to ensure that as the sector begins to play a bigger role in the provision of support to vulnerable groups these systems are in place in all agencies . one way to do so would be for commissioners of services to require these arrangements as a contractual condition . 9.1 in this evaluation , effective joint working ( defined as the pilots ability to achieve its aims and objectives ) was associated not only with an understanding of the purpose of the joint venture , a history of joint working , and clear and effective governance arrangements , but on two other characteristics : the extent and nature of statutory sector participation and whether or not the service is defined , by a history of voluntary sector involvement . those pilots working in service areas with little or no tradition of statutory sector provision for example with sex workers , or where services have developed more recently ( hiv services)appeared to have less difficulty working across organisational or professional boundaries . the hiv sector , for example , has a strong ethos of partnership working across the statutory and voluntary sectors , which appears to lend itself towards a more flexible approach to supporting vulnerable people . as one health partner from the housing support outreach and referral pilot commented , all partners were committed to providing a service for the client group that cuts through the inter - agency bureaucracy and rivalry . committed to providing a service for the client group that cuts through the inter - agency bureaucracy and rivalry . in contrast , although the core partners in pilots working in the fields of learning disabilities and older people services displayed a high level of commitment to joint working , this did not always appear to be as widespread within the agencies concerned . for example , the place to live pilot was based in an integrated team but the different professional groups were not co - located , nor did they meet together on a regular basis or share an understanding of the relationship between housing and well - being . all of these factors are associated with successful joint working and without them , the pilot struggled initially to develop an ethos of joint working . ironically , although there is a long history of community nurses and social workers working in the field of learning disability and , more recently in integrated teams , this seems to have led to the emergence of strong professional boundaries that may inhibit multi - professional working . as one social worker reflected in integrated teams people can get precious about their roles . the pilots demonstrate the important contribution that the voluntary sector can make in supporting vulnerable people to live independently in the community . the involvement of the voluntary sector brought additional credibility to the work of several pilots . as well as harnessing the expertise that exists within the voluntary sector , pilots were able to draw on their networks . for example , the involvement of the terrence higgins trust in the housing support , outreach and referral pilot provided extra credibility because the trust was well known amongst people with hiv and brought with it an extended network of voluntary services that the trust was involved with . consequently the service was able to refer people to a wide range of voluntary organisations , such as community transport services and charitable food projects . as previous research has found it is support with these practical tasks that helps enable service users to live independently . the development of new services in the voluntary sector also provided powerful models of how services could be provided . for example , a health professional involved in the on - track pilot remarked that the pilot had demonstrated how , the traditional ways of providing services from the statutory sector are n't always the best and that other providers can do it more successfully . the traditional ways of providing services from the statutory sector are n't always the best and that other providers can do it more successfully . importantly those pilots that were based outside of the statutory sector demonstrated a high degree of flexibility in the way in which they supported vulnerable people to live independently . pilots worked intensively with individuals to identify what they wanted to address in order to live independently and supported them to achieve the goals they set for themselves . this person - centred approach helped people engage and maintain engagement with services , which they had often failed to do in the past and echoes findings from previous research which suggests flexible and person - centred housing support services are more likely to offer an effective solution to social exclusion . the appropriateness of this approach was alluded to in interviews conducted with people who used services . for example one person supported by the housing support outreach and referral pilot commented i have taken life more seriously now , she ( the project worker ) accompanies me to the alcohol centre and checks how i am doing . i have taken life more seriously now , she ( the project worker ) accompanies me to the alcohol centre and checks how i am doing . similarly another person supported by the pilot described how someone ( the project worker ) is keeping me on track with my appointments . as a result this pilot , like on - track and swan nest , was based within the voluntary sector and suggests that some people , particularly those with chaotic and complex lives , may find it easier to engage with , and remain engaged with voluntary sector services . this may be because they are not based around statutory professions such as social work and community nursing , or because of their enhanced flexibility and responsiveness . this is relevant to current debates regarding the role of the voluntary sector within welfare services . the involvement of voluntary sector agencies in the health pilots was not without difficulty particularly with regard to how professionals from different sectors work together . several pilot workers reported that statutory sector colleagues were reluctant to work in partnership with them . for example , despite the tradition of cross sector working within hiv services some tensions were reported from the housing support outreach and referral pilot . these were most notable amongst staff working in a homeless persons unit who were initially reluctant to identify a link worker between the pilot and the unit . this reluctance appeared to be based on a perception that voluntary sector organisations were not as professional as statutory services . statutory services see us as do - gooders , they do n't see us as a professional service or as an equal . these professional rivalries were most notable when pilots tried to establish effective ways of sharing information with colleagues in the statutory sector . these systems are particularly important when people have complex needs and chaotic lifestyles . in these circumstances services need to be co - ordinated in a timely manner and based on up - to - date information . initially some statutory sector agencies were reported to be unwilling to share information with colleagues in the non - statutory sector . to resolve this problem several pilots , including swan nest and on - track built on local practice , for example adapting existing release of information forms which service users were asked to sign as proof that they had agreed to the pilot contacting other agencies as a means to seek or share relevant information . occasionally the authenticity of these forms became the focus of disagreement between agencies . whilst these disagreements were always resolved they reflect what secker and hill have referred to as a reluctance or structural inability to share information and a lack of clarity about the constraints of confidentiality [ 32 , p 348 ] . these disputes support the need for national guidance and protocols , which would remove the potential for disagreement and would smooth the path of those working at the front line . previous research has consistently emphasised the importance of agencies understanding the basic aims of their joint effort . they illustrate that joint working was most effective when professionals not only understood the aims and objectives of what they were doing , but appreciated the fundamental role of housing as a means to support the individual people they worked with . in essence , dry exhortation to work in partnership was an insufficient catalyst ; the potential benefits of joint working had to be apparent to individual professionals through the experience of the people they worked with . for example , the idea behind the on - track pilot came from professionals working in local health and voluntary services who identified a gap in services for young people with dual diagnosis ( people with mental health and substance misuse needs ) . falling through the net , being passed between youth to adult services and between drug and mental health services . professionals working in different sectors recognised the need for stable housing and intensive support in order to engage young people with dual diagnosis in relevant health care services . they shared an understanding that only rarely could they achieve their own organisational aims and objectives when working in isolation . the pilot was therefore designed to establish a housing related support service that would help young people to engage with appropriate health services . the aim , as one partner described it , was to provide practical help to get a house and then look at their mental health . if housing needs are n't addressed it is unlikely they will address mental health needs . you need to understand that for people with mental health problems everything is connected . to address mental health you have to address housing . if housing needs are n't addressed it is unlikely they will address mental health needs . you need to understand that for people with mental health problems everything is connected . to address mental health you have to address housing . professionals , including front line staff and senior managers , saw the on - track pilot as a positive response to problems identified by those working directly with young people , rather than a service initiated at a strategic level . consequently , it enjoyed the backing of a wide range of agencies that were willing to invest time , energy and resources to establishing and supporting the new service . as one health partner commented we are working together , we are committed to it , we have common goals , common aims and common wins , it 's not simply about achieving outcomes it is about overcoming cultural barriers . we are working together , we are committed to it , we have common goals , common aims and common wins , it 's not simply about achieving outcomes it is about overcoming cultural barriers . this level of understanding about the central role of housing was not initially evident across all pilots . for example , the place to live pilot was inspired by the valuing people white paper , which calls for people with learning difficulties to be given more control over where they chose to live . although originating from discussions within a multi - disciplinary group , the original proposal was written by social care professionals with little or no involvement from community nursing staff whose participation was crucial to ensuring that individuals moving into supported housing had access to appropriate health care service . as one social worker reflected : things should have been done differently during the planning stage ; we needed to take more people on board , it was important for everyone to own the project . things should have been done differently during the planning stage ; we needed to take more people on board , it was important for everyone to own the project . as well as lacking a shared understanding of what the pilot was trying to achieve , few community nurses appeared to recognise the role of housing to well - being . for example , one health professional described how social workers and community nurses had very different ideas about what constituted a few referrals were initially made by community nurses for housing assessments for the people they supported . the place to live pilot also illustrated that staff working in allied services , particularly the homelessness unit , neither appreciated the housing rights of people with learning disabilities nor understood the links between housing and well - being . four of the six pilots identified the need for staff working in homelessness units or hostels to have training about the housing and support needs of vulnerable people . for example , housing department staff associated with the on - track pilot did not appreciate the need to house young people with dual diagnosis away from housing estates known to have a drug problem . training resulted in improved working between these agencies and also improved the support they provided to specific individuals . however , in keeping with previous research this raises serious questions about the role of generic housing officers who , within the current policy framework , have to work with a range of individuals with complex lives and health problems , which have a major impact on their ability to live independently . inevitably this requires housing officers to work more closely with other services but without specific knowledge of the needs of different groups , or a specialist service to provide this support , this type of co - ordination may prove difficult to achieve . for example , at a strategic level the housing support outreach and referral pilot evolved from a long history of partnership working in the field of hiv services between two london boroughs . not only had the boroughs previously commissioned hiv services together but they had also worked in partnership with the primary care trust , which commissioned voluntary sector services . this approach was generally regarded as an effective way of addressing a complex problem . as a result the pilot did not have to spend time developing an ethos of joint working from scratch. the two boroughs commissioned the terrence higgins trust / lighthouse to develop the housing support service . although the organisation had no experience of providing housing support services they had an established record of providing a range of services to people living with hiv and a wealth of experience of working with acute health care providers . i know ( the ) terrence higgins trust , i know their roles , that helps , we expect realistic and appropriate things from each other . i know ( the ) terrence higgins trust , i know their roles , that helps , we expect realistic and appropriate things from each other . the pilot was , therefore , developed within a context that valued joint working as a means to support people living with hiv and benefited from a history of collaboration amongst the central partners . the swan nest pilot also profited from a history of joint working , building on the success of the swan programme , a multi agency initiative originally developed to address community safety issues . as well as providing accommodation and floating tenancy support , pilot staff also assisted women to engage with health services as part of a strategy to exit sex work . all of the agencies involved recognised that they could only set up the scheme by working in partnership and that joint working had been made easier because as one partner reflected , they had had a positive experience of joint working in the past , we trust each other , you will deliver because you have in the past . positive experience of joint working in the past , we trust each other , you will deliver because you have in the past . this shared history , however , was based on individuals who had worked together over a long period of time . reorganisation of statutory and non - statutory agencies was a constant threat and partners recognised that changes in personnel could unsteady the partnership . consequently no one took the partnership for granted and partners were willing to invest considerable time and energy into the development and subsequent management of the new service . one of the key themes to emerge from the evaluation was the need for joint working to be based on clear arrangements in respect of governance and managerial responsibility , both at strategic and operational levels . not only does this help establish the democratic accountability of partnerships but transparent arrangements , agreed by all partners , ensure that staff understand to whom they are accountable and enable the work to be managed effectively . however , as previous research indicates , insufficient thought is often given to the complexity of managing complex initiatives . for example , at several pilots the difficulties of sharing information across organisational boundaries were not resolved because it was not clear where responsibility for this lay . the governance arrangements for the swan nest pilot provide a model for how these complex processes can be made straightforward . progress was reported to the existing swan partnership steering group , which met bi - monthly and comprised of senior representatives from the main partner agencies , including the borough council , the primary care trust , the general practice and the specialist housing support agency . additionally , because the primary care trust held the contract for the pilot with the office of the deputy prime minister , the assistant director for public health reported progress to the primary care trust board . these arrangements ensured not only that one individual took overall responsibility for the pilot but also that there was an effective forum in which to tackle problems across the partnership . the pilot also had an operational manager who provided the link between strategic and front line working . the pilots also demonstrated that operational staff working across organisational boundaries need specialist supervision , as well as managerial supervision . this became evident early on , specifically in pilots that were working with people with particularly complex needs and chaotic life styles such as sex workers and homeless people with hiv . staff at these pilots had to work intensively with individuals in order to link them into a variety of general and specialist health services and other agencies such as housing and probation . not only did this require them to have a detailed knowledge of a range of services it also required them to have an understanding of how best to support individual clients . through the provision of specialist supervision , pilots were able to ensure that the practice of individual workers was effective and safe . it also provided them with time to off load and reflect on the difficult nature of the work they were doing . for example the housing support outreach and referral pilot held regular team meetings , sometimes on a daily basis depending on the cases they were covering . as one of the workers described it , we catch up every day , we are always talking cases over , it 's a tight team . we catch up every day , we are always talking cases over , it 's a tight team . the pilot also used professional supervision to ensure that practice met the expectations of terrence higgins trust / lighthouse . these monthly sessions usually lasted for over an hour and gave the workers an opportunity to deal with the demanding nature of their work . both project workers valued these sessions , as one said a couple of our clients have tried to commit suicide so it is good to have supervision . a couple of our clients have tried to commit suicide so it is good to have supervision . the on - track pilot , for example , did not initially establish formal supervision systems with the result that project workers were anxious about the appropriateness of the support they were providing . these systems play a crucial role in maintaining professional standards and as more integrated services develop agencies will need to provide matrix supervision systems that allow workers to receive professional support for the different elements of their work . after several months the on - track pilot ensured that workers had access to supervision from housing managers as well as from specialist drug agencies . whilst these systems are already common place in many voluntary sector agencies it will be important to ensure that as the sector begins to play a bigger role in the provision of support to vulnerable groups these systems are in place in all agencies . one way to do so would be for commissioners of services to require these arrangements as a contractual condition . 9.1 in this evaluation , effective joint working ( defined as the pilots ability to achieve its aims and objectives ) was associated not only with an understanding of the purpose of the joint venture , a history of joint working , and clear and effective governance arrangements , but on two other characteristics : the extent and nature of statutory sector participation and whether or not the service is defined , by a history of voluntary sector involvement . those pilots working in service areas with little or no tradition of statutory sector provision for example with sex workers , or where services have developed more recently ( hiv services)appeared to have less difficulty working across organisational or professional boundaries . the hiv sector , for example , has a strong ethos of partnership working across the statutory and voluntary sectors , which appears to lend itself towards a more flexible approach to supporting vulnerable people . as one health partner from the housing support outreach and referral pilot commented , all partners were committed to providing a service for the client group that cuts through the inter - agency bureaucracy and rivalry . committed to providing a service for the client group that cuts through the inter - agency bureaucracy and rivalry . in contrast , although the core partners in pilots working in the fields of learning disabilities and older people services displayed a high level of commitment to joint working , this did not always appear to be as widespread within the agencies concerned . for example , the place to live pilot was based in an integrated team but the different professional groups were not co - located , nor did they meet together on a regular basis or share an understanding of the relationship between housing and well - being . all of these factors are associated with successful joint working and without them , the pilot struggled initially to develop an ethos of joint working . ironically , although there is a long history of community nurses and social workers working in the field of learning disability and , more recently in integrated teams , this seems to have led to the emergence of strong professional boundaries that may inhibit multi - professional working . as one social worker reflected in integrated teams people can get precious about their roles . the pilots demonstrate the important contribution that the voluntary sector can make in supporting vulnerable people to live independently in the community . the involvement of the voluntary sector brought additional credibility to the work of several pilots . as well as harnessing the expertise that exists within the voluntary sector , pilots were able to draw on their networks . for example , the involvement of the terrence higgins trust in the housing support , outreach and referral pilot provided extra credibility because the trust was well known amongst people with hiv and brought with it an extended network of voluntary services that the trust was involved with . consequently the service was able to refer people to a wide range of voluntary organisations , such as community transport services and charitable food projects . as previous research has found it is support with these practical tasks that helps enable service users to live independently . the development of new services in the voluntary sector also provided powerful models of how services could be provided . for example , a health professional involved in the on - track pilot remarked that the pilot had demonstrated how , the traditional ways of providing services from the statutory sector are n't always the best and that other providers can do it more successfully . the traditional ways of providing services from the statutory sector are n't always the best and that other providers can do it more successfully . importantly those pilots that were based outside of the statutory sector demonstrated a high degree of flexibility in the way in which they supported vulnerable people to live independently . pilots worked intensively with individuals to identify what they wanted to address in order to live independently and supported them to achieve the goals they set for themselves . this person - centred approach helped people engage and maintain engagement with services , which they had often failed to do in the past and echoes findings from previous research which suggests flexible and person - centred housing support services are more likely to offer an effective solution to social exclusion . the appropriateness of this approach was alluded to in interviews conducted with people who used services . for example one person supported by the housing support outreach and referral pilot commented i have taken life more seriously now , she ( the project worker ) accompanies me to the alcohol centre and checks how i am doing . i have taken life more seriously now , she ( the project worker ) accompanies me to the alcohol centre and checks how i am doing . similarly another person supported by the pilot described how someone ( the project worker ) is keeping me on track with my appointments . as a result this pilot , like on - track and swan nest , was based within the voluntary sector and suggests that some people , particularly those with chaotic and complex lives , may find it easier to engage with , and remain engaged with voluntary sector services . this may be because they are not based around statutory professions such as social work and community nursing , or because of their enhanced flexibility and responsiveness . this is relevant to current debates regarding the role of the voluntary sector within welfare services . the involvement of voluntary sector agencies in the health pilots was not without difficulty particularly with regard to how professionals from different sectors work together . several pilot workers reported that statutory sector colleagues were reluctant to work in partnership with them . for example , despite the tradition of cross sector working within hiv services some tensions were reported from the housing support outreach and referral pilot . these were most notable amongst staff working in a homeless persons unit who were initially reluctant to identify a link worker between the pilot and the unit . this reluctance appeared to be based on a perception that voluntary sector organisations were not as professional as statutory services . statutory services see us as do - gooders , they do n't see us as a professional service or as an equal . these professional rivalries were most notable when pilots tried to establish effective ways of sharing information with colleagues in the statutory sector . these systems are particularly important when people have complex needs and chaotic lifestyles . in these circumstances services need to be co - ordinated in a timely manner and based on up - to - date information . initially some statutory sector agencies were reported to be unwilling to share information with colleagues in the non - statutory sector . to resolve this problem several pilots , including swan nest and on - track built on local practice , for example adapting existing release of information forms which service users were asked to sign as proof that they had agreed to the pilot contacting other agencies as a means to seek or share relevant information . occasionally the authenticity of these forms became the focus of disagreement between agencies . whilst these disagreements were always resolved they reflect what secker and hill have referred to as a reluctance or structural inability to share information and a lack of clarity about the constraints of confidentiality [ 32 , p 348 ] . these disputes support the need for national guidance and protocols , which would remove the potential for disagreement and would smooth the path of those working at the front line . the supporting people health pilot evaluation provided an opportunity to explore joint working between health , social care and housing across a number of different contexts . consequently the evaluation was able to highlight themes that help explain why joint working is successful in relation to some service areas and not others . they suggest for example that whilst some agencies and professionals understood the central role of housing to health and well - being and were willing to work across organisational boundaries to support people to live independently other pilots found it difficult to develop a shared purpose . the place to live pilot established within an integrated learning disability team struggled initially to move beyond what secker and hill have termed rigid demarcations and role boundary conflicts . the provision of training eventually helped to build a sense of common purpose between the different professional groups and improved the co - ordination of work within the team . however , the difficulties suggest that the processes of integration will not in itself remove the historical boundaries between professions and improve joint working . the evaluation drew attention to the impact of voluntary sector involvement in the creation of integrated services to work with people with complex needs . importantly the findings suggest that establishing the pilots in the voluntary sector meant the new services were not constrained by statutory sector models of provision and consequently were more able to respond flexibly to the needs of individuals rather than being controlled by a professional agenda and organisational priorities . whilst current rhetoric emphasises the need for services to focus on the individual circumstances of service - users rather than conforming to a however , in this study those pilots based in the voluntary sector appeared to be less burdened by complex bureaucratic structures and more able than those in the statutory sector to build services around individual need . basing new services in the voluntary sector also brought additional credibility to new services , particularly if it was a national or locally recognised agency and allowed access to networks and expertise that exists outside of the statutory sector . the experience of the pilots suggests that some people , particularly those with complex needs may find it easier to engage with a service primarily because it is based in the voluntary sector . indeed some people find voluntary sector services more accessible because they were not based around statutory professions whilst others need the enhanced responsiveness that they perceive in the voluntary sector . these findings reflect kodner and spreeuwenberg 's assertion that the logic of integration should be determined by the characteristics of specific groups rather than existing organisational structures . the on - track , swan nest and housing support and outreach and referral pilots were not imposed as prescriptive models of joint working . rather they were established because professionals , as well as their managers , identified a gap in provision for these specific groups or recognised existing services were failing to address the complex housing and support needs of these individuals . however , achieving the greater flexibility required in integrated services necessitates changes in strategic and operational management and lines of accountability and the development of a more whole systems way of thinking about service delivery . whilst some pilots were able to develop clear and effective governance arrangements that crossed agency and sector boundaries these developments inevitably pose challenges . for example unequal relationships between statutory commissioners and voluntary provider organisations may undermine the spirit of openness that is required for agencies to develop truly person - centred services and therefore , may have an adverse effect on the ability to form partnerships . indeed hudson suggests that recent reforms to the commissioning process may impede the effectiveness of whole systems working . additionally , there is a danger that the regulation and inspection regime required by commissioners , and new labour more broadly , may restrict the ability of voluntary sector agencies to work flexibly . the supporting people health pilots were established to encourage greater involvement of health and social care professionals in supporting people partnerships , as well as demonstrating the potential benefits to health and social care from joint working . the pilots demonstrate how housing support services can be developed to enable vulnerable people to live independently in the community . they illustrate how agencies and professionals can work across organisational boundaries , ensuring greater access to a wider range of health care services for particularly marginalised groups . the pilots demonstrate that integrating services to support people with complex needs works best when the service is determined by the characteristics of those who use the service rather than pre - existing organisational structures . two new themes emerged which help explain why agencies work together more effectively in some services than in others : the degree to which statutory services are involved in the service and the involvement of voluntary sector agencies . these findings have resonance for broader policy agenda beyond supporting people , particularly recent calls to increase the role of the voluntary sector in health and social care services . , rbs centre for the older person 's agenda , queen margaret university , edinburgh , united kingdom . michael fine , phd , associate professor , department of sociology , deputy director , caroline glendinning , professor , social policy research unit , university of york , united kingdom .

purposethis paper reports the findings of the evaluation of the supporting people health pilots programme , which was established to demonstrate the policy links between housing support services and health and social care services by encouraging the development of integrated services . the paper highlights the challenges of working across housing , health and social care boundaries.methodthe evaluation of the six health pilots rested on two main sources of data collection : quarterly project evaluation reports collected process data as well as reporting progress against aims and objectives . semi - structured interviews conducted across all key professional stakeholder groups and agencies and with people who used services explored their experiences of these new services.resultsthe ability of pilots to work across organisational boundaries to achieve their aims and objectives was associated not only with agencies sharing an understanding of the purpose of the joint venture , a history of joint working and clear and efficient governance arrangements but on two other characteristics : the extent and nature of statutory sector participation and , whether or not the service is defined by a history of voluntary sector involvement . in particular the pilots demonstrated how voluntary sector agencies appeared to be less constrained by organisational priorities and professional agenda and more able to respond flexibly to meet the complex needs of individuals.conclusion and discussionthe pilots demonstrate that integrating services to support people with complex needs works best when the service is determined by the characteristics of those who use the service rather than pre - existing organisational structures .

Introduction The policy context The joint working context The pilots Methodology Results Understanding the aims and objectives Having a history of joint working The management of inter-professional working The role of the voluntary sector Difficulties associated with working across statutory, non-statutory boundaries Discussion Conclusions Reviewers

its significance was reinforced with the launch , in 2003 , of the supporting people programme , which put housing related support at the centre of the government 's strategy to enable vulnerable people to live independently . this article presents some of the findings from our evaluation of six pilots , which was designed to explore how organisations work across boundaries . we highlight what helps and what hinders joint working across agencies and sectors , at both a strategic and operational level , and emphasise the important role that voluntary sector agencies can play in supporting people with complex needs . although the relationship between housing and health is well - established , the structural links between housing services and the nhs have historically been poor . the programme reflects wider policy aims associated with preventive action , tackling social exclusion , co - ordinating services around the needs of individuals , promoting choice and increasing the role of the voluntary sector . the supporting people programme was launched by the then office of the deputy prime minister in april 2003 and is managed by local authorities . it is designed to be delivered through a working partnership of local government , health services , voluntary sector organisations , probation , housing associations and support agencies . to remedy this the office of the deputy prime minister announced its intentions to establish the supporting people health pilot programme in the summer of 2003 , with the intention of demonstrating : the policy links between supporting people and health and social care , the potential benefits from collaboration and how practitioners and agencies could work together to support the health needs of particularly vulnerable groups . policy reform has focused on : enabling the statutory sector to pool budgets and jointly commission services ; establishing new models of provision as a way of ameliorating some of the difficulties of working across organisational boundaries ( for example the creation of care trusts ) , and encouraging the development of integrated services . there is a rich vein of research focusing on joint working between health and social care services [ 1317 ] , that highlights a number of difficulties associated with organisational , cultural and professional and , contextual issues . although the literature on joint working between housing and health is less well - developed , similar difficulties have been noted [ 3 , 1821 ] . this conceptualisation mirrors the basic aims of the supporting people health pilot programme , which sought to co - ordinate services across housing , health and social care boundaries as a means to support vulnerable people to live independently in the community . attention initially focused on housing adaptation and the development of independent living as a means of helping specific groups of people older people and people with learning disabilities to live independently in the community . although the relationship between housing and health is well - established , the structural links between housing services and the nhs have historically been poor . the programme reflects wider policy aims associated with preventive action , tackling social exclusion , co - ordinating services around the needs of individuals , promoting choice and increasing the role of the voluntary sector . the supporting people programme was launched by the then office of the deputy prime minister in april 2003 and is managed by local authorities . to remedy this the office of the deputy prime minister announced its intentions to establish the supporting people health pilot programme in the summer of 2003 , with the intention of demonstrating : the policy links between supporting people and health and social care , the potential benefits from collaboration and how practitioners and agencies could work together to support the health needs of particularly vulnerable groups . policy reform has focused on : enabling the statutory sector to pool budgets and jointly commission services ; establishing new models of provision as a way of ameliorating some of the difficulties of working across organisational boundaries ( for example the creation of care trusts ) , and encouraging the development of integrated services . there is a rich vein of research focusing on joint working between health and social care services [ 1317 ] , that highlights a number of difficulties associated with organisational , cultural and professional and , contextual issues . this conceptualisation mirrors the basic aims of the supporting people health pilot programme , which sought to co - ordinate services across housing , health and social care boundaries as a means to support vulnerable people to live independently in the community . the office of the deputy prime minister received bids from 122 partnerships in england wishing to be health pilots . the six pilots selected by the office of the deputy prime minister represented a wide range of people who use supporting people services and a range of agencies from the statutory , independent and voluntary sectors . the evaluation sought to identify common themes and issues as the basis of an overall evaluation of the initiative per se , but took a tailored approach to the evaluation of each individual project . two main sources of data collection were used : quarterly project evaluation reports from each pilot and semi - structured interviews with key stakeholders , including people who use services . the project reports provided data about process and implementation as well as reporting progress against aims and objectives related to health target(s ) which pilots had been required to identify . during these visits we conducted interviews with between six and eight interviewees across all key professional stakeholder groups , including commissioners , managers of services , representatives of partner agencies as well as project workers . interviewees were selected on the basis that they were involved in the development of the pilot and/or that they were centrally involved in the work of the pilot . interviewees were asked their views on whether or not the pilot was achieving its aims and objectives and to describe the factors that supported or hindered efforts to work across housing , health and social care boundaries . the interviews allowed us to explore the development of the health pilots from a range of perspectives . interviews were transcribed and analysed thematically , emerging themes were discussed within the research team and , as a means to increase the saliency of the analysis , we checked the authenticity of our findings with representatives of the pilots at regular workshops . in the following sections we discuss the themes emerging from the data , highlighting the challenges entailed in providing integrated care across housing and health and social care services . they illustrate that joint working was most effective when professionals not only understood the aims and objectives of what they were doing , but appreciated the fundamental role of housing as a means to support the individual people they worked with . in essence , dry exhortation to work in partnership was an insufficient catalyst ; the potential benefits of joint working had to be apparent to individual professionals through the experience of the people they worked with . they shared an understanding that only rarely could they achieve their own organisational aims and objectives when working in isolation . for example , the place to live pilot was inspired by the valuing people white paper , which calls for people with learning difficulties to be given more control over where they chose to live . as well as lacking a shared understanding of what the pilot was trying to achieve , few community nurses appeared to recognise the role of housing to well - being . four of the six pilots identified the need for staff working in homelessness units or hostels to have training about the housing and support needs of vulnerable people . however , in keeping with previous research this raises serious questions about the role of generic housing officers who , within the current policy framework , have to work with a range of individuals with complex lives and health problems , which have a major impact on their ability to live independently . inevitably this requires housing officers to work more closely with other services but without specific knowledge of the needs of different groups , or a specialist service to provide this support , this type of co - ordination may prove difficult to achieve . not only had the boroughs previously commissioned hiv services together but they had also worked in partnership with the primary care trust , which commissioned voluntary sector services . although the organisation had no experience of providing housing support services they had an established record of providing a range of services to people living with hiv and a wealth of experience of working with acute health care providers . the pilot was , therefore , developed within a context that valued joint working as a means to support people living with hiv and benefited from a history of collaboration amongst the central partners . the swan nest pilot also profited from a history of joint working , building on the success of the swan programme , a multi agency initiative originally developed to address community safety issues . all of the agencies involved recognised that they could only set up the scheme by working in partnership and that joint working had been made easier because as one partner reflected , they had had a positive experience of joint working in the past , we trust each other , you will deliver because you have in the past . one of the key themes to emerge from the evaluation was the need for joint working to be based on clear arrangements in respect of governance and managerial responsibility , both at strategic and operational levels . the pilots also demonstrated that operational staff working across organisational boundaries need specialist supervision , as well as managerial supervision . this became evident early on , specifically in pilots that were working with people with particularly complex needs and chaotic life styles such as sex workers and homeless people with hiv . not only did this require them to have a detailed knowledge of a range of services it also required them to have an understanding of how best to support individual clients . 9.1 in this evaluation , effective joint working ( defined as the pilots ability to achieve its aims and objectives ) was associated not only with an understanding of the purpose of the joint venture , a history of joint working , and clear and effective governance arrangements , but on two other characteristics : the extent and nature of statutory sector participation and whether or not the service is defined , by a history of voluntary sector involvement . those pilots working in service areas with little or no tradition of statutory sector provision for example with sex workers , or where services have developed more recently ( hiv services)appeared to have less difficulty working across organisational or professional boundaries . for example , the place to live pilot was based in an integrated team but the different professional groups were not co - located , nor did they meet together on a regular basis or share an understanding of the relationship between housing and well - being . all of these factors are associated with successful joint working and without them , the pilot struggled initially to develop an ethos of joint working . ironically , although there is a long history of community nurses and social workers working in the field of learning disability and , more recently in integrated teams , this seems to have led to the emergence of strong professional boundaries that may inhibit multi - professional working . the pilots demonstrate the important contribution that the voluntary sector can make in supporting vulnerable people to live independently in the community . as well as harnessing the expertise that exists within the voluntary sector , pilots were able to draw on their networks . for example , the involvement of the terrence higgins trust in the housing support , outreach and referral pilot provided extra credibility because the trust was well known amongst people with hiv and brought with it an extended network of voluntary services that the trust was involved with . consequently the service was able to refer people to a wide range of voluntary organisations , such as community transport services and charitable food projects . the development of new services in the voluntary sector also provided powerful models of how services could be provided . for example , a health professional involved in the on - track pilot remarked that the pilot had demonstrated how , the traditional ways of providing services from the statutory sector are n't always the best and that other providers can do it more successfully . this person - centred approach helped people engage and maintain engagement with services , which they had often failed to do in the past and echoes findings from previous research which suggests flexible and person - centred housing support services are more likely to offer an effective solution to social exclusion . the appropriateness of this approach was alluded to in interviews conducted with people who used services . the involvement of voluntary sector agencies in the health pilots was not without difficulty particularly with regard to how professionals from different sectors work together . initially some statutory sector agencies were reported to be unwilling to share information with colleagues in the non - statutory sector . they illustrate that joint working was most effective when professionals not only understood the aims and objectives of what they were doing , but appreciated the fundamental role of housing as a means to support the individual people they worked with . in essence , dry exhortation to work in partnership was an insufficient catalyst ; the potential benefits of joint working had to be apparent to individual professionals through the experience of the people they worked with . they shared an understanding that only rarely could they achieve their own organisational aims and objectives when working in isolation . for example , the place to live pilot was inspired by the valuing people white paper , which calls for people with learning difficulties to be given more control over where they chose to live . as well as lacking a shared understanding of what the pilot was trying to achieve , few community nurses appeared to recognise the role of housing to well - being . however , in keeping with previous research this raises serious questions about the role of generic housing officers who , within the current policy framework , have to work with a range of individuals with complex lives and health problems , which have a major impact on their ability to live independently . inevitably this requires housing officers to work more closely with other services but without specific knowledge of the needs of different groups , or a specialist service to provide this support , this type of co - ordination may prove difficult to achieve . not only had the boroughs previously commissioned hiv services together but they had also worked in partnership with the primary care trust , which commissioned voluntary sector services . although the organisation had no experience of providing housing support services they had an established record of providing a range of services to people living with hiv and a wealth of experience of working with acute health care providers . the pilot was , therefore , developed within a context that valued joint working as a means to support people living with hiv and benefited from a history of collaboration amongst the central partners . the swan nest pilot also profited from a history of joint working , building on the success of the swan programme , a multi agency initiative originally developed to address community safety issues . one of the key themes to emerge from the evaluation was the need for joint working to be based on clear arrangements in respect of governance and managerial responsibility , both at strategic and operational levels . the pilots also demonstrated that operational staff working across organisational boundaries need specialist supervision , as well as managerial supervision . this became evident early on , specifically in pilots that were working with people with particularly complex needs and chaotic life styles such as sex workers and homeless people with hiv . not only did this require them to have a detailed knowledge of a range of services it also required them to have an understanding of how best to support individual clients . 9.1 in this evaluation , effective joint working ( defined as the pilots ability to achieve its aims and objectives ) was associated not only with an understanding of the purpose of the joint venture , a history of joint working , and clear and effective governance arrangements , but on two other characteristics : the extent and nature of statutory sector participation and whether or not the service is defined , by a history of voluntary sector involvement . those pilots working in service areas with little or no tradition of statutory sector provision for example with sex workers , or where services have developed more recently ( hiv services)appeared to have less difficulty working across organisational or professional boundaries . for example , the place to live pilot was based in an integrated team but the different professional groups were not co - located , nor did they meet together on a regular basis or share an understanding of the relationship between housing and well - being . all of these factors are associated with successful joint working and without them , the pilot struggled initially to develop an ethos of joint working . the pilots demonstrate the important contribution that the voluntary sector can make in supporting vulnerable people to live independently in the community . as well as harnessing the expertise that exists within the voluntary sector , pilots were able to draw on their networks . for example , the involvement of the terrence higgins trust in the housing support , outreach and referral pilot provided extra credibility because the trust was well known amongst people with hiv and brought with it an extended network of voluntary services that the trust was involved with . consequently the service was able to refer people to a wide range of voluntary organisations , such as community transport services and charitable food projects . the development of new services in the voluntary sector also provided powerful models of how services could be provided . for example , a health professional involved in the on - track pilot remarked that the pilot had demonstrated how , the traditional ways of providing services from the statutory sector are n't always the best and that other providers can do it more successfully . this person - centred approach helped people engage and maintain engagement with services , which they had often failed to do in the past and echoes findings from previous research which suggests flexible and person - centred housing support services are more likely to offer an effective solution to social exclusion . the appropriateness of this approach was alluded to in interviews conducted with people who used services . the involvement of voluntary sector agencies in the health pilots was not without difficulty particularly with regard to how professionals from different sectors work together . this reluctance appeared to be based on a perception that voluntary sector organisations were not as professional as statutory services . the supporting people health pilot evaluation provided an opportunity to explore joint working between health , social care and housing across a number of different contexts . they suggest for example that whilst some agencies and professionals understood the central role of housing to health and well - being and were willing to work across organisational boundaries to support people to live independently other pilots found it difficult to develop a shared purpose . the evaluation drew attention to the impact of voluntary sector involvement in the creation of integrated services to work with people with complex needs . importantly the findings suggest that establishing the pilots in the voluntary sector meant the new services were not constrained by statutory sector models of provision and consequently were more able to respond flexibly to the needs of individuals rather than being controlled by a professional agenda and organisational priorities . whilst current rhetoric emphasises the need for services to focus on the individual circumstances of service - users rather than conforming to a however , in this study those pilots based in the voluntary sector appeared to be less burdened by complex bureaucratic structures and more able than those in the statutory sector to build services around individual need . basing new services in the voluntary sector also brought additional credibility to new services , particularly if it was a national or locally recognised agency and allowed access to networks and expertise that exists outside of the statutory sector . the experience of the pilots suggests that some people , particularly those with complex needs may find it easier to engage with a service primarily because it is based in the voluntary sector . these findings reflect kodner and spreeuwenberg 's assertion that the logic of integration should be determined by the characteristics of specific groups rather than existing organisational structures . rather they were established because professionals , as well as their managers , identified a gap in provision for these specific groups or recognised existing services were failing to address the complex housing and support needs of these individuals . however , achieving the greater flexibility required in integrated services necessitates changes in strategic and operational management and lines of accountability and the development of a more whole systems way of thinking about service delivery . whilst some pilots were able to develop clear and effective governance arrangements that crossed agency and sector boundaries these developments inevitably pose challenges . additionally , there is a danger that the regulation and inspection regime required by commissioners , and new labour more broadly , may restrict the ability of voluntary sector agencies to work flexibly . the supporting people health pilots were established to encourage greater involvement of health and social care professionals in supporting people partnerships , as well as demonstrating the potential benefits to health and social care from joint working . the pilots demonstrate how housing support services can be developed to enable vulnerable people to live independently in the community . they illustrate how agencies and professionals can work across organisational boundaries , ensuring greater access to a wider range of health care services for particularly marginalised groups . the pilots demonstrate that integrating services to support people with complex needs works best when the service is determined by the characteristics of those who use the service rather than pre - existing organisational structures . two new themes emerged which help explain why agencies work together more effectively in some services than in others : the degree to which statutory services are involved in the service and the involvement of voluntary sector agencies . these findings have resonance for broader policy agenda beyond supporting people , particularly recent calls to increase the role of the voluntary sector in health and social care services .

the general design of the hunt study and the basis for the present analyses are illustrated in fig . 1 . hunt is a population - based study , conducted in the county of nord - trndelag in norway . three surveys were conducted from 1984 to 2008 . all inhabitants aged 20 years in the county the investigations carried out are presented in detail on the hunt website ( http://www.hunt.ntnu.no/index.php?side=english ) . in brief , in hunt1 , 19841986 , a total of 76,885 ( 90.3% ) of the eligible individuals participated . participants completed questionnaires on health and lifestyle and also underwent a clinical examination , including anthropometrical measurements ( 12 ) . in hunt2 , 19951997 , a total of 66,140 of 92,936 eligible individuals ( 71.2% ) participated . a total of 37,004 individuals constituting 56% of hunt2 participants and 40% of participants in hunt1 ( the present study is based on individuals who participated in at least two hunt surveys . our study population included 90,819 individuals , free of diabetes at baseline ( hunt1 or hunt2 ) with complete baseline information on smoking . the hunt study is approved by the norwegian data inspectorate and regional committee for medical research ethics . the participants provided informed consent . a chart presentation for the hunt study , 19842008 . each participant s record at hunt is linked to his or her exclusive 11-digit personal identification number , which allows linkage with other health registries . for the current study , we acquired date of death from the national mortality registry ( 13 ) . all with self - reported diabetes at hunt2 or hunt3 were given an appointment for fasting blood sampling . samples were analyzed for glucose , c - peptide , and antibodies to glutamic decarboxylase ( gada ) . patients aged 35 years at diagnosis of diabetes were classified as having type 2 diabetes if , in addition , they were gada - negative ( < 0.08 ) ( n = 1,860 ) . we further classified patients as having autoimmune diabetes if they were gada - positive ( 0.08 ) and were 35 years old at onset of diabetes ( n = 140 ) . as a further criterion for lada , we used information on insulin treatment ( available for 82% of the participants ) to separate lada from classical type 1 diabetes . individuals were classified as lada if they were gada - positive and did not receive insulin treatment during the first year after diagnosis of diabetes ( n = 100 ) . individuals were classified as having classical type 1 diabetes if insulin treatment was started during the first year after onset and gada was either positive or negative . in the cases of negative gada , a low level of c - peptide ( < 150 gada was analyzed at aker university hospital , oslo , norway , by a previously validated method ( 14 ) . the sensitivity and specificity of the assay at the cutoff level of > 0.08 were 0.64 and 1.00 , respectively , according to results obtained in the diabetes antibody standardization program . the cutoff index of 0.08 was equivalent to 43 world health organization units / ml ( 16 ) . c - peptide was measured by radioimmunoassay ( diagnostic system laboratories , webster , tx ) . fasting serum levels of glucose were measured by hemocue at the central laboratory of levanger hospital ( levanger , norway ) ( 13 ) . homeostasis model assessment for insulin resistance ( homa - ir ) and -cell function ( homa-%b ) were calculated using the updated model ( 17 ) . current smoking was defined as current daily smoking of at least 1 g of tobacco in the form of cigarettes , cigars , or pipe . current and former smokers were inquired at age of starting smoking , years of smoking , and average number of cigarettes smoked per day . the intensity of smoking among current and former smokers was assessed by two categories : light smokers ( < 20 cigarettes / day ) and heavy smokers ( 20 cigarettes / day ) . pack - years were calculated according to the formula : cigarettes per day/20 years smoked . cumulative quantity of active smoking was assessed in three categories ( < 6 , 612 , and 13 pack - years ) . p values for means and proportions were calculated using one - way anova and f test . we used cox proportional hazards models to estimate hazard ratios ( hr ) of type 2 diabetes and autoimmune diabetes in relation to smoking habits with 95% cis ( sas 9.2 ; sas institute inc , cary , nc ) . person - years of follow - up were calculated from the age that the participants entered the study ( hunt1 or hunt2 ) until age of onset of diabetes , death , or the end of the follow - up period at hunt2 or hunt3 ( 1997 or 2008 , respectively ) , whichever came first . confounders controlled for age ( in years , as underlying time scale in cox model ) , sex , and baseline information ( from either hunt1 or hunt2 depending on when the participant entered the study ) , bmi ( calculated as weight [ kg]/height [ m ] , continuous ) , education ( primary school , upper secondary school , or university ) , and physical activity [ physically active or inactive , categorization details are described elsewhere ( 18 ) ] , unless otherwise specified . additional adjustment for alcohol consumption and family history of diabetes did not change the results ( change in hr < 10% ) . the analyses were time - dependent , which means that for individuals with information on smoking ( or any of the covariates ) from more than one point in time ( i.e. , both hunt1 and hunt2 ) , information was updated during follow - up . in all analyses , we used never - smokers as a reference group , unless otherwise specified . for evaluating interaction between bmi and smoking habits , participants were categorized as : 1 ) bmi < 25 kg / m and never smoking ( ) ( as reference group in analyses ) , 2 ) bmi < 25 kg / m and heavy smoking ( + ) , 3 ) bmi 25 kg / m and never smoking ( + ) , and 4 ) bmi 25 kg / m and heavy smoking ( + + ) . to estimate additive interaction , we calculated the relative excess risk due to the interaction ( reri ) using reri = hr++ hr+ hr+ + 1 and attributable proportion ( ap ) due to interaction as ap = reri / hr++ ( 19 ) . correlation between smoking ( pack - years ) and homa indices ( loghoma2-ir and loghoma2-%b , logarithmic transformation was applied due to skewing of variables ) were assessed with pearson correlation coefficient . the general design of the hunt study and the basis for the present analyses are illustrated in fig . 1 . hunt is a population - based study , conducted in the county of nord - trndelag in norway . three surveys were conducted from 1984 to 2008 . all inhabitants aged 20 years in the county the investigations carried out are presented in detail on the hunt website ( http://www.hunt.ntnu.no/index.php?side=english ) . in brief , in hunt1 , 19841986 , a total of 76,885 ( 90.3% ) of the eligible individuals participated . participants completed questionnaires on health and lifestyle and also underwent a clinical examination , including anthropometrical measurements ( 12 ) . in hunt2 , 19951997 , a total of 66,140 of 92,936 eligible individuals ( 71.2% ) participated . a total of 37,004 individuals constituting 56% of hunt2 participants and 40% of participants in hunt1 ( the present study is based on individuals who participated in at least two hunt surveys . our study population included 90,819 individuals , free of diabetes at baseline ( hunt1 or hunt2 ) with complete baseline information on smoking . the hunt study is approved by the norwegian data inspectorate and regional committee for medical research ethics . the participants provided informed consent . a chart presentation for the hunt study , 19842008 . each participant s record at hunt is linked to his or her exclusive 11-digit personal identification number , which allows linkage with other health registries . for the current study , we acquired date of death from the national mortality registry ( 13 ) . all with self - reported diabetes at hunt2 or hunt3 were given an appointment for fasting blood sampling . samples were analyzed for glucose , c - peptide , and antibodies to glutamic decarboxylase ( gada ) . patients aged 35 years at diagnosis of diabetes were classified as having type 2 diabetes if , in addition , they were gada - negative ( < 0.08 ) ( n = 1,860 ) . we further classified patients as having autoimmune diabetes if they were gada - positive ( 0.08 ) and were 35 years old at onset of diabetes ( n = 140 ) . as a further criterion for lada , we used information on insulin treatment ( available for 82% of the participants ) to separate lada from classical type 1 diabetes . individuals were classified as lada if they were gada - positive and did not receive insulin treatment during the first year after diagnosis of diabetes ( n = 100 ) . individuals were classified as having classical type 1 diabetes if insulin treatment was started during the first year after onset and gada was either positive or negative . in the cases of negative gada , a low level of c - peptide ( < 150 gada was analyzed at aker university hospital , oslo , norway , by a previously validated method ( 14 ) . the sensitivity and specificity of the assay at the cutoff level of > 0.08 were 0.64 and 1.00 , respectively , according to results obtained in the diabetes antibody standardization program . the cutoff index of 0.08 was equivalent to 43 world health organization units / ml ( 16 ) . c - peptide was measured by radioimmunoassay ( diagnostic system laboratories , webster , tx ) . fasting serum levels of glucose were measured by hemocue at the central laboratory of levanger hospital ( levanger , norway ) ( 13 ) . homeostasis model assessment for insulin resistance ( homa - ir ) and -cell function ( homa-%b ) were calculated using the updated model ( 17 ) . current smoking was defined as current daily smoking of at least 1 g of tobacco in the form of cigarettes , cigars , or pipe . current and former smokers were inquired at age of starting smoking , years of smoking , and average number of cigarettes smoked per day . the intensity of smoking among current and former smokers was assessed by two categories : light smokers ( < 20 cigarettes / day ) and heavy smokers ( 20 cigarettes / day ) . pack - years were calculated according to the formula : cigarettes per day/20 years smoked . cumulative quantity of active smoking was assessed in three categories ( < 6 , 612 , and 13 pack - years ) . p values for means and proportions were calculated using one - way anova and f test . we used cox proportional hazards models to estimate hazard ratios ( hr ) of type 2 diabetes and autoimmune diabetes in relation to smoking habits with 95% cis ( sas 9.2 ; sas institute inc , cary , nc ) . person - years of follow - up were calculated from the age that the participants entered the study ( hunt1 or hunt2 ) until age of onset of diabetes , death , or the end of the follow - up period at hunt2 or hunt3 ( 1997 or 2008 , respectively ) , whichever came first . confounders controlled for age ( in years , as underlying time scale in cox model ) , sex , and baseline information ( from either hunt1 or hunt2 depending on when the participant entered the study ) , bmi ( calculated as weight [ kg]/height [ m ] , continuous ) , education ( primary school , upper secondary school , or university ) , and physical activity [ physically active or inactive , categorization details are described elsewhere ( 18 ) ] , unless otherwise specified . additional adjustment for alcohol consumption and family history of diabetes did not change the results ( change in hr < 10% ) . the analyses were time - dependent , which means that for individuals with information on smoking ( or any of the covariates ) from more than one point in time ( i.e. , both hunt1 and hunt2 ) , information was updated during follow - up . in all analyses , we used never - smokers as a reference group , unless otherwise specified . for evaluating interaction between bmi and smoking habits , participants were categorized as : 1 ) bmi < 25 kg / m and never smoking ( ) ( as reference group in analyses ) , 2 ) bmi < 25 kg / m and heavy smoking ( + ) , 3 ) bmi 25 kg / m and never smoking ( + ) , and 4 ) bmi 25 kg / m and heavy smoking ( + + ) . to estimate additive interaction , we calculated the relative excess risk due to the interaction ( reri ) using reri = hr++ hr+ hr+ + 1 and attributable proportion ( ap ) due to interaction as ap = reri / hr++ ( 19 ) . correlation between smoking ( pack - years ) and homa indices ( loghoma2-ir and loghoma2-%b , logarithmic transformation was applied due to skewing of variables ) were assessed with pearson correlation coefficient . baseline characteristics of the study population according to smoking status at enrollment in the hunt study , 19841997 by most characteristics , cases of autoimmune and type 2 diabetes were similar ( supplementary table 1 ) . compared with individuals without diabetes , those with autoimmune and type 2 diabetes alike tended to be older , heavier , and less physically active . also , the frequency of family history of diabetes was higher . current smokers displayed a reduced risk of autoimmune diabetes compared with never smokers ( hr 0.52 [ 95% ci 0.300.89 ] ; table 2 ) . this effect was also seen when the analysis was restricted to heavy smokers ( 0.42 [ 0.180.98 ] ) . stratifying the results by sex did not reveal differences ( e.g. , in men , hr for 13 pack - years was 0.45 [ 0.210.96 ] and in women , 0.29 [ 0.071.23 ] ) . the reduced risk by smoking was upheld when we divided cases with autoimmune diabetes into lada and classical type 1 diabetes . for cases of lada , the hr was 0.31 ( 95% ci 0.140.71 ) for 13 pack - years . for cases ( few ) of classical type 1 diabetes , hr of autoimmune diabetes in adults in relation to smoking : results from the hunt study , 19842008 the reduced risk of autoimmune diabetes in relation to current smoking ( all smokers ) was not different between normal weight and overweight individuals ( hr 0.53 [ 95% ci 0.191.45 ] for bmi < 25 kg / m and 0.53 [ 0.280.99 ] for bmi 25 kg / m ) . we analyzed levels of gada and c - peptide among diabetic patients across categories of smoking . a decrease in levels of gada was seen across pack - years categories ( 0.068 [ < 6 pack - years ] , 0.065 [ 612 pack - years ] , 0.022 [ 13 pack - years ] ; p = 0.01 ) ; additional adjustment for diabetes duration did not change the results ( p = 0.01 ) . in line with this , heavy smokers ( 20 cigarettes / day ) had substantially lower levels of gada ( 0.009 vs. 0.056 ; p = 0.001 ) compared with never smokers . current smokers compared with never smokers had higher levels of c - peptide ( 964 vs. 886 ; p = 0.03 ) . this same tendency persisted after adjustment for diabetes duration and also after excluding patients with glucose values < 7 mmol / l ( c - peptide = 942 vs. 856 ; p = 0.056 ) . current smoking ( all smokers ) was positively associated with incidence of type 2 diabetes ( table 3 ) . the highest risk was observed in those who smoked 20 cigarettes / day ( hr 1.32 [ 95% ci 1.111.56 ] and in those who reported 13 pack - years smoking ( 1.20 [ 1.051.37 ] ) . hr of type 2 diabetes in relation to smoking and bmi : results from the hunt study , 19842008 stratifying the results by bmi and sex indicated that smoking was associated with an increased risk of type 2 diabetes in overweight men ( 25 kg / m ) ( hr 1.33 [ 95% ci 1.101.6 ] in current smokers ) . in individuals with bmi < 25 kg / m , current smoking was , in contrast , associated with a reduced risk ( 0.60 [ 0.380.96 ] in men and 0.60 [ 0.361.00 ] in women ) . being overweight was associated with an increased risk of type 2 diabetes across all categories of smoking but only in men ( demonstrated in supplementary fig . heavy smoking was associated with a fivefold elevated risk of type 2 diabetes ( hr 5.62 [ 95% ci 3.948.02 ] ) when combined with overweight and a 12-fold excess risk ( 12.54 [ 8.5318.44 ] ) when combined with obesity ( 30 kg / m ) . the relative excess risk due to interaction was estimated at 2.24 ( 95% ci 1.013.46 ) , and the ap was estimated at 0.40 ( 0.230.57 ) , implying that 40% of the cases among heavy smokers with bmi 25 kg / m were caused by the interaction between these two risk factors . there was a positive correlation between number of pack - years of smoking and homa2-ir in type 2 diabetes cases with bmi 25 kg / m ( r = 0.11 ; p = 0.014 ) there was no clear correlation neither in lean and normal weight type 2 diabetes cases ( r = 0.068 ; p = 0.576 ) nor in cases with autoimmune diabetes ( r = 0.181 ; p = 0.281 ) . baseline characteristics of the study population according to smoking status at enrollment in the hunt study , 19841997 by most characteristics , cases of autoimmune and type 2 diabetes were similar ( supplementary table 1 ) . compared with individuals without diabetes , those with autoimmune and type 2 diabetes alike tended to be older , heavier , and less physically active . current smokers displayed a reduced risk of autoimmune diabetes compared with never smokers ( hr 0.52 [ 95% ci 0.300.89 ] ; table 2 ) . this effect was also seen when the analysis was restricted to heavy smokers ( 0.42 [ 0.180.98 ] ) . stratifying the results by sex did not reveal differences ( e.g. , in men , hr for 13 pack - years was 0.45 [ 0.210.96 ] and in women , 0.29 [ 0.071.23 ] ) . the reduced risk by smoking was upheld when we divided cases with autoimmune diabetes into lada and classical type 1 diabetes . for cases of lada , the hr was 0.31 ( 95% ci 0.140.71 ) for 13 pack - years . for cases ( few ) of classical type 1 diabetes , hr of autoimmune diabetes in adults in relation to smoking : results from the hunt study , 19842008 the reduced risk of autoimmune diabetes in relation to current smoking ( all smokers ) was not different between normal weight and overweight individuals ( hr 0.53 [ 95% ci 0.191.45 ] for bmi < 25 kg / m and 0.53 [ 0.280.99 ] for bmi 25 kg / m ) . we analyzed levels of gada and c - peptide among diabetic patients across categories of smoking . a decrease in levels of gada was seen across pack - years categories ( 0.068 [ < 6 pack - years ] , 0.065 [ 612 pack - years ] , 0.022 [ 13 pack - years ] ; p = 0.01 ) ; additional adjustment for diabetes duration did not change the results ( p = 0.01 ) . in line with this , heavy smokers ( 20 cigarettes / day ) had substantially lower levels of gada ( 0.009 vs. 0.056 ; p = 0.001 ) compared with never smokers . current smokers compared with never smokers had higher levels of c - peptide ( 964 vs. 886 ; p = 0.03 ) . this same tendency persisted after adjustment for diabetes duration and also after excluding patients with glucose values < 7 mmol / l ( c - peptide = 942 vs. 856 ; p = 0.056 ) . current smoking ( all smokers ) was positively associated with incidence of type 2 diabetes ( table 3 ) . the highest risk was observed in those who smoked 20 cigarettes / day ( hr 1.32 [ 95% ci 1.111.56 ] and in those who reported 13 pack - years smoking ( 1.20 [ 1.051.37 ] ) . hr of type 2 diabetes in relation to smoking and bmi : results from the hunt study , 19842008 stratifying the results by bmi and sex indicated that smoking was associated with an increased risk of type 2 diabetes in overweight men ( 25 kg / m ) ( hr 1.33 [ 95% ci 1.101.6 ] in current smokers ) . in individuals with bmi < 25 kg / m , current smoking was , in contrast , associated with a reduced risk ( 0.60 [ 0.380.96 ] in men and 0.60 [ 0.361.00 ] in women ) . being overweight was associated with an increased risk of type 2 diabetes across all categories of smoking but only in men ( demonstrated in supplementary fig . heavy smoking was associated with a fivefold elevated risk of type 2 diabetes ( hr 5.62 [ 95% ci 3.948.02 ] ) when combined with overweight and a 12-fold excess risk ( 12.54 [ 8.5318.44 ] ) when combined with obesity ( 30 kg / m ) . the relative excess risk due to interaction was estimated at 2.24 ( 95% ci 1.013.46 ) , and the ap was estimated at 0.40 ( 0.230.57 ) , implying that 40% of the cases among heavy smokers with bmi 25 kg / m were caused by the interaction between these two risk factors . there was a positive correlation between number of pack - years of smoking and homa2-ir in type 2 diabetes cases with bmi 25 kg / m ( r = 0.11 ; p = 0.014 ) there was no clear correlation neither in lean and normal weight type 2 diabetes cases ( r = 0.068 ; p = 0.576 ) nor in cases with autoimmune diabetes ( r = 0.181 ; p = 0.281 ) . the risk reduction was seen both in men and women across categories of bmi and was related both to development of lada and classical type 1 diabetes ( although the analyses regarding type 1 diabetes were admittedly based on very small numbers ) . to our knowledge , our previous report ( 4 ) of risk reduction by of smoking in relation to autoimmune diabetes is up to now the only one demonstrating in humans this , by its beneficial nature , potentially controversial effect . we note that our observations agree with those in an animal study ( 3 ) and are consistent with those from some previous studies , in which a reduced risk of type 1 diabetes was seen in the offspring of smokers ( 2022 ) . it seems important that we can now confirm and extend our previous findings by providing a larger sample and a longer time of follow - up of the hunt cohort . our incidence data are in line with an inhibitory effect of smoking on autoimmune activity . this notion is supported by our observation showing lower levels of gada in long - time heavy smokers than in other lada patients . in this context , one can not rule out the possibility that the association with gada is restricted to alteration of the time dynamics of gada . gada levels are known to rise and then fall in pediatric patients with type 1 diabetes ( 23,24 ) ; however , evidence indicates that gada are more persistent in lada ( 25,26 ) , at least in those who display high titers . in a previous study ( 25 ) , it was shown that a majority of those who developed lada between hunt2 and hunt3 were gada - positive already at hunt2 ( i.e. , during prediabetes ) with no significant change in gada between hunt2 and 3 . these findings do not support the possibility of smoking affecting merely the time course of gada . which component of smoking is important ? in animal and human studies , exposure to nicotine , the major active component of cigarettes , can exert immunosuppressive ( 3,27 ) as well as anti - inflammatory effects ( 1,2,28 ) . hence , it is likely , but not proven , that nicotine is the component of cigarette smoking behind the risk reduction in autoimmune diabetes . with regard to type 2 diabetes , our findings confirm a similar increase in risk associated with heavy smoking as in other studies ( 2933 ) . in contrast to most previous studies ( 5 ) , the increased risk of type 2 diabetes was limited to men , and this finding is similar to those in a french cohort study ( 34 ) . sex differences in inhalation practice ( 35 ) could be a factor . also , so far unknown risk factors present in men but not in women could be operative . a further distinction in our study was the limitation of risk with smoking to men with bmi 25 kg / m , whereas a reduced risk was actually seen in lean and normal weight smokers . one explanation for the reduced risk in lean smokers could relate to the rise in metabolic rate caused by smoking ( 3638 ) . such a rise would counteract insulin resistance , and this beneficial effect could perhaps , in light smokers with low bmi , more than outweigh any countering influence by smoking - induced insulin resistance ( for which the underlying mechanisms are not known ) . previous studies have suggested a possible interaction between high bmi and smoking in the development of type 2 diabetes ( 11 ) . in confirmation of this , the highest risk of type 2 diabetes was seen in men exposed both to overweight and heavy smoking . smoking was associated with homa - ir and higher levels of c - peptide , findings that are compatible with an insulin resistance effect . as mentioned above , an insulin resistance effect as found in this study is in line with previous observations as mentioned above . one explanation behind the bmi and smoking interaction could be that exposure to both these sources of insulin resistance accelerates exhaustion of -cells with subsequent inability to maintain glucose homeostasis . the strengths of this study include an all - population area - based sample , a long follow - up , and data on gada and c - peptide . also , it was possible to control for potential sociodemographic , lifestyle , and anthropometric confounders . information on smoking was collected by several questions and updated during follow - up . however , there are some potential biases to be considered . this method has been shown to correctly classify > 95% of cases ( 39 ) , but will miss cases of undiagnosed diabetes . also , we relied on self - reports of smoking habits . such socially undesirable behaviors can be afflicted by underreporting ( 40 ) . due to prospective nature of this study , any misclassification of smoking can however be assumed to be nondifferential . in conclusion , smoking was associated with a reduced risk of autoimmune diabetes in adults ; an effect is probably exerted by inhibition of the autoimmune process . we further find that the increased risk by smoking in type 2 diabetes is limited to overweight men and that the combination of overweight and smoking is an especially potent combination of risk factors for type 2 diabetes .

objectiveto investigate the association between smoking habits and risk of autoimmune diabetes in adults and of type 2 diabetes.research design and methodswe used data from the three surveys of the nord - trndelag health study , spanning 19842008 and including a cohort of 90,819 norwegian men ( 48% ) and women ( 52% ) aged 20 years . incident cases of diabetes were identified by questionnaire and classified as type 2 diabetes ( n = 1,860 ) and autoimmune diabetes ( n = 140 ) based on antibodies to glutamic decarboxylase ( gada ) and age at onset of diabetes . hazard ratios ( hrs ) adjusted for confounders were estimated by cox proportional hazards regression models.resultsthe risk of autoimmune diabetes was reduced by 48% ( hr 0.52 [ 95% ci 0.300.89 ] ) in current smokers and 58% in heavy smokers ( 0.42 [ 0.180.98 ] ) . the reduced risk was positively associated with number of pack - years . heavy smoking was associated with lower levels of gada ( p = 0.001 ) and higher levels of c - peptide ( 964 vs. 886 pmol / l ; p = 0.03 ) . in contrast , smoking was associated with an increased risk of type 2 diabetes , restricted to overweight men ( 1.33 [ 1.101.61 ] ) . attributable proportion due to an interaction between overweight and heavy smoking was estimated to 0.40 ( 95% ci 0.230.57).conclusionsin this epidemiological study , smoking is associated with a reduced risk of autoimmune diabetes , possibly linked to an inhibitory effect on the autoimmune process . an increased risk of type 2 diabetes was restricted to overweight men .

RESEARCH DESIGN AND METHODS Study population and design Identification and classification of diabetes Biochemical analysis Assessment of smoking habits Statistical analysis RESULTS Baseline characteristics Smoking attenuates the incidence of autoimmune diabetes Smoking increases the incidence of type 2 diabetes in overweight men Increased risk by smoking in type 2 diabetes associates with insulin resistance CONCLUSIONS Supplementary Material

the general design of the hunt study and the basis for the present analyses are illustrated in fig . hunt is a population - based study , conducted in the county of nord - trndelag in norway . all inhabitants aged 20 years in the county the investigations carried out are presented in detail on the hunt website ( http://www.hunt.ntnu.no/index.php?side=english ) . for the current study , we acquired date of death from the national mortality registry ( 13 ) . samples were analyzed for glucose , c - peptide , and antibodies to glutamic decarboxylase ( gada ) . patients aged 35 years at diagnosis of diabetes were classified as having type 2 diabetes if , in addition , they were gada - negative ( < 0.08 ) ( n = 1,860 ) . we further classified patients as having autoimmune diabetes if they were gada - positive ( 0.08 ) and were 35 years old at onset of diabetes ( n = 140 ) . as a further criterion for lada , we used information on insulin treatment ( available for 82% of the participants ) to separate lada from classical type 1 diabetes . individuals were classified as lada if they were gada - positive and did not receive insulin treatment during the first year after diagnosis of diabetes ( n = 100 ) . in the cases of negative gada , a low level of c - peptide ( < 150 gada was analyzed at aker university hospital , oslo , norway , by a previously validated method ( 14 ) . c - peptide was measured by radioimmunoassay ( diagnostic system laboratories , webster , tx ) . fasting serum levels of glucose were measured by hemocue at the central laboratory of levanger hospital ( levanger , norway ) ( 13 ) . the intensity of smoking among current and former smokers was assessed by two categories : light smokers ( < 20 cigarettes / day ) and heavy smokers ( 20 cigarettes / day ) . pack - years were calculated according to the formula : cigarettes per day/20 years smoked . cumulative quantity of active smoking was assessed in three categories ( < 6 , 612 , and 13 pack - years ) . we used cox proportional hazards models to estimate hazard ratios ( hr ) of type 2 diabetes and autoimmune diabetes in relation to smoking habits with 95% cis ( sas 9.2 ; sas institute inc , cary , nc ) . person - years of follow - up were calculated from the age that the participants entered the study ( hunt1 or hunt2 ) until age of onset of diabetes , death , or the end of the follow - up period at hunt2 or hunt3 ( 1997 or 2008 , respectively ) , whichever came first . for evaluating interaction between bmi and smoking habits , participants were categorized as : 1 ) bmi < 25 kg / m and never smoking ( ) ( as reference group in analyses ) , 2 ) bmi < 25 kg / m and heavy smoking ( + ) , 3 ) bmi 25 kg / m and never smoking ( + ) , and 4 ) bmi 25 kg / m and heavy smoking ( + + ) . to estimate additive interaction , we calculated the relative excess risk due to the interaction ( reri ) using reri = hr++ hr+ hr+ + 1 and attributable proportion ( ap ) due to interaction as ap = reri / hr++ ( 19 ) . correlation between smoking ( pack - years ) and homa indices ( loghoma2-ir and loghoma2-%b , logarithmic transformation was applied due to skewing of variables ) were assessed with pearson correlation coefficient . hunt is a population - based study , conducted in the county of nord - trndelag in norway . all inhabitants aged 20 years in the county the investigations carried out are presented in detail on the hunt website ( http://www.hunt.ntnu.no/index.php?side=english ) . in brief , in hunt1 , 19841986 , a total of 76,885 ( 90.3% ) of the eligible individuals participated . for the current study , we acquired date of death from the national mortality registry ( 13 ) . samples were analyzed for glucose , c - peptide , and antibodies to glutamic decarboxylase ( gada ) . patients aged 35 years at diagnosis of diabetes were classified as having type 2 diabetes if , in addition , they were gada - negative ( < 0.08 ) ( n = 1,860 ) . we further classified patients as having autoimmune diabetes if they were gada - positive ( 0.08 ) and were 35 years old at onset of diabetes ( n = 140 ) . individuals were classified as lada if they were gada - positive and did not receive insulin treatment during the first year after diagnosis of diabetes ( n = 100 ) . in the cases of negative gada , a low level of c - peptide ( < 150 gada was analyzed at aker university hospital , oslo , norway , by a previously validated method ( 14 ) . c - peptide was measured by radioimmunoassay ( diagnostic system laboratories , webster , tx ) . current smoking was defined as current daily smoking of at least 1 g of tobacco in the form of cigarettes , cigars , or pipe . the intensity of smoking among current and former smokers was assessed by two categories : light smokers ( < 20 cigarettes / day ) and heavy smokers ( 20 cigarettes / day ) . pack - years were calculated according to the formula : cigarettes per day/20 years smoked . cumulative quantity of active smoking was assessed in three categories ( < 6 , 612 , and 13 pack - years ) . we used cox proportional hazards models to estimate hazard ratios ( hr ) of type 2 diabetes and autoimmune diabetes in relation to smoking habits with 95% cis ( sas 9.2 ; sas institute inc , cary , nc ) . person - years of follow - up were calculated from the age that the participants entered the study ( hunt1 or hunt2 ) until age of onset of diabetes , death , or the end of the follow - up period at hunt2 or hunt3 ( 1997 or 2008 , respectively ) , whichever came first . additional adjustment for alcohol consumption and family history of diabetes did not change the results ( change in hr < 10% ) . for evaluating interaction between bmi and smoking habits , participants were categorized as : 1 ) bmi < 25 kg / m and never smoking ( ) ( as reference group in analyses ) , 2 ) bmi < 25 kg / m and heavy smoking ( + ) , 3 ) bmi 25 kg / m and never smoking ( + ) , and 4 ) bmi 25 kg / m and heavy smoking ( + + ) . to estimate additive interaction , we calculated the relative excess risk due to the interaction ( reri ) using reri = hr++ hr+ hr+ + 1 and attributable proportion ( ap ) due to interaction as ap = reri / hr++ ( 19 ) . correlation between smoking ( pack - years ) and homa indices ( loghoma2-ir and loghoma2-%b , logarithmic transformation was applied due to skewing of variables ) were assessed with pearson correlation coefficient . baseline characteristics of the study population according to smoking status at enrollment in the hunt study , 19841997 by most characteristics , cases of autoimmune and type 2 diabetes were similar ( supplementary table 1 ) . compared with individuals without diabetes , those with autoimmune and type 2 diabetes alike tended to be older , heavier , and less physically active . also , the frequency of family history of diabetes was higher . current smokers displayed a reduced risk of autoimmune diabetes compared with never smokers ( hr 0.52 [ 95% ci 0.300.89 ] ; table 2 ) . this effect was also seen when the analysis was restricted to heavy smokers ( 0.42 [ 0.180.98 ] ) . , in men , hr for 13 pack - years was 0.45 [ 0.210.96 ] and in women , 0.29 [ 0.071.23 ] ) . the reduced risk by smoking was upheld when we divided cases with autoimmune diabetes into lada and classical type 1 diabetes . for cases of lada , the hr was 0.31 ( 95% ci 0.140.71 ) for 13 pack - years . for cases ( few ) of classical type 1 diabetes , hr of autoimmune diabetes in adults in relation to smoking : results from the hunt study , 19842008 the reduced risk of autoimmune diabetes in relation to current smoking ( all smokers ) was not different between normal weight and overweight individuals ( hr 0.53 [ 95% ci 0.191.45 ] for bmi < 25 kg / m and 0.53 [ 0.280.99 ] for bmi 25 kg / m ) . we analyzed levels of gada and c - peptide among diabetic patients across categories of smoking . a decrease in levels of gada was seen across pack - years categories ( 0.068 [ < 6 pack - years ] , 0.065 [ 612 pack - years ] , 0.022 [ 13 pack - years ] ; p = 0.01 ) ; additional adjustment for diabetes duration did not change the results ( p = 0.01 ) . in line with this , heavy smokers ( 20 cigarettes / day ) had substantially lower levels of gada ( 0.009 vs. 0.056 ; p = 0.001 ) compared with never smokers . current smokers compared with never smokers had higher levels of c - peptide ( 964 vs. 886 ; p = 0.03 ) . this same tendency persisted after adjustment for diabetes duration and also after excluding patients with glucose values < 7 mmol / l ( c - peptide = 942 vs. 856 ; p = 0.056 ) . current smoking ( all smokers ) was positively associated with incidence of type 2 diabetes ( table 3 ) . the highest risk was observed in those who smoked 20 cigarettes / day ( hr 1.32 [ 95% ci 1.111.56 ] and in those who reported 13 pack - years smoking ( 1.20 [ 1.051.37 ] ) . hr of type 2 diabetes in relation to smoking and bmi : results from the hunt study , 19842008 stratifying the results by bmi and sex indicated that smoking was associated with an increased risk of type 2 diabetes in overweight men ( 25 kg / m ) ( hr 1.33 [ 95% ci 1.101.6 ] in current smokers ) . in individuals with bmi < 25 kg / m , current smoking was , in contrast , associated with a reduced risk ( 0.60 [ 0.380.96 ] in men and 0.60 [ 0.361.00 ] in women ) . being overweight was associated with an increased risk of type 2 diabetes across all categories of smoking but only in men ( demonstrated in supplementary fig . heavy smoking was associated with a fivefold elevated risk of type 2 diabetes ( hr 5.62 [ 95% ci 3.948.02 ] ) when combined with overweight and a 12-fold excess risk ( 12.54 [ 8.5318.44 ] ) when combined with obesity ( 30 kg / m ) . the relative excess risk due to interaction was estimated at 2.24 ( 95% ci 1.013.46 ) , and the ap was estimated at 0.40 ( 0.230.57 ) , implying that 40% of the cases among heavy smokers with bmi 25 kg / m were caused by the interaction between these two risk factors . there was a positive correlation between number of pack - years of smoking and homa2-ir in type 2 diabetes cases with bmi 25 kg / m ( r = 0.11 ; p = 0.014 ) there was no clear correlation neither in lean and normal weight type 2 diabetes cases ( r = 0.068 ; p = 0.576 ) nor in cases with autoimmune diabetes ( r = 0.181 ; p = 0.281 ) . baseline characteristics of the study population according to smoking status at enrollment in the hunt study , 19841997 by most characteristics , cases of autoimmune and type 2 diabetes were similar ( supplementary table 1 ) . compared with individuals without diabetes , those with autoimmune and type 2 diabetes alike tended to be older , heavier , and less physically active . current smokers displayed a reduced risk of autoimmune diabetes compared with never smokers ( hr 0.52 [ 95% ci 0.300.89 ] ; table 2 ) . this effect was also seen when the analysis was restricted to heavy smokers ( 0.42 [ 0.180.98 ] ) . , in men , hr for 13 pack - years was 0.45 [ 0.210.96 ] and in women , 0.29 [ 0.071.23 ] ) . the reduced risk by smoking was upheld when we divided cases with autoimmune diabetes into lada and classical type 1 diabetes . for cases of lada , the hr was 0.31 ( 95% ci 0.140.71 ) for 13 pack - years . for cases ( few ) of classical type 1 diabetes , hr of autoimmune diabetes in adults in relation to smoking : results from the hunt study , 19842008 the reduced risk of autoimmune diabetes in relation to current smoking ( all smokers ) was not different between normal weight and overweight individuals ( hr 0.53 [ 95% ci 0.191.45 ] for bmi < 25 kg / m and 0.53 [ 0.280.99 ] for bmi 25 kg / m ) . we analyzed levels of gada and c - peptide among diabetic patients across categories of smoking . a decrease in levels of gada was seen across pack - years categories ( 0.068 [ < 6 pack - years ] , 0.065 [ 612 pack - years ] , 0.022 [ 13 pack - years ] ; p = 0.01 ) ; additional adjustment for diabetes duration did not change the results ( p = 0.01 ) . in line with this , heavy smokers ( 20 cigarettes / day ) had substantially lower levels of gada ( 0.009 vs. 0.056 ; p = 0.001 ) compared with never smokers . current smokers compared with never smokers had higher levels of c - peptide ( 964 vs. 886 ; p = 0.03 ) . this same tendency persisted after adjustment for diabetes duration and also after excluding patients with glucose values < 7 mmol / l ( c - peptide = 942 vs. 856 ; p = 0.056 ) . current smoking ( all smokers ) was positively associated with incidence of type 2 diabetes ( table 3 ) . the highest risk was observed in those who smoked 20 cigarettes / day ( hr 1.32 [ 95% ci 1.111.56 ] and in those who reported 13 pack - years smoking ( 1.20 [ 1.051.37 ] ) . hr of type 2 diabetes in relation to smoking and bmi : results from the hunt study , 19842008 stratifying the results by bmi and sex indicated that smoking was associated with an increased risk of type 2 diabetes in overweight men ( 25 kg / m ) ( hr 1.33 [ 95% ci 1.101.6 ] in current smokers ) . in individuals with bmi < 25 kg / m , current smoking was , in contrast , associated with a reduced risk ( 0.60 [ 0.380.96 ] in men and 0.60 [ 0.361.00 ] in women ) . being overweight was associated with an increased risk of type 2 diabetes across all categories of smoking but only in men ( demonstrated in supplementary fig . heavy smoking was associated with a fivefold elevated risk of type 2 diabetes ( hr 5.62 [ 95% ci 3.948.02 ] ) when combined with overweight and a 12-fold excess risk ( 12.54 [ 8.5318.44 ] ) when combined with obesity ( 30 kg / m ) . the relative excess risk due to interaction was estimated at 2.24 ( 95% ci 1.013.46 ) , and the ap was estimated at 0.40 ( 0.230.57 ) , implying that 40% of the cases among heavy smokers with bmi 25 kg / m were caused by the interaction between these two risk factors . there was a positive correlation between number of pack - years of smoking and homa2-ir in type 2 diabetes cases with bmi 25 kg / m ( r = 0.11 ; p = 0.014 ) there was no clear correlation neither in lean and normal weight type 2 diabetes cases ( r = 0.068 ; p = 0.576 ) nor in cases with autoimmune diabetes ( r = 0.181 ; p = 0.281 ) . the risk reduction was seen both in men and women across categories of bmi and was related both to development of lada and classical type 1 diabetes ( although the analyses regarding type 1 diabetes were admittedly based on very small numbers ) . to our knowledge , our previous report ( 4 ) of risk reduction by of smoking in relation to autoimmune diabetes is up to now the only one demonstrating in humans this , by its beneficial nature , potentially controversial effect . we note that our observations agree with those in an animal study ( 3 ) and are consistent with those from some previous studies , in which a reduced risk of type 1 diabetes was seen in the offspring of smokers ( 2022 ) . our incidence data are in line with an inhibitory effect of smoking on autoimmune activity . this notion is supported by our observation showing lower levels of gada in long - time heavy smokers than in other lada patients . in this context , one can not rule out the possibility that the association with gada is restricted to alteration of the time dynamics of gada . gada levels are known to rise and then fall in pediatric patients with type 1 diabetes ( 23,24 ) ; however , evidence indicates that gada are more persistent in lada ( 25,26 ) , at least in those who display high titers . with regard to type 2 diabetes , our findings confirm a similar increase in risk associated with heavy smoking as in other studies ( 2933 ) . in contrast to most previous studies ( 5 ) , the increased risk of type 2 diabetes was limited to men , and this finding is similar to those in a french cohort study ( 34 ) . a further distinction in our study was the limitation of risk with smoking to men with bmi 25 kg / m , whereas a reduced risk was actually seen in lean and normal weight smokers . one explanation for the reduced risk in lean smokers could relate to the rise in metabolic rate caused by smoking ( 3638 ) . previous studies have suggested a possible interaction between high bmi and smoking in the development of type 2 diabetes ( 11 ) . in confirmation of this , the highest risk of type 2 diabetes was seen in men exposed both to overweight and heavy smoking . smoking was associated with homa - ir and higher levels of c - peptide , findings that are compatible with an insulin resistance effect . the strengths of this study include an all - population area - based sample , a long follow - up , and data on gada and c - peptide . this method has been shown to correctly classify > 95% of cases ( 39 ) , but will miss cases of undiagnosed diabetes . due to prospective nature of this study , any misclassification of smoking can however be assumed to be nondifferential . in conclusion , smoking was associated with a reduced risk of autoimmune diabetes in adults ; an effect is probably exerted by inhibition of the autoimmune process . we further find that the increased risk by smoking in type 2 diabetes is limited to overweight men and that the combination of overweight and smoking is an especially potent combination of risk factors for type 2 diabetes .

randomised controlled trials ( rcts ) in attention deficit hyperactivity disorder ( adhd ) have shown that long - acting methylphenidate ( mph ) preparations have similar efficacy to multiple doses of short - acting , immediate - release ( ir ) formulations , but with the advantage of convenient once - daily dosing [ 4 , 15 , 16 ] . patients receiving long - acting or modified - release ( mr ) , formulations may have better treatment adherence than those receiving mph - ir . however , this is still a matter of debate as rcts of mph are usually only short - term ( i.e. a few weeks duration ) and the patient population is highly selected ; therefore , results from rcts do not fully represent long - term treatment or the heterogeneous population of patients with adhd found in daily clinical practice . to learn more about adequate individualised treatment with mph preparations in a real - world setting , and to obtain a clearer picture of effectiveness ( is it of use ? ) and efficiency ( how much benefit at what cost ? ) , studies of treatment approaches beyond the rct setting are necessary . the general trend for increasing use of mph - mr preparations in adhd [ 23 , 34 ] means that post - rct observational studies are required to ensure well - monitored , up - to - date drug management . furthermore , there is a need for an evidence - based , long - term perspective for patients . in paediatric psychopathology , the boundaries between observational and experimental studies are considered indistinct , with observational studies generally used predominantly in epidemiology and less so for clinical purposes . however , clinical observational studies may be useful in generating additional , often more definitive , conclusions about treatment guidance and safety effects ( especially over the long - term ) , or the natural history of a disorder . such findings can complement the results obtained from the experimental studies , which are a pre - condition for drug treatment evaluation . thus , rcts and observational studies can each make a useful contribution to the evaluation of treatment effectiveness . the rct is seen by many as the gold standard as it should , by design , ensure that patients being compared differ only in their exposure to the intervention under study . in fact , both randomised and non - randomised trial designs have their own particular limitations . issues related to the process of randomisation that may affect the validity of conclusions drawn from the results of rcts and non - randomised studies were explored in a systematic review . here , non - randomised studies were considered to include quasi - experiments , natural experiments and prospective , observational , cohort studies . results showed that rcts and non - randomised studies can produce different results but the direction of the difference is not consistent opposing opinions about the relative merits of rcts and non - randomised studies may be underpinned by differences in threats to validity between the two study designs , which can be external ( the extent to which the results are generalisable to all potential recipients ) , or internal ( whether differences in observed effects can be attributed to differences in the intervention ) . rcts may lack external validity in that those who meet eligibility criteria , or are invited , or agree to participate are significantly different to the population to whom the results of the study will be applied , and so the clinical usefulness of the study may be limited . although evidence is limited , there is a tendency for settings of non - randomised studies to be slightly more representative of those who are eligible to be included . non - randomised studies also include other real - world effects , for example patients preferences [ 25 , 30 ] , or the doctor patient relationship , increasing their external validity . however , a major criticism of non - randomised studies is the possibility that groups being compared differ prognostically in important characteristics , affecting the internal validity of such trials . clearly , for best effect , internal and external validity need to be balanced against each other . interestingly , despite their methodological differences , the systematic review found that the differences in results between rcts and non - randomised studies are frequently smaller than those between rcts or between non - randomised studies . as observational studies are broader in scope and less rigorous than rcts , they may be prone to certain pitfalls . observational studies , including those sponsored by pharmaceutical companies , should be designed in such a way that the scientific merit of the study has the highest priority and secondary aspects are minimised . to avoid methodological shortcomings , several points need to be considered , including : selection bias ( e.g. is the sample representative for the population in mind ? are subgroups comparable?)information bias ( e.g. is all information gathered in a similar way?)measurement errors ( e.g. are inventories psychometrically sound ? are subgroups comparable ? ) information bias ( e.g. is all information gathered in a similar way ? ) measurement errors ( e.g. are inventories psychometrically sound ? evaluation problems ( e.g. is the study design appropriate ? is quality control of data sampling included ? awareness of these possible pitfalls should mean that they can be avoided by the use of an appropriate study design , or at least that any weaknesses and limitations can be considered when reporting and interpreting the data . therefore , it would be advantageous for investigators to consider all 22 points of the strobe ( strengthening the reporting of observational studies in epidemiology ) consortium checklist [ 1 , 32 ] when planning an observational study as well as when reporting it . aside from study design , the guidelines governing what data can be collected in observational studies vary between countries , so what may require ethics approval in one country would not in another . this also means that reporting these studies can be problematic if , for example , the regulations in the country in which the journal is published require ethics approval even though the country in which the data were collected did not . treatment with mph may improve symptoms of adhd in about 75% of patients , and response rates of up to 90% can be achieved by switching unresponsive patients to other stimulants ( e.g. amphetamine sulphate ) . this leaves at least 10% of patients who do not benefit from stimulants , and for whom other medications ( e.g. atomoxetine , guanfacine ) may be considered . in addition , it is still not clear how much of an improvement can be achieved if one mph preparation is replaced by another at an equivalent dose . this question is of particular interest when considering a switch from mph - ir to mph - mr , with the potential for more convenient dosing , less stigmatisation and potentially better adherence as mph - mr only has to be administered once daily in the morning . good adherence to therapy is of utmost importance , especially in chronic mental health conditions such as adhd , and must be considered in addition to the effectiveness and efficiency of an intervention . during treatment with mph - ir preparations in adhd , poor adherence is a critical issue , ranging from 20 to 80% after 1 year , with adherence rates of 4050% after 35 years . following parental complaints about social stigmatisation , and the inconvenience of handling multiple doses during the day , it was hoped that adherence would be improved by developing once - daily mph preparations . however , better rates of adherence do not necessarily follow from switching to an improved formulation , as there could be other clinical and social factors involved that are associated with the disorganised and oppositional behaviours of patients with adhd and their families . for this reason , it is important to investigate all drug- and non - drug - related aspects of adherence when using mph - mr preparations for the treatment of adhd in the daily clinical setting . although previous rcts have compared mph - ir , mph - mr and placebo [ 15 , 16 ] , such trials do not provide any information about the clinical effects resulting from switching from mph - ir to mph - mr during the study protocol . observational studies may be better suited to investigating these effects , but to date only a few observational studies of switching to mph - mr formulations have been reported , investigating the switch to treatment with oros mph ( concerta , janssen - cilag , uk ) , mph - sodas ( ritalin - la , novartis , us ) , medikinet retard / xl ( medice , germany ) and equasym xl1 ( shire pharmaceuticals ireland limited , ireland ) [ 11 , 12 ] . one of the open - label studies was a multicentre , european study of oros mph , which included 150 children ( aged 616 years ) with adhd who were stable on mph - ir treatment before switching . the german subgroup of 50 patients was increased afterwards to 221 patients and data were analysed separately [ 20 , 21 ] . in the first 3-week phase ( european and german study ) , children benefited from switching formulations , but iowa conners scores improved only for parent ratings ; teachers did not recognise a change in behaviour . the global effectiveness evaluation was positive , i.e. very good or good ( german study : teachers 55% , investigators 77% , parents 79% ) . tolerability of oros mph was also good , with about 90% of parents favouring continuation of the mph switch , a good predictor for better adherence in the long - term . these results are notable because the preceding treatment with mph - ir had already reduced the symptoms of adhd . for comparison , a score of 10 is usual on the parent iowa conners scale inattention / hyperactivity for unmedicated children with adhd . in the oros mph study , the baseline score ( i.e. after treatment with mph - ir , but before switching to mr ) was 6.4 , which decreased further to 4.2 after 21 days of treatment with oros mph . however , one should be cautious about drawing firm conclusions : this was an open study , the starting dose was heterogeneous and there was a tendency to increase doses while switching ( which may be interpreted as dose optimisation ) . also , the discrepancy between parent and teacher evaluation in the iowa conners scale may be due to both a pharmacokinetic weakness of the oros mph formulation in the morning ( lower mph availability ) and a pharmacokinetic strength in the afternoon ( better symptom control ) . in summary the results of this german sample suggest that children and adolescents with adhd achieve at least as good control of central adhd symptoms if there is a switch from mph - ir to oros mph. in the framework of the above - mentioned international study , 89/101 ( 88% ) patients with adhd continued to receive open but controlled ( visit every 2nd month ) treatment during a 12 month extension . a total of 56 children ( 63% ) continued treatment until the end of the extension period . compliance with treatment was assessed using questionnaires for parents and investigators , but no iowa conners data are available . tolerability of treatment was still good and parents ( ~ 50% ) and investigators ( ~ 75% ) gave positive feedback . unfortunately , adherence decreased by about half during the year , indicating that drop - out rates for this mph - mr preparation differed little compared with the usual drop - out rates for ir preparations [ 17 , 33 ] . hence , the predicted long - lasting improvement in adherence with mph - mr preparations was not observed in this study . however , it is important to also take into account the other benefits of switching from mph - ir to mph - mr preparations ; e.g. it has been suggested that mph - mr may be less prone to misuse or abuse compared with mph - ir . in an observational study in brazil , patients with adhd were switched from clinically stable mph - ir treatment to mph - sodas . with its 8-week duration , the study provided information about effectiveness and satisfaction with mph - sodas , but not about long - term adherence . as for the above - mentioned study with oros mph , no information was given about the reason for switching . it is probable that clinically stable patients were just asked if they would like to take part in the planned study , so that both of these observational trials were this is in contrast to the obseer ( observation of safety and effectiveness of equasym xl in routine care ) study reported in this supplement , the adore study , and a study on the effects of atomoxetine on adhd in clinical paediatric treatment settings , which can all be considered as naturalistic it was up to the physician which patients with adhd were included in the study . the mph - sodas switch study included only a small sample of children ( n = 31 ) and adults ( n = 23 ) , with a protocol adherence of 7080% after 8 weeks . there were no significant changes in behavioural parameters and side - effects on mph - sodas , but the majority of patients were reported to be satisfied with switching ( 74% ) . the authors stated that the latter may reflect the convenience of once - daily dosing of this mph - extended release , as speculated in previous studies , referring to a study in taiwan . further publications from this taiwanese research group [ 9 , 17 ] looked at children with adhd who adhered poorly to mph - ir treatment and who were offered the chance to switch to mph - oros . of 607 children with adhd aged 516 years , 240 ( 40% ) were poorly adherent to treatment . a final subgroup of 124 children ( originally 137 ; 13 were lost to follow - up ) switched to mph - oros and showed superior adherence and improved efficacy over a period of 3 weeks in an intra - individual comparison of mph - ir versus mph - oros use . this shows in principle that such a switch appears to work , at least in the short - term ( 3 weeks ) ; however , only about half of the poor adherents to medication seemed to accept the offer . reasons for , and determinants of , poor adherence may partly explain this observation ( table 1 ) . this underlines the idea that the switch of mph preparations itself ( from mph - ir to mph - mr in this case ) is not the only factor affecting treatment adherence . furthermore , switching preparations is usually accompanied by dosage increases and closer monitoring.table 1reasons for , and determinants of , poor adherence to mph - ir treatmentreasons ( patients / parents report)determinants ( univariate analysis)forgetting medicationolder ageside effects / safety concernsincreased frequency of drug administrationlack of perceived effectolder age at onset / diagnosisprivacy issuesfamily history of adhdbitter tastehigher paternal educationteacher objectionhigher mean dose of mphswitch to mph - oros was based mainly on the decision of the investigator after mutual discussion with patients and their parents ( according to gau et al . ) reasons for , and determinants of , poor adherence to mph - ir treatment switch to mph - oros was based mainly on the decision of the investigator after mutual discussion with patients and their parents ( according to gau et al . ) a switch to medikinet retard / xl ( long - acting mph with 50% mph - ir ) was explored in an open - label study of 447 patients ( aged 617 years ) who were either untreated ( but considered suitable for such treatment ) or currently receiving maintenance treatment with an approved mph dosage form ( ir or mr ) . primary outcome measures were adhd severity and side effects , which were evaluated by physicians and parents at the time of the medication changeover and 46 weeks later . adhd symptom severity declined significantly , and oppositional behaviour and side effects , as assessed by parents , were also reduced . as expected , the strongest effects were found in patients without prior pharmacotherapy , but significant improvements were also observed in patients with once - daily or more than once - daily mph - ir as prior medication , as well as concerta . similarly , a switch to equasym xl was analysed in another open - label study performed in children aged 617 years , either untreated or currently receiving maintenance treatment with a different mph formulation . the majority ( 65% ) of the 308 patients enrolled demonstrated a positive response , which was also observed within the group who were previously treated with mph - ir ( 64% ) and those treated with a different mph - mr formulation ( 55% ) . further results supporting a switch to mph - mr formulations were obtained in the german multicentre , prospective , observational , naturalistic obseer study on the safety and effectiveness of equasym xl , and are reported in this supplement [ 6 , 12 , 14 , 28 ] . obseer , planned and reported in line with the strobe criteria ( table 2 ) , collected behavioural data over a period of about 12 weeks from 822 children aged 617 years with adhd . a total of 574 ( 70% ) children had been treated previously with other mph formulations such as mph - ir or mph - mr . the treating physician decided independently , on the basis of his clinical assessment and experience , if treatment with equasym xl should be started . therefore , the children did not need to be clinically stable on mph - ir , as in the other switching studies described above . a subgroup of children ( n = 371 , 45% ) was switched from mph - ir ( once - daily : n = 101 , 12% ; repeated [ i.e. administered several times per day ] : n = 270 , 33% ) to mph - mr . these children showed an improvement in all parameters investigated , including symptom reduction , satisfaction with treatment and quality of life [ 14 , 28 ] . the mph - ir once - daily prior treatment subgroup showed improvements in adherence rates during equasym xl treatment that were similar to the other subgroups ( prior treatment with mph - mr or other / not specified ) , and a particular advantage was noted for the mph - ir repeated prior treatment subgroup . the positive effects were reflected by information from parents , teachers , children and physicians , and results were stable all day long . as in the other mph - ir to mr switch studies , interestingly , improvements were also noted in the subgroup of patients who switched from a different mph - mr formulation to equasym xl , although these were less marked than for the mph - ir subgroups , as might be expected [ 14 , 28].table 2strobe criteria and confirmation of items included for the obseer cohort studyitem norecommendationtitle and abstract1(a ) indicate the study s design with a commonly used term in the title or the abstract(b ) provide in the abstract an informative and balanced summary of what was done and what was foundintroduction background / rationale2explain the scientific background and rationale for the investigation being reported objectives3state specific objectives , including any prespecified hypothesesmethods study design4present key elements of study design early in the paper setting5describe the setting , locations , and relevant dates , including periods of recruitment , exposure , follow - up , and data collection participants6(a ) cohort study give the eligibility criteria , and the sources and methods of selection of participants . describe methods of follow - upcase control study give the eligibility criteria , and the sources and methods of case ascertainment and control selection . give the rationale for the choice of cases and controlscross - sectional study give the eligibility criteria , and the sources and methods of selection of participants(b ) cohort study for matched studies , give matching criteria and number of exposed and unexposedcase control study for matched studies , give matching criteria and the number of controls per case variables7clearly define all outcomes , exposures , predictors , potential confounders , and effect modifiers . give diagnostic criteria , if applicable data sources / measurement8for each variable of interest , give sources of data and details of methods of assessment ( measurement ) . describe comparability of assessment methods if there is more than one group bias9describe any efforts to address potential sources of bias study size10explain how the study size was arrived at quantitative variables11explain how quantitative variables were handled in the analyses . if applicable , describe which groupings were chosen and why statistical methods12(a ) describe all statistical methods , including those used to control for confounding(b ) describe any methods used to examine subgroups and interactions(c ) explain how missing data were addressed(d ) cohort study if applicable , explain how matching of cases and controls was addressedcross - sectional study if applicable , describe analytical methods taking account of sampling strategyna(e ) describe any sensitivity analysesnaresults participants13(a ) report numbers of individuals at each stage of study numbers potentially eligible , examined for eligibility , confirmed eligible , included in the study , completing follow - up , and analysed(b ) give reasons for non - participation at each stage(c ) consider use of a flow diagram descriptive data14(a ) give characteristics of study participants ( e.g. demographic , clinical , social ) and information on exposures and potential confounders(b ) indicate number of participants with missing data for each variable of interest(c ) cohort study summarise follow - up time ( e.g. average and total amount) outcome data15cohort study report numbers of outcome events or summary measures over timecase control study report numbers in each exposure category , or summary measures of exposurecross - sectional study report numbers of outcome events or summary measures main results16(a ) give unadjusted estimates and , if applicable , confounder - adjusted estimates and their precision ( e.g. 95% confidence interval ) . make clear which confounders were adjusted for and why they were included(b ) report category boundaries when continuous variables were categorized(c ) if relevant , consider translating estimates of relative risk into absolute risk for a meaningful time period other analyses17report other analyses done e.g . analyses of subgroups and interactions , and sensitivity analysesdiscussion key results18summarise key results with reference to study objectives limitations19discuss limitations of the study , taking into account sources of potential bias or imprecision . discuss both direction and magnitude of any potential bias interpretation20give a cautious overall interpretation of results considering objectives , limitations , multiplicity of analyses , results from similar studies , and other relevant evidence generalisability21discuss the generalisability ( external validity ) of the study resultsother information funding22give the source of funding and the role of the funders for the present study and , if applicable , for the original study on which the present article is basedstrobe statement checklist of items that should be included in reports of observational studiesan explanation and elaboration article discusses each checklist item and gives methodological background and published examples of transparent reporting . the strobe checklist is best used in conjunction with this article ( freely available on the web sites of plos medicine at http://www.plosmedicine.org/ , annals of internal medicine at http://www.annals.org/ , and epidemiology at http://www.epidem.com/ ) . information on the strobe initiative is available at www.strobe-statement.orggiven information separately for cases and controls in case control studies and , if applicable , for exposed and unexposed groups in cohort and cross - sectional studies strobe criteria and confirmation of items included for the obseer cohort study strobe statement checklist of items that should be included in reports of observational studies an explanation and elaboration article discusses each checklist item and gives methodological background and published examples of transparent reporting . the strobe checklist is best used in conjunction with this article ( freely available on the web sites of plos medicine at http://www.plosmedicine.org/ , annals of internal medicine at http://www.annals.org/ , and epidemiology at http://www.epidem.com/ ) . information on the strobe initiative is available at www.strobe-statement.org given information separately for cases and controls in case control studies and , if applicable , for exposed and unexposed groups in cohort and cross - sectional studies data from observational studies indicate that switching from one mph preparation to another , specifically from mph - ir to mph - mr , but even from one mph - mr formulation to another , appears to be a valid clinical approach that may contribute to treatment success . the observed improvement in various treatment outcomes might be best explained by the following factors : first , by the increased ( and thus optimised ) dose of mph ; second , the shorter intervals between visits directly after switching , leading to more intense psycho education and guidance ; third , a positive expectation of improvement by all participants ; and fourth , there might be an improvement in adherence in the long - term ( short - term improvements have been shown [ 9 , 17 ] ) accompanied by better general success of treatment , but this requires clarification in controlled studies . in summary , within the framework of successful drug management of children and adolescents with adhd , observational studies are necessary to refine the results and recommendations derived from rcts , and enable further progress towards individualised medication .

patients with adhd may have better adherence to treatment with modified - release methylphenidate ( mph - mr ) formulations , which are taken once daily , compared with immediate - release ( ir ) formulations , which need to be taken several times a day . data on long - term outcomes such as adherence may be lacking from randomised controlled trials as these are usually only short - term . observational studies , if performed and reported appropriately , can provide valuable long - term data on such outcomes , as well as additional information on effectiveness and efficiency , from a real - life setting . by reviewing previous observational studies that have investigated switching treatment from mph - ir to mph - mr , results from a new , naturalistic observational study , the obseer study , are put into context . we conclude that , based on observational trial data , switching from mph - ir to mph - mr is a valid clinical approach , with the potential for improved clinical outcome and treatment adherence .

Introduction Strengths and weaknesses of RCTs and observational trials Avoiding pitfalls in observational trials Switching between ADHD drug treatments and patient adherence Switching of MPH preparations in observational studies Conclusions

randomised controlled trials ( rcts ) in attention deficit hyperactivity disorder ( adhd ) have shown that long - acting methylphenidate ( mph ) preparations have similar efficacy to multiple doses of short - acting , immediate - release ( ir ) formulations , but with the advantage of convenient once - daily dosing [ 4 , 15 , 16 ] . patients receiving long - acting or modified - release ( mr ) , formulations may have better treatment adherence than those receiving mph - ir . however , this is still a matter of debate as rcts of mph are usually only short - term ( i.e. a few weeks duration ) and the patient population is highly selected ; therefore , results from rcts do not fully represent long - term treatment or the heterogeneous population of patients with adhd found in daily clinical practice . to learn more about adequate individualised treatment with mph preparations in a real - world setting , and to obtain a clearer picture of effectiveness ( is it of use ? ) and efficiency ( how much benefit at what cost ? ) the general trend for increasing use of mph - mr preparations in adhd [ 23 , 34 ] means that post - rct observational studies are required to ensure well - monitored , up - to - date drug management . furthermore , there is a need for an evidence - based , long - term perspective for patients . in paediatric psychopathology , the boundaries between observational and experimental studies are considered indistinct , with observational studies generally used predominantly in epidemiology and less so for clinical purposes . however , clinical observational studies may be useful in generating additional , often more definitive , conclusions about treatment guidance and safety effects ( especially over the long - term ) , or the natural history of a disorder . such findings can complement the results obtained from the experimental studies , which are a pre - condition for drug treatment evaluation . thus , rcts and observational studies can each make a useful contribution to the evaluation of treatment effectiveness . results showed that rcts and non - randomised studies can produce different results but the direction of the difference is not consistent opposing opinions about the relative merits of rcts and non - randomised studies may be underpinned by differences in threats to validity between the two study designs , which can be external ( the extent to which the results are generalisable to all potential recipients ) , or internal ( whether differences in observed effects can be attributed to differences in the intervention ) . although evidence is limited , there is a tendency for settings of non - randomised studies to be slightly more representative of those who are eligible to be included . clearly , for best effect , internal and external validity need to be balanced against each other . as observational studies are broader in scope and less rigorous than rcts , they may be prone to certain pitfalls . observational studies , including those sponsored by pharmaceutical companies , should be designed in such a way that the scientific merit of the study has the highest priority and secondary aspects are minimised . to avoid methodological shortcomings , several points need to be considered , including : selection bias ( e.g. therefore , it would be advantageous for investigators to consider all 22 points of the strobe ( strengthening the reporting of observational studies in epidemiology ) consortium checklist [ 1 , 32 ] when planning an observational study as well as when reporting it . aside from study design , the guidelines governing what data can be collected in observational studies vary between countries , so what may require ethics approval in one country would not in another . this also means that reporting these studies can be problematic if , for example , the regulations in the country in which the journal is published require ethics approval even though the country in which the data were collected did not . this question is of particular interest when considering a switch from mph - ir to mph - mr , with the potential for more convenient dosing , less stigmatisation and potentially better adherence as mph - mr only has to be administered once daily in the morning . good adherence to therapy is of utmost importance , especially in chronic mental health conditions such as adhd , and must be considered in addition to the effectiveness and efficiency of an intervention . during treatment with mph - ir preparations in adhd , poor adherence is a critical issue , ranging from 20 to 80% after 1 year , with adherence rates of 4050% after 35 years . however , better rates of adherence do not necessarily follow from switching to an improved formulation , as there could be other clinical and social factors involved that are associated with the disorganised and oppositional behaviours of patients with adhd and their families . for this reason , it is important to investigate all drug- and non - drug - related aspects of adherence when using mph - mr preparations for the treatment of adhd in the daily clinical setting . although previous rcts have compared mph - ir , mph - mr and placebo [ 15 , 16 ] , such trials do not provide any information about the clinical effects resulting from switching from mph - ir to mph - mr during the study protocol . observational studies may be better suited to investigating these effects , but to date only a few observational studies of switching to mph - mr formulations have been reported , investigating the switch to treatment with oros mph ( concerta , janssen - cilag , uk ) , mph - sodas ( ritalin - la , novartis , us ) , medikinet retard / xl ( medice , germany ) and equasym xl1 ( shire pharmaceuticals ireland limited , ireland ) [ 11 , 12 ] . one of the open - label studies was a multicentre , european study of oros mph , which included 150 children ( aged 616 years ) with adhd who were stable on mph - ir treatment before switching . in the first 3-week phase ( european and german study ) , children benefited from switching formulations , but iowa conners scores improved only for parent ratings ; teachers did not recognise a change in behaviour . tolerability of oros mph was also good , with about 90% of parents favouring continuation of the mph switch , a good predictor for better adherence in the long - term . these results are notable because the preceding treatment with mph - ir had already reduced the symptoms of adhd . for comparison , a score of 10 is usual on the parent iowa conners scale inattention / hyperactivity for unmedicated children with adhd . in the oros mph study , the baseline score ( i.e. after treatment with mph - ir , but before switching to mr ) was 6.4 , which decreased further to 4.2 after 21 days of treatment with oros mph . however , one should be cautious about drawing firm conclusions : this was an open study , the starting dose was heterogeneous and there was a tendency to increase doses while switching ( which may be interpreted as dose optimisation ) . also , the discrepancy between parent and teacher evaluation in the iowa conners scale may be due to both a pharmacokinetic weakness of the oros mph formulation in the morning ( lower mph availability ) and a pharmacokinetic strength in the afternoon ( better symptom control ) . in summary the results of this german sample suggest that children and adolescents with adhd achieve at least as good control of central adhd symptoms if there is a switch from mph - ir to oros mph. in the framework of the above - mentioned international study , 89/101 ( 88% ) patients with adhd continued to receive open but controlled ( visit every 2nd month ) treatment during a 12 month extension . unfortunately , adherence decreased by about half during the year , indicating that drop - out rates for this mph - mr preparation differed little compared with the usual drop - out rates for ir preparations [ 17 , 33 ] . hence , the predicted long - lasting improvement in adherence with mph - mr preparations was not observed in this study . however , it is important to also take into account the other benefits of switching from mph - ir to mph - mr preparations ; e.g. it has been suggested that mph - mr may be less prone to misuse or abuse compared with mph - ir . in an observational study in brazil , patients with adhd were switched from clinically stable mph - ir treatment to mph - sodas . with its 8-week duration , the study provided information about effectiveness and satisfaction with mph - sodas , but not about long - term adherence . it is probable that clinically stable patients were just asked if they would like to take part in the planned study , so that both of these observational trials were this is in contrast to the obseer ( observation of safety and effectiveness of equasym xl in routine care ) study reported in this supplement , the adore study , and a study on the effects of atomoxetine on adhd in clinical paediatric treatment settings , which can all be considered as naturalistic it was up to the physician which patients with adhd were included in the study . the mph - sodas switch study included only a small sample of children ( n = 31 ) and adults ( n = 23 ) , with a protocol adherence of 7080% after 8 weeks . there were no significant changes in behavioural parameters and side - effects on mph - sodas , but the majority of patients were reported to be satisfied with switching ( 74% ) . the authors stated that the latter may reflect the convenience of once - daily dosing of this mph - extended release , as speculated in previous studies , referring to a study in taiwan . further publications from this taiwanese research group [ 9 , 17 ] looked at children with adhd who adhered poorly to mph - ir treatment and who were offered the chance to switch to mph - oros . of 607 children with adhd aged 516 years , 240 ( 40% ) were poorly adherent to treatment . a final subgroup of 124 children ( originally 137 ; 13 were lost to follow - up ) switched to mph - oros and showed superior adherence and improved efficacy over a period of 3 weeks in an intra - individual comparison of mph - ir versus mph - oros use . this shows in principle that such a switch appears to work , at least in the short - term ( 3 weeks ) ; however , only about half of the poor adherents to medication seemed to accept the offer . reasons for , and determinants of , poor adherence may partly explain this observation ( table 1 ) . this underlines the idea that the switch of mph preparations itself ( from mph - ir to mph - mr in this case ) is not the only factor affecting treatment adherence . furthermore , switching preparations is usually accompanied by dosage increases and closer monitoring.table 1reasons for , and determinants of , poor adherence to mph - ir treatmentreasons ( patients / parents report)determinants ( univariate analysis)forgetting medicationolder ageside effects / safety concernsincreased frequency of drug administrationlack of perceived effectolder age at onset / diagnosisprivacy issuesfamily history of adhdbitter tastehigher paternal educationteacher objectionhigher mean dose of mphswitch to mph - oros was based mainly on the decision of the investigator after mutual discussion with patients and their parents ( according to gau et al . ) reasons for , and determinants of , poor adherence to mph - ir treatment switch to mph - oros was based mainly on the decision of the investigator after mutual discussion with patients and their parents ( according to gau et al . ) a switch to medikinet retard / xl ( long - acting mph with 50% mph - ir ) was explored in an open - label study of 447 patients ( aged 617 years ) who were either untreated ( but considered suitable for such treatment ) or currently receiving maintenance treatment with an approved mph dosage form ( ir or mr ) . primary outcome measures were adhd severity and side effects , which were evaluated by physicians and parents at the time of the medication changeover and 46 weeks later . as expected , the strongest effects were found in patients without prior pharmacotherapy , but significant improvements were also observed in patients with once - daily or more than once - daily mph - ir as prior medication , as well as concerta . similarly , a switch to equasym xl was analysed in another open - label study performed in children aged 617 years , either untreated or currently receiving maintenance treatment with a different mph formulation . the majority ( 65% ) of the 308 patients enrolled demonstrated a positive response , which was also observed within the group who were previously treated with mph - ir ( 64% ) and those treated with a different mph - mr formulation ( 55% ) . further results supporting a switch to mph - mr formulations were obtained in the german multicentre , prospective , observational , naturalistic obseer study on the safety and effectiveness of equasym xl , and are reported in this supplement [ 6 , 12 , 14 , 28 ] . obseer , planned and reported in line with the strobe criteria ( table 2 ) , collected behavioural data over a period of about 12 weeks from 822 children aged 617 years with adhd . a total of 574 ( 70% ) children had been treated previously with other mph formulations such as mph - ir or mph - mr . the treating physician decided independently , on the basis of his clinical assessment and experience , if treatment with equasym xl should be started . therefore , the children did not need to be clinically stable on mph - ir , as in the other switching studies described above . a subgroup of children ( n = 371 , 45% ) was switched from mph - ir ( once - daily : n = 101 , 12% ; repeated [ i.e. administered several times per day ] : n = 270 , 33% ) to mph - mr . the mph - ir once - daily prior treatment subgroup showed improvements in adherence rates during equasym xl treatment that were similar to the other subgroups ( prior treatment with mph - mr or other / not specified ) , and a particular advantage was noted for the mph - ir repeated prior treatment subgroup . as in the other mph - ir to mr switch studies , interestingly , improvements were also noted in the subgroup of patients who switched from a different mph - mr formulation to equasym xl , although these were less marked than for the mph - ir subgroups , as might be expected [ 14 , 28].table 2strobe criteria and confirmation of items included for the obseer cohort studyitem norecommendationtitle and abstract1(a ) indicate the study s design with a commonly used term in the title or the abstract(b ) provide in the abstract an informative and balanced summary of what was done and what was foundintroduction background / rationale2explain the scientific background and rationale for the investigation being reported objectives3state specific objectives , including any prespecified hypothesesmethods study design4present key elements of study design early in the paper setting5describe the setting , locations , and relevant dates , including periods of recruitment , exposure , follow - up , and data collection participants6(a ) cohort study give the eligibility criteria , and the sources and methods of selection of participants . give the rationale for the choice of cases and controlscross - sectional study give the eligibility criteria , and the sources and methods of selection of participants(b ) cohort study for matched studies , give matching criteria and number of exposed and unexposedcase control study for matched studies , give matching criteria and the number of controls per case variables7clearly define all outcomes , exposures , predictors , potential confounders , and effect modifiers . give diagnostic criteria , if applicable data sources / measurement8for each variable of interest , give sources of data and details of methods of assessment ( measurement ) . if applicable , describe which groupings were chosen and why statistical methods12(a ) describe all statistical methods , including those used to control for confounding(b ) describe any methods used to examine subgroups and interactions(c ) explain how missing data were addressed(d ) cohort study if applicable , explain how matching of cases and controls was addressedcross - sectional study if applicable , describe analytical methods taking account of sampling strategyna(e ) describe any sensitivity analysesnaresults participants13(a ) report numbers of individuals at each stage of study numbers potentially eligible , examined for eligibility , confirmed eligible , included in the study , completing follow - up , and analysed(b ) give reasons for non - participation at each stage(c ) consider use of a flow diagram descriptive data14(a ) give characteristics of study participants ( e.g. demographic , clinical , social ) and information on exposures and potential confounders(b ) indicate number of participants with missing data for each variable of interest(c ) cohort study summarise follow - up time ( e.g. average and total amount) outcome data15cohort study report numbers of outcome events or summary measures over timecase control study report numbers in each exposure category , or summary measures of exposurecross - sectional study report numbers of outcome events or summary measures main results16(a ) give unadjusted estimates and , if applicable , confounder - adjusted estimates and their precision ( e.g. analyses of subgroups and interactions , and sensitivity analysesdiscussion key results18summarise key results with reference to study objectives limitations19discuss limitations of the study , taking into account sources of potential bias or imprecision . discuss both direction and magnitude of any potential bias interpretation20give a cautious overall interpretation of results considering objectives , limitations , multiplicity of analyses , results from similar studies , and other relevant evidence generalisability21discuss the generalisability ( external validity ) of the study resultsother information funding22give the source of funding and the role of the funders for the present study and , if applicable , for the original study on which the present article is basedstrobe statement checklist of items that should be included in reports of observational studiesan explanation and elaboration article discusses each checklist item and gives methodological background and published examples of transparent reporting . information on the strobe initiative is available at www.strobe-statement.orggiven information separately for cases and controls in case control studies and , if applicable , for exposed and unexposed groups in cohort and cross - sectional studies strobe criteria and confirmation of items included for the obseer cohort study strobe statement checklist of items that should be included in reports of observational studies an explanation and elaboration article discusses each checklist item and gives methodological background and published examples of transparent reporting . information on the strobe initiative is available at www.strobe-statement.org given information separately for cases and controls in case control studies and , if applicable , for exposed and unexposed groups in cohort and cross - sectional studies data from observational studies indicate that switching from one mph preparation to another , specifically from mph - ir to mph - mr , but even from one mph - mr formulation to another , appears to be a valid clinical approach that may contribute to treatment success . the observed improvement in various treatment outcomes might be best explained by the following factors : first , by the increased ( and thus optimised ) dose of mph ; second , the shorter intervals between visits directly after switching , leading to more intense psycho education and guidance ; third , a positive expectation of improvement by all participants ; and fourth , there might be an improvement in adherence in the long - term ( short - term improvements have been shown [ 9 , 17 ] ) accompanied by better general success of treatment , but this requires clarification in controlled studies . in summary , within the framework of successful drug management of children and adolescents with adhd , observational studies are necessary to refine the results and recommendations derived from rcts , and enable further progress towards individualised medication .

osteoarthritis ( oa ) is among the diseases with the highest rates of comorbidity.1,2 comorbidity can be defined as any distinct additional clinical entity that has existed or that may occur during the clinical course of a patient who has the index disease under study.3 common comorbidities in oa include cardiovascular diseases , diabetes , obesity chronic obstructive pulmonary disease ( copd ) , chronic pain , depression , and visual and hearing impairments.4 comorbidity in older adults with oa is associated with more pain , greater limitations in daily activities , and a worse prognosis with respect to these limitations.5,6 performing exercises is one of the key recommendations in current guidelines for the management of knee oa;7,8 this has been found to relieve pain and to reduce activity limitations.9 comorbidity may interfere with the application of exercise therapy in oa , however;10 for example , in persons with heart failure , only moderate - intensity resistance training is recommended , and the last repetitions should not be straining.11 furthermore , the warming - up and cooling - down sessions should be prolonged ; perceived exertion and/or dyspnea scales should take precedence over heart rate and work rate targets ; and isometric exercises should be avoided.12 because comorbidities have a significant influence on prognosis6 and may influence treatment , they should be routinely taken into account.13 unfortunately , there is no evidence - based protocol available for the treatment of patients with knee oa and comorbidity.14 current oa guidelines do not offer specific recommendations concerning comorbidity - associated exercise adaptations.7,8,15 it is often not feasible to combine different disease - specific treatment guidelines , since one treatment might interact negatively with another treatment or affect the natural course of a coexisting disease.16 furthermore , in clinical practice , older adults with knee oa and ( severe ) comorbidity are seldom referred for exercise therapy ; often drop out at an early stage of the treatment ; or may be treated inadequately ( eg , therapists may reduce the intensity of treatment to an ineffective level ) . when dealing with comorbidity , a patient - centered rather than a disease - oriented approach , in which the process of decision making should be based on clinical reasoning , is preferred.16 the hypothesis - oriented algorithm for clinicians ( hoac ) ii17 describes a framework for clinical decision making in physical therapy ; it addresses examination , evaluation , diagnosis , prognosis , and intervention in a specific patient . although the hoac ii gives general direction in clinical reasoning , specific advice concerning comorbidity - adapted oa exercise therapy and comorbidity is not available in the literature . therefore , there is a need for comorbidity - adapted protocols for exercise therapy in older adults with knee oa and comorbidity . these protocols are expected to improve the application of oa - specific exercise therapy , may help to avoid adverse events , and may improve the outcome of exercise therapy . the evaluation of complex interventions requires a phased approach , because of specific difficulties in developing , identifying , documenting , and reproducing the intervention . to design a complex intervention , we used the medical research council ( mrc ) framework , which was developed to help researchers to define clearly where they are in the research process.18 the framework describes four phases in the design and evaluation of complex interventions : the preclinical or theoretical phase ; phase i , or the modeling phase ; phase ii , or the exploratory trial ; and phase iii , or definitive randomized controlled trial . in the preclinical or theoretical phase , the evidence that the intervention might have the desired effect is identified . the theoretical basis for the intervention is reviewed and potentially active ingredients are identified . in phase i , or the modeling phase , the components of the intervention are defined and tested , using qualitative techniques ( eg , case studies ) . in phase ii , or the exploratory trial , the optimum intervention is developed , based on the information gathered in phase i. phase iii consists of the definitive randomized controlled trial , and phase iv the long - term implementation of the intervention.18 in a previous study , restrictions and contraindications for exercise therapy for patients with knee oa and comorbidity ( theoretical phase ) were identified in the literature.10 the purpose of the present study was to develop comorbidity - adapted exercise protocols for older adults with knee oa and comorbidity ( phase i , modeling phase ) . first , based on previous work,4 we selected comorbidities in oa that 1 ) are common ( present > 5% ) , and 2 ) have impact on pain and/or affect daily functioning . the following comorbidities were selected : cardiac diseases ; hypertension ; type 2 diabetes ; obesity ; copd ; low back pain ; chronic pain ; depression ; and visual or hearing impairments.4 second , a literature search in the pubmed ( publication date range 19662009 ) database was conducted to make an inventory of restrictions and contraindications for exercise therapy in patients with oa of the knee and highly prevalent comorbidities . the method and the results of this search based on the results of the first two steps , comorbidity - related adaptations to the diagnosis and treatment of oa were described . guidelines on exercise therapy in each comorbidity ( eg , cardiac disease , diabetes , copd , and nonspecific low back pain ) were consulted.1923 if there was no exercise therapy guideline available for a specific comorbidity , an available medical guideline was used ( eg , guidelines for depression or hypertension).24,25 the principles described in these guidelines were incorporated into the adapted protocols for exercise therapy in oa of the knee . fourth , the preliminary versions of the protocols were discussed with clinical experts in the fields of each comorbid disease and , subsequently , based on their feedback , further improved . the experts had extensive experience in the fields of cardiac rehabilitation , diabetes , copd rehabilitation , chronic nonspecific pain , and visual and hearing impairments . advice was sought on the treatment of each comorbidity and on how the principles of exercise therapy and training of the comorbid diseases should be incorporated into the exercise regimen for oa of the knee . after optimizing the protocols , the clinical experts were consulted again for the collection of feedback and to gain final consensus on the protocols . fifth , the draft protocols were field - tested in a pilot study in patients with knee oa and the target comorbidities . thereafter , the protocols were further improved , based on the feedback from therapists and patients , leading to a final version of the protocols . the method for field - testing of the protocols in this pilot study participants were referred to our rehabilitation center by their general practitioner because of persistent knee problems . physical measurements were carried out by a research assistant and questionnaires were filled out by the participants . the study was approved by the medical ethical review board of the slotervaart hospital and reade , amsterdam , the netherlands . all participants gave written informed consent and the study was conducted in accordance with the handbook for good clinical research practice of the world health organization and declaration of helsinki principles.26 fourteen participants were recruited . inclusion criteria were : 1 ) diagnosis of knee oa according to the clinical american college of rheumatology criteria;27 2 ) presence of at least one of the target comorbidities , ie , coronary disease , heart failure , hypertension , type 2 diabetes , obesity , copd , chronic pain , nonspecific low back pain , depression , and vision and/or hearing impairment ( diagnosed by a medical specialist ) ; 3 ) severity score 2 of the comorbidity on the cumulative illness rating scale,28 indicating that the comorbidity has an impact on daily activities ; and 4 ) the primary treatment goal should be oa related ( instead of comorbidity related ) . exclusion criteria were : 1 ) indication for total knee replacement ; 2 ) inability to participate in treatment , eg , due to transport problems ; 3 ) insufficient capacity in the dutch language . the protocols were applied and evaluated by three qualified physical therapists with extensive experience ( 3 , 8 , and 12 years ) in knee / hip rehabilitation oa treatment . in addition , two of the three therapists were members of the committee for hip and knee oa guideline development for the royal dutch society of physical therapy . to evaluate the treatment process , the therapists completed a weekly registration form , providing information about the duration of the treatment period , content of the treatment , adaptations in the treatment due to the comorbidity , and any problems encountered in applying the protocols . adverse events , defined as any undesirable experience occurring in a subject during the study ( regardless of whether or not this was related to the treatment ) , were registered . to evaluate the feasibility of the protocols , semi - structured interviews were held by the first author ( mdr ) along with therapists and participants . topic lists were used to structure the interview ( see table 1 ) . to evaluate patient outcome after treatment , performance - based tests were performed and self - reported questionnaires were filled in by participants at baseline and directly after treatment . the western ontario and mcmaster universities osteoarthritis index ( womac ) is a disease - specific , self - administered questionnaire , developed to study patients with hip and knee oa.29,30 the womac consists of 24 questions grouped into three subscales ( pain : five questions ; stiffness : two questions ; and physical function : 17 questions ) and scaled in a 5-point likert ( lk ) scale . the maximum score in the lk scale is 20 points for pain , 8 points for stiffness , and 68 points for physical function . the womac is widely used in clinical research , and has been shown to be reliable , valid , and responsive for use in patients with oa.2931 the patient - specific functioning scale was used to evaluate limitations in activities of the individual patient.32 patients were asked , which activities do you perceive as important and were hampered by knee pain during the last week ? a list of activity suggestions was offered to support recall , and patients were allowed to provide other limited activities that were not on the list . of these activities , the difficulty of performance of the main activities were scored by self - assessment on a numeric rating scale ( [ nrs from 010 ] 0= no problems to perform the activity ; 10= impossible to perform the activity ) . patient - specific functional scale is an efficient and valid measure for assessing limitations in activities and change in limitations in persons with knee dysfunction.32 the get up and go test33,34 was performed with subjects seated on a high standard chair ( seat height 49 cm ) . the subjects were instructed to stand up without the help of the arms on the command go and walk 15 m along an unobstructed corridor as fast as possible without running . patients who normally used walking devices were allowed to use them during the test . a longer time taken to perform the test the total distance covered in meters during 6 minutes of walking was scored.35,36 quality of life was assessed with the 36-item short - form health survey ( sf-36).37 the sf-36 is a widely applied generic instrument for measuring health status and consists of eight dimensions : physical functioning , social functioning , physical role , emotional role , mental health , energy , pain and general health perception . the sf-36 gives scores on a 0100 scale , with higher scores indicating better health . the reliability ( median reliability coefficient 0.85 for all subscales ) for the sf-36 has been established,3840 and its validity has been shown in an elderly population , in which the instrument distinguished between those with and without poor health.41 psychological functioning was assessed with the hospital anxiety and depression scale ( hads).42 the hads is a self - report rating scale of 14 items on a 4-point lk scale ( range : 03 ) . it is designed to measure anxiety and depression ( seven items for each subscale ) . the total score is the sum of the 14 items , and , for each subscale , the score is the sum of the respective seven items ( ranging from 021 ) . the hads is widely used in clinical research and has been shown to be reliable , valid , and responsive for use as a screening tool in patients with oa.43 pain was assessed with a subscale of the womac . muscle strength was assessed for flexion and extension of the knee using an isokinetic dynamometer ( enknee ; enraf - nonius b.v . , patients performed a maximum of three test repetitions to measure the strength of the quadriceps and hamstrings for each knee . mean muscle strength per leg was calculated to obtain a measure of overall leg muscle strength ( in nm ) . subsequently , mean muscle strength was divided by the patient s weight to control for the correlation between muscle strength and weight . excellent intra - rater reliability ( intraclass correlation coefficient 0.93 ) has been reported for this measure in knee oa patients.45 at the end of the treatment , patients were asked to rate global perceived effect ( gpe ) of the treatment46 on a scale of 19 , with a score of 1 meaning much better , 5 meaning no change , and 9 meaning much worse . patient satisfaction with the kind of treatment was measured by the nrs ( 010 ) , with higher scores indicating greater satisfaction . additional data recorded were age , sex , and duration of complaints . the weight ( kg ) and height ( m ) were measured in standing position . comorbidity was scored with the cumulative illness rating scale.28 radiographs of the knee were scored using the grading scales proposed by kellgren and lawrence ( k&l).47 in order to evaluate the treatment process , a descriptive analysis of the treatment registration forms was performed . the feasibility of the protocols was evaluated by analyzing the notes that were taken during the interviews with the therapists and participants . a faithful depiction of the experiences of the participants and therapists was achieved by verifying with the participant or therapist whether the remarks were interpreted in a correct way by giving a summary at the end of the interview . to analyze the patient outcomes after treatment , because the data were not normally distributed pre- and posttreatment scores were analyzed with a nonparametric wilcoxon signed - rank test ( p - value < 0.05 ) . first , based on previous work,4 we selected comorbidities in oa that 1 ) are common ( present > 5% ) , and 2 ) have impact on pain and/or affect daily functioning . the following comorbidities were selected : cardiac diseases ; hypertension ; type 2 diabetes ; obesity ; copd ; low back pain ; chronic pain ; depression ; and visual or hearing impairments.4 second , a literature search in the pubmed ( publication date range 19662009 ) database was conducted to make an inventory of restrictions and contraindications for exercise therapy in patients with oa of the knee and highly prevalent comorbidities . the method and the results of this search based on the results of the first two steps , comorbidity - related adaptations to the diagnosis and treatment of oa were described . guidelines on exercise therapy in each comorbidity ( eg , cardiac disease , diabetes , copd , and nonspecific low back pain ) were consulted.1923 if there was no exercise therapy guideline available for a specific comorbidity , an available medical guideline was used ( eg , guidelines for depression or hypertension).24,25 the principles described in these guidelines were incorporated into the adapted protocols for exercise therapy in oa of the knee . fourth , the preliminary versions of the protocols were discussed with clinical experts in the fields of each comorbid disease and , subsequently , based on their feedback , further improved . the experts had extensive experience in the fields of cardiac rehabilitation , diabetes , copd rehabilitation , chronic nonspecific pain , and visual and hearing impairments . advice was sought on the treatment of each comorbidity and on how the principles of exercise therapy and training of the comorbid diseases should be incorporated into the exercise regimen for oa of the knee . after optimizing the protocols , the clinical experts were consulted again for the collection of feedback and to gain final consensus on the protocols . fifth , the draft protocols were field - tested in a pilot study in patients with knee oa and the target comorbidities . thereafter , the protocols were further improved , based on the feedback from therapists and patients , leading to a final version of the protocols . the method for field - testing of the protocols in this pilot study participants were referred to our rehabilitation center by their general practitioner because of persistent knee problems . physical measurements were carried out by a research assistant and questionnaires were filled out by the participants . the study was approved by the medical ethical review board of the slotervaart hospital and reade , amsterdam , the netherlands . all participants gave written informed consent and the study was conducted in accordance with the handbook for good clinical research practice of the world health organization and declaration of helsinki principles.26 fourteen participants were recruited . inclusion criteria were : 1 ) diagnosis of knee oa according to the clinical american college of rheumatology criteria;27 2 ) presence of at least one of the target comorbidities , ie , coronary disease , heart failure , hypertension , type 2 diabetes , obesity , copd , chronic pain , nonspecific low back pain , depression , and vision and/or hearing impairment ( diagnosed by a medical specialist ) ; 3 ) severity score 2 of the comorbidity on the cumulative illness rating scale,28 indicating that the comorbidity has an impact on daily activities ; and 4 ) the primary treatment goal should be oa related ( instead of comorbidity related ) . exclusion criteria were : 1 ) indication for total knee replacement ; 2 ) inability to participate in treatment , eg , due to transport problems ; 3 ) insufficient capacity in the dutch language . the protocols were applied and evaluated by three qualified physical therapists with extensive experience ( 3 , 8 , and 12 years ) in knee / hip rehabilitation oa treatment . in addition , two of the three therapists were members of the committee for hip and knee oa guideline development for the royal dutch society of physical therapy . to evaluate the treatment process , the therapists completed a weekly registration form , providing information about the duration of the treatment period , content of the treatment , adaptations in the treatment due to the comorbidity , and any problems encountered in applying the protocols . adverse events , defined as any undesirable experience occurring in a subject during the study ( regardless of whether or not this was related to the treatment ) , were registered . to evaluate the feasibility of the protocols , semi - structured interviews were held by the first author ( mdr ) along with therapists and participants . topic lists were used to structure the interview ( see table 1 ) . to evaluate patient outcome after treatment , performance - based tests were performed and self - reported questionnaires were filled in by participants at baseline and directly after treatment . the western ontario and mcmaster universities osteoarthritis index ( womac ) is a disease - specific , self - administered questionnaire , developed to study patients with hip and knee oa.29,30 the womac consists of 24 questions grouped into three subscales ( pain : five questions ; stiffness : two questions ; and physical function : 17 questions ) and scaled in a 5-point likert ( lk ) scale . the maximum score in the lk scale is 20 points for pain , 8 points for stiffness , and 68 points for physical function . the womac is widely used in clinical research , and has been shown to be reliable , valid , and responsive for use in patients with oa.2931 the patient - specific functioning scale was used to evaluate limitations in activities of the individual patient.32 patients were asked , which activities do you perceive as important and were hampered by knee pain during the last week ? a list of activity suggestions was offered to support recall , and patients were allowed to provide other limited activities that were not on the list . of these activities , the patient selected three main activities and ranked them in order of importance . the difficulty of performance of the main activities were scored by self - assessment on a numeric rating scale ( [ nrs from 010 ] 0= no problems to perform the activity ; 10= impossible to perform the activity ) . patient - specific functional scale is an efficient and valid measure for assessing limitations in activities and change in limitations in persons with knee dysfunction.32 the get up and go test33,34 was performed with subjects seated on a high standard chair ( seat height 49 cm ) . the subjects were instructed to stand up without the help of the arms on the command go and walk 15 m along an unobstructed corridor as fast as possible without running . patients who normally used walking devices were allowed to use them during the test . a longer time taken to perform the test the total distance covered in meters during 6 minutes of walking was scored.35,36 quality of life was assessed with the 36-item short - form health survey ( sf-36).37 the sf-36 is a widely applied generic instrument for measuring health status and consists of eight dimensions : physical functioning , social functioning , physical role , emotional role , mental health , energy , pain and general health perception . the sf-36 gives scores on a 0100 scale , with higher scores indicating better health . the reliability ( median reliability coefficient 0.85 for all subscales ) for the sf-36 has been established,3840 and its validity has been shown in an elderly population , in which the instrument distinguished between those with and without poor health.41 psychological functioning was assessed with the hospital anxiety and depression scale ( hads).42 the hads is a self - report rating scale of 14 items on a 4-point lk scale ( range : 03 ) . it is designed to measure anxiety and depression ( seven items for each subscale ) . the total score is the sum of the 14 items , and , for each subscale , the score is the sum of the respective seven items ( ranging from 021 ) . the hads is widely used in clinical research and has been shown to be reliable , valid , and responsive for use as a screening tool in patients with oa.43 pain was assessed with a subscale of the womac . muscle strength was assessed for flexion and extension of the knee using an isokinetic dynamometer ( enknee ; enraf - nonius b.v . , patients performed a maximum of three test repetitions to measure the strength of the quadriceps and hamstrings for each knee . mean muscle strength per leg was calculated to obtain a measure of overall leg muscle strength ( in nm ) . subsequently , mean muscle strength was divided by the patient s weight to control for the correlation between muscle strength and weight . excellent intra - rater reliability ( intraclass correlation coefficient 0.93 ) has been reported for this measure in knee oa patients.45 at the end of the treatment , patients were asked to rate global perceived effect ( gpe ) of the treatment46 on a scale of 19 , with a score of 1 meaning much better , 5 meaning no change , and 9 meaning much worse . patient satisfaction with the kind of treatment was measured by the nrs ( 010 ) , with higher scores indicating greater satisfaction . additional data recorded were age , sex , and duration of complaints . the weight ( kg ) and height ( m ) were measured in standing position . comorbidity was scored with the cumulative illness rating scale.28 radiographs of the knee were scored using the grading scales proposed by kellgren and lawrence ( k&l).47 in order to evaluate the treatment process , a descriptive analysis of the treatment registration forms was performed . the feasibility of the protocols was evaluated by analyzing the notes that were taken during the interviews with the therapists and participants . a faithful depiction of the experiences of the participants and therapists was achieved by verifying with the participant or therapist whether the remarks were interpreted in a correct way by giving a summary at the end of the interview . to analyze the patient outcomes after treatment , change scores were determined by subtracting the baseline scores from the posttreatment scores . because the data were not normally distributed pre- and posttreatment scores were analyzed with a nonparametric wilcoxon signed - rank test ( p - value < 0.05 ) . participants were referred to our rehabilitation center by their general practitioner because of persistent knee problems . physical measurements were carried out by a research assistant and questionnaires were filled out by the participants . the study was approved by the medical ethical review board of the slotervaart hospital and reade , amsterdam , the netherlands . all participants gave written informed consent and the study was conducted in accordance with the handbook for good clinical research practice of the world health organization and declaration of helsinki principles.26 inclusion criteria were : 1 ) diagnosis of knee oa according to the clinical american college of rheumatology criteria;27 2 ) presence of at least one of the target comorbidities , ie , coronary disease , heart failure , hypertension , type 2 diabetes , obesity , copd , chronic pain , nonspecific low back pain , depression , and vision and/or hearing impairment ( diagnosed by a medical specialist ) ; 3 ) severity score 2 of the comorbidity on the cumulative illness rating scale,28 indicating that the comorbidity has an impact on daily activities ; and 4 ) the primary treatment goal should be oa related ( instead of comorbidity related ) . exclusion criteria were : 1 ) indication for total knee replacement ; 2 ) inability to participate in treatment , eg , due to transport problems ; 3 ) insufficient capacity in the dutch language . the protocols were applied and evaluated by three qualified physical therapists with extensive experience ( 3 , 8 , and 12 years ) in knee / hip rehabilitation oa treatment . in addition , two of the three therapists were members of the committee for hip and knee oa guideline development for the royal dutch society of physical therapy . to evaluate the treatment process , the therapists completed a weekly registration form , providing information about the duration of the treatment period , content of the treatment , adaptations in the treatment due to the comorbidity , and any problems encountered in applying the protocols . adverse events , defined as any undesirable experience occurring in a subject during the study ( regardless of whether or not this was related to the treatment ) , were registered . to evaluate the feasibility of the protocols , semi - structured interviews were held by the first author ( mdr ) along with therapists and participants . topic lists were used to structure the interview ( see table 1 ) . to evaluate patient outcome after treatment , performance - based tests were performed and self - reported questionnaires were filled in by participants at baseline and directly after treatment . the western ontario and mcmaster universities osteoarthritis index ( womac ) is a disease - specific , self - administered questionnaire , developed to study patients with hip and knee oa.29,30 the womac consists of 24 questions grouped into three subscales ( pain : five questions ; stiffness : two questions ; and physical function : 17 questions ) and scaled in a 5-point likert ( lk ) scale . the maximum score in the lk scale is 20 points for pain , 8 points for stiffness , and 68 points for physical function . the womac is widely used in clinical research , and has been shown to be reliable , valid , and responsive for use in patients with oa.2931 the patient - specific functioning scale was used to evaluate limitations in activities of the individual patient.32 patients were asked , which activities do you perceive as important and were hampered by knee pain during the last week ? a list of activity suggestions was offered to support recall , and patients were allowed to provide other limited activities that were not on the list . of these activities , the patient selected three main activities and ranked them in order of importance . the difficulty of performance of the main activities were scored by self - assessment on a numeric rating scale ( [ nrs from 010 ] 0= no problems to perform the activity ; 10= impossible to perform the activity ) . patient - specific functional scale is an efficient and valid measure for assessing limitations in activities and change in limitations in persons with knee dysfunction.32 the get up and go test33,34 was performed with subjects seated on a high standard chair ( seat height 49 cm ) . the subjects were instructed to stand up without the help of the arms on the command go and walk 15 m along an unobstructed corridor as fast as possible without running . patients who normally used walking devices were allowed to use them during the test . a longer time taken to perform the test the total distance covered in meters during 6 minutes of walking was scored.35,36 quality of life was assessed with the 36-item short - form health survey ( sf-36).37 the sf-36 is a widely applied generic instrument for measuring health status and consists of eight dimensions : physical functioning , social functioning , physical role , emotional role , mental health , energy , pain and general health perception . the sf-36 gives scores on a 0100 scale , with higher scores indicating better health . the reliability ( median reliability coefficient 0.85 for all subscales ) for the sf-36 has been established,3840 and its validity has been shown in an elderly population , in which the instrument distinguished between those with and without poor health.41 psychological functioning was assessed with the hospital anxiety and depression scale ( hads).42 the hads is a self - report rating scale of 14 items on a 4-point lk scale ( range : 03 ) . it is designed to measure anxiety and depression ( seven items for each subscale ) . the total score is the sum of the 14 items , and , for each subscale , the score is the sum of the respective seven items ( ranging from 021 ) . the hads is widely used in clinical research and has been shown to be reliable , valid , and responsive for use as a screening tool in patients with oa.43 pain was assessed with a subscale of the womac . muscle strength was assessed for flexion and extension of the knee using an isokinetic dynamometer ( enknee ; enraf - nonius b.v . , patients performed a maximum of three test repetitions to measure the strength of the quadriceps and hamstrings for each knee . mean muscle strength per leg was calculated to obtain a measure of overall leg muscle strength ( in nm ) . subsequently , mean muscle strength was divided by the patient s weight to control for the correlation between muscle strength and weight . excellent intra - rater reliability ( intraclass correlation coefficient 0.93 ) has been reported for this measure in knee oa patients.45 at the end of the treatment , patients were asked to rate global perceived effect ( gpe ) of the treatment46 on a scale of 19 , with a score of 1 meaning much better , 5 meaning no change , and 9 meaning much worse . patient satisfaction with the kind of treatment was measured by the nrs ( 010 ) , with higher scores indicating greater satisfaction . the weight ( kg ) and height ( m ) were measured in standing position . comorbidity was scored with the cumulative illness rating scale.28 radiographs of the knee were scored using the grading scales proposed by kellgren and lawrence ( k&l).47 in order to evaluate the treatment process , a descriptive analysis of the treatment registration forms was performed . the feasibility of the protocols was evaluated by analyzing the notes that were taken during the interviews with the therapists and participants . a faithful depiction of the experiences of the participants and therapists was achieved by verifying with the participant or therapist whether the remarks were interpreted in a correct way by giving a summary at the end of the interview . to analyze the patient outcomes after treatment , change scores were determined by subtracting the baseline scores from the posttreatment scores . because the data were not normally distributed pre- and posttreatment scores were analyzed with a nonparametric wilcoxon signed - rank test ( p - value < 0.05 ) . eleven draft protocols for exercise therapy in persons with knee oa and comorbidity were developed based on our literature search and consultation with experts . regular oa exercise therapy as recommended in oa guidelines7,8,15 was the basis of the protocols . in the protocols , it was made explicit 1 ) how comorbidity compromises the regular application of exercise therapy in oa of the knee , and 2 ) how the therapist should consider the whole system , consisting of integrated body structures / functions and activities instead of separate organs , for all phases of treatment ( examination , evaluation , diagnosis , prognosis , and intervention ) . the hoac ii framework was used to incorporate principles of clinical reasoning into the protocols.17 the protocols on exercise therapy in persons with knee oa and comorbidities consist of a diagnostic phase and an intervention phase . each step in the protocols encourages clinical reasoning in order to tailor the diagnostic and intervention phase to the individual person . to facilitate this process , we designed a flowchart for the diagnostic and intervention phase ( figure 1 ) . the diagnostic phase includes an anamnesis , physical examination , establishment of treatment goals , and determination of the treatment strategy . during the anamnesis , oa - related problems , comorbidity - related restrictions and contraindications for exercise therapy thereafter , a clinical decision is made as to whether physical examination is possible , or whether the referring physician needs to be consulted because of contraindications for physical examination or the need for further medical information . with respect to the latter , test results of a maximum symptom - limited exercise test may be required ( for example , for persons with heart failure ) to establish an appropriate training intensity . if there are no contraindications for physical examination , comorbidity - related examination is performed according to the protocols ( eg , foot examination in patients with type 2 diabetes ) . subsequently , a decision is made as to whether there are contraindications or restrictions for exercise therapy . in case of a contraindication , if there are comorbidity - related restrictions for exercise therapy , a comorbidity - adapted program is indicated . in this phase , the therapist also considers whether referral to professionals in other disciplines ( eg , a dietician , psychologist , or occupational therapist ) is indicated . with regard to the intervention phase , the basic intervention in persons with knee oa consists of regular exercise therapy , according to the royal dutch society for physical therapy s guideline for physical therapy in patients with knee oa,15 which is similar to international guidelines.7,8 regular exercises for patients with oa comprise exercises aiming at improvement of muscle strength , aerobic capacity , flexibility , and ability to perform daily weight - bearing activities such as walking , stair climbing , and transfers ( eg , sitting down or standing up from a chair ) . individual therapy is given two times per week for between 30 to 60 minutes per session . the training intensity is increased from 40%85% of the maximum oxygen uptake ( vo2max ) or the heart rate reserve . the increase of training intensity is monitored by using the borg rpe scale ( 620 ) or heart rate frequency . the treatment ends when treatment goals are achieved or when no further improvement is feasible . the regular oa exercises are adapted to the comorbidity by changes in the duration , frequency , intensity , and type ( content ) of exercise therapy . the exact adaptations depend on restrictions for exercise therapy identified by the therapist in the diagnostic phase ( anamnesis and physical examination ) . the specific options for adaptations to oa exercises are listed in the protocols and summarized in table 1 . three participants dropped out , one because of ocular problems due to diabetes and two others because of comorbidities not included in this study ( hemochromatosis and cancer ) . nine of the eleven remaining participants had two or more comorbidities ( table 2 ) . the normal duration of the treatment in persons with knee oa without comorbidity in our center is 12 weeks . there were no adverse events reported during the study . in participants with comorbidities resulting in physiological impairments ( coronary disease , heart failure , type 2 diabetes , copd , and obesity ; n=6 ) , four were referred back to the general practitioner or specialist because of a high or fluctuating blood pressure . in these cases , while medication was adjusted , aerobic and strength exercises were postponed during the first 4 to 6 weeks of treatment . whole - body training or arm training was applied when loadability of the lower extremities was extremely low . this occurred mostly in participants with more than two comorbidities . in participants with comorbidities resulting in behavioral impairments ( chronic pain and nonspecific low back pain ; n=4 ) , adaptations were made by using a combined behavioral approach with regular oa exercises . in a time - contingent manner , the amount of physical activity was gradually increased combined with a gradual increase in the level of regular oa exercise , such as strengthening exercises of the lower limbs . adaptations were made by giving extra attention to providing positive feedback , stimulating a positive attitude toward physical activities , and gradually increasing the level of physical activity . in one participant with low vision , environmental restrictions led to adaptations in training equipment , training conditions ( eg , lighting ) , and treatment location . no specific adaptations of the oa exercise program were needed in participants with hearing impairments ( two of eleven participants ) . with regard to the feasibility of the protocols , the physical therapists who tested the protocols found that they offered guidance in setting up a treatment plan / strategy , making clinical decisions , and adapting the treatment to the comorbid disease ( table 3 ) . the following quote is from one of the physical therapists : by using the protocol i had more knowledge about the physical capabilities of the person with oa and this specific comorbidity . because of this i was able to design a more adequate training program and to better estimate the training intensity . this enabled me to treat the patient more intensively than i would have done without the use of the protocol . all physical therapists indicated that the list of restrictions for exercise therapy was a conveniently arranged checklist for the diagnostic and treatment phases . the list was also helpful in the process of clinical decision making , especially when more than one comorbidity was present . if more than one comorbidity was present , more emphasis was placed on the protocol(s ) for the comorbidity with the highest impact on physical functioning . importantly , the therapists agreed with the suggestion to increase feasibility by reducing the protocols to three main protocols . protocol a concerned physiological adaptations ( for persons with coronary artery disease , heart failure , hypertension , type 2 diabetes , obesity , and/or copd ) . protocol b concerned behavioral adaptations ( for persons with chronic pain , nonspecific low back pain , and/or depression ) . three of eleven participants would have been excluded from treatment in the absence of the protocols . they tailored the programs according to the individual s capacity , hereby preventing adverse events . the protocols were feasible in persons with mild ( k&l grade 1 ) to severe ( k&l grade 4 ) oa . all participants were satisfied with the applicability of the protocols , as indicated by a mean score of 8 points ( range : 710 ) on the nrs of satisfaction . nine of eleven participants mentioned that the therapists appeared to have a good level of knowledge about their health condition(s ) , which gave them more confidence in performing exercises . the following quote is from one participant : i felt more confident in performing exercises and was less afraid to get hypoglycemia during or after the training , because the therapist had more knowledge about my diseases and training possibilities . when i was treated in primary care for my knee complaints , i dropped out in an early phase of the treatment because my knee pain was getting worse due to the high training intensity at the beginning of the program . in addition , i was afraid when feeling an increase in my heart rate during the exercises and of becoming hypoglycemic . patient outcomes after treatment are presented in table 4 . on the womac physical functioning scale , a statistically significant improvement ( p<0.05 ) was found with an average increase of 18% above the baseline score . for the 6-minute walking test , the average increase was 13% above the baseline score ( p<0.05 ) . there was also a statistically significant decrease in pain , as measured with the womac pain subscale , where the average was 16% above the baseline score ( p<0.05 ) . the main activity limitation ( as ranked by each participant as being most important ) on the patient - specific functioning scale questionnaire also showed a statistically significant improvement ( p<0.05 ) . with regard to the extent to which symptoms changed over the period of treatment ( global perceived effect scale ) , four patients indicated that they were much or moderately improved after treatment ; four patients reported little improvement after the treatment ; and two patients reported no change in symptoms after treatment . eleven draft protocols for exercise therapy in persons with knee oa and comorbidity were developed based on our literature search and consultation with experts . regular oa exercise therapy as recommended in oa guidelines7,8,15 was the basis of the protocols . in the protocols , it was made explicit 1 ) how comorbidity compromises the regular application of exercise therapy in oa of the knee , and 2 ) how the therapist should consider the whole system , consisting of integrated body structures / functions and activities instead of separate organs , for all phases of treatment ( examination , evaluation , diagnosis , prognosis , and intervention ) . the hoac ii framework was used to incorporate principles of clinical reasoning into the protocols.17 the protocols on exercise therapy in persons with knee oa and comorbidities consist of a diagnostic phase and an intervention phase . each step in the protocols encourages clinical reasoning in order to tailor the diagnostic and intervention phase to the individual person . to facilitate this process , we designed a flowchart for the diagnostic and intervention phase ( figure 1 ) . the diagnostic phase includes an anamnesis , physical examination , establishment of treatment goals , and determination of the treatment strategy . during the anamnesis , oa - related problems , comorbidity - related restrictions and contraindications for exercise therapy thereafter , a clinical decision is made as to whether physical examination is possible , or whether the referring physician needs to be consulted because of contraindications for physical examination or the need for further medical information . with respect to the latter , test results of a maximum symptom - limited exercise test may be required ( for example , for persons with heart failure ) to establish an appropriate training intensity . if there are no contraindications for physical examination , comorbidity - related examination is performed according to the protocols ( eg , foot examination in patients with type 2 diabetes ) . subsequently , a decision is made as to whether there are contraindications or restrictions for exercise therapy . in case of a contraindication , if there are comorbidity - related restrictions for exercise therapy , a comorbidity - adapted program is indicated . in this phase , the therapist also considers whether referral to professionals in other disciplines ( eg , a dietician , psychologist , or occupational therapist ) is indicated . with regard to the intervention phase , the basic intervention in persons with knee oa consists of regular exercise therapy , according to the royal dutch society for physical therapy s guideline for physical therapy in patients with knee oa,15 which is similar to international guidelines.7,8 regular exercises for patients with oa comprise exercises aiming at improvement of muscle strength , aerobic capacity , flexibility , and ability to perform daily weight - bearing activities such as walking , stair climbing , and transfers ( eg , sitting down or standing up from a chair ) . individual therapy is given two times per week for between 30 to 60 minutes per session . the training intensity is increased from 40%85% of the maximum oxygen uptake ( vo2max ) or the heart rate reserve . the increase of training intensity is monitored by using the borg rpe scale ( 620 ) or heart rate frequency . the treatment ends when treatment goals are achieved or when no further improvement is feasible . the regular oa exercises are adapted to the comorbidity by changes in the duration , frequency , intensity , and type ( content ) of exercise therapy . the exact adaptations depend on restrictions for exercise therapy identified by the therapist in the diagnostic phase ( anamnesis and physical examination ) . the specific options for adaptations to oa exercises are listed in the protocols and summarized in table 1 . three participants dropped out , one because of ocular problems due to diabetes and two others because of comorbidities not included in this study ( hemochromatosis and cancer ) . nine of the eleven remaining participants had two or more comorbidities ( table 2 ) . the normal duration of the treatment in persons with knee oa without comorbidity in our center is 12 weeks . there were no adverse events reported during the study . in participants with comorbidities resulting in physiological impairments ( coronary disease , heart failure , type 2 diabetes , copd , and obesity ; n=6 ) , four were referred back to the general practitioner or specialist because of a high or fluctuating blood pressure . in these cases , while medication was adjusted , aerobic and strength exercises were postponed during the first 4 to 6 weeks of treatment . whole - body training or arm training was applied when loadability of the lower extremities was extremely low . this occurred mostly in participants with more than two comorbidities . in participants with comorbidities resulting in behavioral impairments ( chronic pain and nonspecific low back pain ; n=4 ) , adaptations were made by using a combined behavioral approach with regular oa exercises . in a time - contingent manner , the amount of physical activity was gradually increased combined with a gradual increase in the level of regular oa exercise , such as strengthening exercises of the lower limbs . adaptations were made by giving extra attention to providing positive feedback , stimulating a positive attitude toward physical activities , and gradually increasing the level of physical activity . in one participant with low vision , environmental restrictions led to adaptations in training equipment , training conditions ( eg , lighting ) , and treatment location . no specific adaptations of the oa exercise program were needed in participants with hearing impairments ( two of eleven participants ) . with regard to the feasibility of the protocols , the physical therapists who tested the protocols found that they offered guidance in setting up a treatment plan / strategy , making clinical decisions , and adapting the treatment to the comorbid disease ( table 3 ) . the following quote is from one of the physical therapists : by using the protocol i had more knowledge about the physical capabilities of the person with oa and this specific comorbidity . because of this i was able to design a more adequate training program and to better estimate the training intensity . this enabled me to treat the patient more intensively than i would have done without the use of the protocol . all physical therapists indicated that the list of restrictions for exercise therapy was a conveniently arranged checklist for the diagnostic and treatment phases . the list was also helpful in the process of clinical decision making , especially when more than one comorbidity was present . if more than one comorbidity was present , more emphasis was placed on the protocol(s ) for the comorbidity with the highest impact on physical functioning . importantly , the therapists agreed with the suggestion to increase feasibility by reducing the protocols to three main protocols . protocol a concerned physiological adaptations ( for persons with coronary artery disease , heart failure , hypertension , type 2 diabetes , obesity , and/or copd ) . protocol b concerned behavioral adaptations ( for persons with chronic pain , nonspecific low back pain , and/or depression ) . three of eleven participants would have been excluded from treatment in the absence of the protocols . they tailored the programs according to the individual s capacity , hereby preventing adverse events . the protocols were feasible in persons with mild ( k&l grade 1 ) to severe ( k&l grade 4 ) oa . all participants were satisfied with the applicability of the protocols , as indicated by a mean score of 8 points ( range : 710 ) on the nrs of satisfaction . nine of eleven participants mentioned that the therapists appeared to have a good level of knowledge about their health condition(s ) , which gave them more confidence in performing exercises . the following quote is from one participant : i felt more confident in performing exercises and was less afraid to get hypoglycemia during or after the training , because the therapist had more knowledge about my diseases and training possibilities . when i was treated in primary care for my knee complaints , i dropped out in an early phase of the treatment because my knee pain was getting worse due to the high training intensity at the beginning of the program . in addition , i was afraid when feeling an increase in my heart rate during the exercises and of becoming hypoglycemic . patient outcomes after treatment are presented in table 4 . on the womac physical functioning scale , a statistically significant improvement ( p<0.05 ) was found with an average increase of 18% above the baseline score . for the 6-minute walking test , there was also a statistically significant decrease in pain , as measured with the womac pain subscale , where the average was 16% above the baseline score ( p<0.05 ) . the main activity limitation ( as ranked by each participant as being most important ) on the patient - specific functioning scale questionnaire also showed a statistically significant improvement ( p<0.05 ) . no significant changes were found for the other measurements . with regard to the extent to which symptoms changed over the period of treatment ( global perceived effect scale ) , four patients indicated that they were much or moderately improved after treatment ; four patients reported little improvement after the treatment ; and two patients reported no change in symptoms after treatment . oa . nevertheless , no evidence - based recommendations are available concerning comorbidity - adapted exercises in patients with knee oa . the present study concerns the development of comorbidity - adapted exercise protocols in patients with knee oa . the protocols were found to be feasible and helpful in clinical reasoning and adapting oa exercises . to our knowledge , this is the first time that comorbidity - adapted protocols have been developed for exercise therapy in patients with oa of the knee and comorbidity . evidence - based diagnostics and treatment strategies generally overlook comorbidity.13,14 the interacting effects of diseases and their management require more complex and individualized care than simply the sum of separate guideline components . eleven comorbidity - adapted exercise protocols were developed for patients with knee oa and comorbidity . the protocols were found to provide guidance in clinical reasoning to direct both the diagnostic and treatment phases in persons with oa and complex , comorbidity - related health problems . the results of our field - testing revealed that the eleven protocols could be reduced to three main protocols due to overlap in diagnostics and/or treatment - related adaptations of the comorbidities and to improve user - friendliness . protocol a concerned physiological adaptations ( for persons with coronary artery disease , heart failure , hypertension , type 2 diabetes , obesity , and/or copd ) . protocol b concerned behavioral adaptations ( for persons with chronic pain , nonspecific low back pain , and/or depression ) . protocol c concerned environmental adaptations ( for persons with visual and/or hearing impairments ) . the protocols encourage physical therapists to think in advance about 1 ) how comorbidity compromises the regular application of exercise therapy by using the list of restrictions for exercise therapy of the comorbid disease and 2 ) how to adapt the exercise . as expected , in participants with physiological impairments ( eg , coronary disease ) , the training intensity and frequency and type of exercises were adapted to the comorbidity . in participants with behavioral impairments ( eg , chronic pain ) , a combination of regular oa exercises with a behavioral approach was preferred , in which the level of physical activity was gradually increased in a time - contingent manner . in one participant with visual impairments , environmental adaptations were applied ( eg , adapting the lighting in the exercise hall ) . an average increase of 18% on the physical functioning subscale and a decrease of 16% on the pain subscale were found with womac , which can be regarded as clinically important , relevant change.48 the treatment was safe and , by using the protocols , more patients with oa and comorbidity could participate in the exercise therapy . when taking comorbidity into account , adequate clinical reasoning is essential in order to deal with persons with a complex health status . physical therapists need to be alert to changes in health conditions that may necessitate further adaptations of the exercises . physical therapists with experience in dealing with chronic conditions may have an advantage in clinical reasoning and in the adaption of exercise programs in accordance with the comorbidity . physical therapists need to have an advanced understanding of complex system interrelationships regarding multiple morbidities . therefore , therapists should receive specific training to increase their knowledge about various comorbidities and their effects on oa exercise therapy . a number of remarks can be made about the usage and further development of the protocols . first , part of the results are based on personal opinions of three physical therapists working in a rehabilitation setting . to make the protocols broadly applicable , testing among various physical therapists practicing in different settings second , the protocols were tested on eleven patients with knee oa and various comorbidities . to compare the effectiveness of the protocols to usual care , a randomized clinical trial should be performed . comorbidity - adapted exercise protocols for patients with knee oa were developed that can provide guidance in clinical reasoning with regard to diagnostics and treatment . to evaluate the effectiveness of treatment in line with our protocols , a randomized clinical trial should be performed .

backgroundexercise therapy is generally recommended for patients with osteoarthritis ( oa ) of the knee . comorbidity , which is highly prevalent in oa , may interfere with exercise therapy . to date , there is no evidence - based protocol for the treatment of patients with knee oa and comorbidity . special protocols adapted to the comorbidity may facilitate the application of exercise therapy in patients with knee oa and one or more comorbidities.purposethe purpose of this study was to develop comorbidity - adapted exercise protocols for patients with knee oa and comorbidity.methodseveral steps were undertaken to develop comorbidity - adapted protocols : selection of highly prevalent comorbidities in oa , a literature search to identify restrictions and contraindications for exercise therapy for the various comorbid diseases , consultation of experts on each comorbid disease , and field testing of the protocol in eleven patients with knee oa and comorbidity.resultsbased on literature and expert opinion , comorbidity - adapted protocols were developed for highly prevalent comorbidities in oa . field testing showed that the protocols provided guidance in clinical decision making in both the diagnostic and the treatment phase . because of overlap , the number of exercise protocols could be reduced to three : one for physiological adaptations ( coronary disease , heart failure , hypertension , diabetes type 2 , chronic obstructive pulmonary diseases , obesity ) , one for behavioral adaptations ( chronic a - specific pain , nonspecific low back pain , depression ) , and one for environmental adaptations ( visual or hearing impairments ) . evaluation of patient outcome after treatment showed significant ( p<0.05 ) and clinically relevant improvements in activity limitations and pain.conclusioncomorbidity-adapted exercise protocols for patients with knee oa were developed , providing guidance in clinical reasoning with regard to diagnostics and treatment . to evaluate the effectiveness of treatment in line with our protocols , a randomized clinical trial should be performed .

Introduction Methods Development of comorbidity-adapted protocols Field-testing Procedure Participants Therapists Measurements Analysis Results Results of the development of the protocols Results from the field-testing Discussion Conclusion

osteoarthritis ( oa ) is among the diseases with the highest rates of comorbidity.1,2 comorbidity can be defined as any distinct additional clinical entity that has existed or that may occur during the clinical course of a patient who has the index disease under study.3 common comorbidities in oa include cardiovascular diseases , diabetes , obesity chronic obstructive pulmonary disease ( copd ) , chronic pain , depression , and visual and hearing impairments.4 comorbidity in older adults with oa is associated with more pain , greater limitations in daily activities , and a worse prognosis with respect to these limitations.5,6 performing exercises is one of the key recommendations in current guidelines for the management of knee oa;7,8 this has been found to relieve pain and to reduce activity limitations.9 comorbidity may interfere with the application of exercise therapy in oa , however;10 for example , in persons with heart failure , only moderate - intensity resistance training is recommended , and the last repetitions should not be straining.11 furthermore , the warming - up and cooling - down sessions should be prolonged ; perceived exertion and/or dyspnea scales should take precedence over heart rate and work rate targets ; and isometric exercises should be avoided.12 because comorbidities have a significant influence on prognosis6 and may influence treatment , they should be routinely taken into account.13 unfortunately , there is no evidence - based protocol available for the treatment of patients with knee oa and comorbidity.14 current oa guidelines do not offer specific recommendations concerning comorbidity - associated exercise adaptations.7,8,15 it is often not feasible to combine different disease - specific treatment guidelines , since one treatment might interact negatively with another treatment or affect the natural course of a coexisting disease.16 furthermore , in clinical practice , older adults with knee oa and ( severe ) comorbidity are seldom referred for exercise therapy ; often drop out at an early stage of the treatment ; or may be treated inadequately ( eg , therapists may reduce the intensity of treatment to an ineffective level ) . when dealing with comorbidity , a patient - centered rather than a disease - oriented approach , in which the process of decision making should be based on clinical reasoning , is preferred.16 the hypothesis - oriented algorithm for clinicians ( hoac ) ii17 describes a framework for clinical decision making in physical therapy ; it addresses examination , evaluation , diagnosis , prognosis , and intervention in a specific patient . although the hoac ii gives general direction in clinical reasoning , specific advice concerning comorbidity - adapted oa exercise therapy and comorbidity is not available in the literature . therefore , there is a need for comorbidity - adapted protocols for exercise therapy in older adults with knee oa and comorbidity . these protocols are expected to improve the application of oa - specific exercise therapy , may help to avoid adverse events , and may improve the outcome of exercise therapy . in phase ii , or the exploratory trial , the optimum intervention is developed , based on the information gathered in phase i. phase iii consists of the definitive randomized controlled trial , and phase iv the long - term implementation of the intervention.18 in a previous study , restrictions and contraindications for exercise therapy for patients with knee oa and comorbidity ( theoretical phase ) were identified in the literature.10 the purpose of the present study was to develop comorbidity - adapted exercise protocols for older adults with knee oa and comorbidity ( phase i , modeling phase ) . the following comorbidities were selected : cardiac diseases ; hypertension ; type 2 diabetes ; obesity ; copd ; low back pain ; chronic pain ; depression ; and visual or hearing impairments.4 second , a literature search in the pubmed ( publication date range 19662009 ) database was conducted to make an inventory of restrictions and contraindications for exercise therapy in patients with oa of the knee and highly prevalent comorbidities . the method and the results of this search based on the results of the first two steps , comorbidity - related adaptations to the diagnosis and treatment of oa were described . guidelines on exercise therapy in each comorbidity ( eg , cardiac disease , diabetes , copd , and nonspecific low back pain ) were consulted.1923 if there was no exercise therapy guideline available for a specific comorbidity , an available medical guideline was used ( eg , guidelines for depression or hypertension).24,25 the principles described in these guidelines were incorporated into the adapted protocols for exercise therapy in oa of the knee . fourth , the preliminary versions of the protocols were discussed with clinical experts in the fields of each comorbid disease and , subsequently , based on their feedback , further improved . advice was sought on the treatment of each comorbidity and on how the principles of exercise therapy and training of the comorbid diseases should be incorporated into the exercise regimen for oa of the knee . fifth , the draft protocols were field - tested in a pilot study in patients with knee oa and the target comorbidities . inclusion criteria were : 1 ) diagnosis of knee oa according to the clinical american college of rheumatology criteria;27 2 ) presence of at least one of the target comorbidities , ie , coronary disease , heart failure , hypertension , type 2 diabetes , obesity , copd , chronic pain , nonspecific low back pain , depression , and vision and/or hearing impairment ( diagnosed by a medical specialist ) ; 3 ) severity score 2 of the comorbidity on the cumulative illness rating scale,28 indicating that the comorbidity has an impact on daily activities ; and 4 ) the primary treatment goal should be oa related ( instead of comorbidity related ) . to evaluate the treatment process , the therapists completed a weekly registration form , providing information about the duration of the treatment period , content of the treatment , adaptations in the treatment due to the comorbidity , and any problems encountered in applying the protocols . the womac is widely used in clinical research , and has been shown to be reliable , valid , and responsive for use in patients with oa.2931 the patient - specific functioning scale was used to evaluate limitations in activities of the individual patient.32 patients were asked , which activities do you perceive as important and were hampered by knee pain during the last week ? the following comorbidities were selected : cardiac diseases ; hypertension ; type 2 diabetes ; obesity ; copd ; low back pain ; chronic pain ; depression ; and visual or hearing impairments.4 second , a literature search in the pubmed ( publication date range 19662009 ) database was conducted to make an inventory of restrictions and contraindications for exercise therapy in patients with oa of the knee and highly prevalent comorbidities . the method and the results of this search based on the results of the first two steps , comorbidity - related adaptations to the diagnosis and treatment of oa were described . guidelines on exercise therapy in each comorbidity ( eg , cardiac disease , diabetes , copd , and nonspecific low back pain ) were consulted.1923 if there was no exercise therapy guideline available for a specific comorbidity , an available medical guideline was used ( eg , guidelines for depression or hypertension).24,25 the principles described in these guidelines were incorporated into the adapted protocols for exercise therapy in oa of the knee . advice was sought on the treatment of each comorbidity and on how the principles of exercise therapy and training of the comorbid diseases should be incorporated into the exercise regimen for oa of the knee . fifth , the draft protocols were field - tested in a pilot study in patients with knee oa and the target comorbidities . inclusion criteria were : 1 ) diagnosis of knee oa according to the clinical american college of rheumatology criteria;27 2 ) presence of at least one of the target comorbidities , ie , coronary disease , heart failure , hypertension , type 2 diabetes , obesity , copd , chronic pain , nonspecific low back pain , depression , and vision and/or hearing impairment ( diagnosed by a medical specialist ) ; 3 ) severity score 2 of the comorbidity on the cumulative illness rating scale,28 indicating that the comorbidity has an impact on daily activities ; and 4 ) the primary treatment goal should be oa related ( instead of comorbidity related ) . to evaluate the treatment process , the therapists completed a weekly registration form , providing information about the duration of the treatment period , content of the treatment , adaptations in the treatment due to the comorbidity , and any problems encountered in applying the protocols . the womac is widely used in clinical research , and has been shown to be reliable , valid , and responsive for use in patients with oa.2931 the patient - specific functioning scale was used to evaluate limitations in activities of the individual patient.32 patients were asked , which activities do you perceive as important and were hampered by knee pain during the last week ? comorbidity was scored with the cumulative illness rating scale.28 radiographs of the knee were scored using the grading scales proposed by kellgren and lawrence ( k&l).47 in order to evaluate the treatment process , a descriptive analysis of the treatment registration forms was performed . all participants gave written informed consent and the study was conducted in accordance with the handbook for good clinical research practice of the world health organization and declaration of helsinki principles.26 inclusion criteria were : 1 ) diagnosis of knee oa according to the clinical american college of rheumatology criteria;27 2 ) presence of at least one of the target comorbidities , ie , coronary disease , heart failure , hypertension , type 2 diabetes , obesity , copd , chronic pain , nonspecific low back pain , depression , and vision and/or hearing impairment ( diagnosed by a medical specialist ) ; 3 ) severity score 2 of the comorbidity on the cumulative illness rating scale,28 indicating that the comorbidity has an impact on daily activities ; and 4 ) the primary treatment goal should be oa related ( instead of comorbidity related ) . to evaluate the treatment process , the therapists completed a weekly registration form , providing information about the duration of the treatment period , content of the treatment , adaptations in the treatment due to the comorbidity , and any problems encountered in applying the protocols . the womac is widely used in clinical research , and has been shown to be reliable , valid , and responsive for use in patients with oa.2931 the patient - specific functioning scale was used to evaluate limitations in activities of the individual patient.32 patients were asked , which activities do you perceive as important and were hampered by knee pain during the last week ? comorbidity was scored with the cumulative illness rating scale.28 radiographs of the knee were scored using the grading scales proposed by kellgren and lawrence ( k&l).47 in order to evaluate the treatment process , a descriptive analysis of the treatment registration forms was performed . eleven draft protocols for exercise therapy in persons with knee oa and comorbidity were developed based on our literature search and consultation with experts . in the protocols , it was made explicit 1 ) how comorbidity compromises the regular application of exercise therapy in oa of the knee , and 2 ) how the therapist should consider the whole system , consisting of integrated body structures / functions and activities instead of separate organs , for all phases of treatment ( examination , evaluation , diagnosis , prognosis , and intervention ) . the hoac ii framework was used to incorporate principles of clinical reasoning into the protocols.17 the protocols on exercise therapy in persons with knee oa and comorbidities consist of a diagnostic phase and an intervention phase . during the anamnesis , oa - related problems , comorbidity - related restrictions and contraindications for exercise therapy thereafter , a clinical decision is made as to whether physical examination is possible , or whether the referring physician needs to be consulted because of contraindications for physical examination or the need for further medical information . if there are no contraindications for physical examination , comorbidity - related examination is performed according to the protocols ( eg , foot examination in patients with type 2 diabetes ) . with regard to the intervention phase , the basic intervention in persons with knee oa consists of regular exercise therapy , according to the royal dutch society for physical therapy s guideline for physical therapy in patients with knee oa,15 which is similar to international guidelines.7,8 regular exercises for patients with oa comprise exercises aiming at improvement of muscle strength , aerobic capacity , flexibility , and ability to perform daily weight - bearing activities such as walking , stair climbing , and transfers ( eg , sitting down or standing up from a chair ) . the regular oa exercises are adapted to the comorbidity by changes in the duration , frequency , intensity , and type ( content ) of exercise therapy . in participants with comorbidities resulting in physiological impairments ( coronary disease , heart failure , type 2 diabetes , copd , and obesity ; n=6 ) , four were referred back to the general practitioner or specialist because of a high or fluctuating blood pressure . with regard to the feasibility of the protocols , the physical therapists who tested the protocols found that they offered guidance in setting up a treatment plan / strategy , making clinical decisions , and adapting the treatment to the comorbid disease ( table 3 ) . all physical therapists indicated that the list of restrictions for exercise therapy was a conveniently arranged checklist for the diagnostic and treatment phases . protocol a concerned physiological adaptations ( for persons with coronary artery disease , heart failure , hypertension , type 2 diabetes , obesity , and/or copd ) . protocol b concerned behavioral adaptations ( for persons with chronic pain , nonspecific low back pain , and/or depression ) . with regard to the extent to which symptoms changed over the period of treatment ( global perceived effect scale ) , four patients indicated that they were much or moderately improved after treatment ; four patients reported little improvement after the treatment ; and two patients reported no change in symptoms after treatment . eleven draft protocols for exercise therapy in persons with knee oa and comorbidity were developed based on our literature search and consultation with experts . in the protocols , it was made explicit 1 ) how comorbidity compromises the regular application of exercise therapy in oa of the knee , and 2 ) how the therapist should consider the whole system , consisting of integrated body structures / functions and activities instead of separate organs , for all phases of treatment ( examination , evaluation , diagnosis , prognosis , and intervention ) . the hoac ii framework was used to incorporate principles of clinical reasoning into the protocols.17 the protocols on exercise therapy in persons with knee oa and comorbidities consist of a diagnostic phase and an intervention phase . during the anamnesis , oa - related problems , comorbidity - related restrictions and contraindications for exercise therapy thereafter , a clinical decision is made as to whether physical examination is possible , or whether the referring physician needs to be consulted because of contraindications for physical examination or the need for further medical information . if there are no contraindications for physical examination , comorbidity - related examination is performed according to the protocols ( eg , foot examination in patients with type 2 diabetes ) . with regard to the intervention phase , the basic intervention in persons with knee oa consists of regular exercise therapy , according to the royal dutch society for physical therapy s guideline for physical therapy in patients with knee oa,15 which is similar to international guidelines.7,8 regular exercises for patients with oa comprise exercises aiming at improvement of muscle strength , aerobic capacity , flexibility , and ability to perform daily weight - bearing activities such as walking , stair climbing , and transfers ( eg , sitting down or standing up from a chair ) . the regular oa exercises are adapted to the comorbidity by changes in the duration , frequency , intensity , and type ( content ) of exercise therapy . in participants with comorbidities resulting in physiological impairments ( coronary disease , heart failure , type 2 diabetes , copd , and obesity ; n=6 ) , four were referred back to the general practitioner or specialist because of a high or fluctuating blood pressure . with regard to the feasibility of the protocols , the physical therapists who tested the protocols found that they offered guidance in setting up a treatment plan / strategy , making clinical decisions , and adapting the treatment to the comorbid disease ( table 3 ) . all physical therapists indicated that the list of restrictions for exercise therapy was a conveniently arranged checklist for the diagnostic and treatment phases . protocol a concerned physiological adaptations ( for persons with coronary artery disease , heart failure , hypertension , type 2 diabetes , obesity , and/or copd ) . protocol b concerned behavioral adaptations ( for persons with chronic pain , nonspecific low back pain , and/or depression ) . for the 6-minute walking test , there was also a statistically significant decrease in pain , as measured with the womac pain subscale , where the average was 16% above the baseline score ( p<0.05 ) . with regard to the extent to which symptoms changed over the period of treatment ( global perceived effect scale ) , four patients indicated that they were much or moderately improved after treatment ; four patients reported little improvement after the treatment ; and two patients reported no change in symptoms after treatment . nevertheless , no evidence - based recommendations are available concerning comorbidity - adapted exercises in patients with knee oa . the present study concerns the development of comorbidity - adapted exercise protocols in patients with knee oa . to our knowledge , this is the first time that comorbidity - adapted protocols have been developed for exercise therapy in patients with oa of the knee and comorbidity . eleven comorbidity - adapted exercise protocols were developed for patients with knee oa and comorbidity . the protocols were found to provide guidance in clinical reasoning to direct both the diagnostic and treatment phases in persons with oa and complex , comorbidity - related health problems . the results of our field - testing revealed that the eleven protocols could be reduced to three main protocols due to overlap in diagnostics and/or treatment - related adaptations of the comorbidities and to improve user - friendliness . protocol a concerned physiological adaptations ( for persons with coronary artery disease , heart failure , hypertension , type 2 diabetes , obesity , and/or copd ) . protocol b concerned behavioral adaptations ( for persons with chronic pain , nonspecific low back pain , and/or depression ) . the protocols encourage physical therapists to think in advance about 1 ) how comorbidity compromises the regular application of exercise therapy by using the list of restrictions for exercise therapy of the comorbid disease and 2 ) how to adapt the exercise . as expected , in participants with physiological impairments ( eg , coronary disease ) , the training intensity and frequency and type of exercises were adapted to the comorbidity . an average increase of 18% on the physical functioning subscale and a decrease of 16% on the pain subscale were found with womac , which can be regarded as clinically important , relevant change.48 the treatment was safe and , by using the protocols , more patients with oa and comorbidity could participate in the exercise therapy . to make the protocols broadly applicable , testing among various physical therapists practicing in different settings second , the protocols were tested on eleven patients with knee oa and various comorbidities . to compare the effectiveness of the protocols to usual care , a randomized clinical trial should be performed . comorbidity - adapted exercise protocols for patients with knee oa were developed that can provide guidance in clinical reasoning with regard to diagnostics and treatment . to evaluate the effectiveness of treatment in line with our protocols , a randomized clinical trial should be performed .

increasing salinization of agricultural soils is one of the most challenging issues faced by modern agriculture . in excess of 30% of cultivated soils are affected by salinity ( epstein et al . , 1980 ; zhu et al . , 1997 much of this salinization is attributable to the infiltration and accumulation of nacl ( zhu , 2001 ; munns , 2005 ) , often resulting in soil na concentrations above 40 mm , and growth suppression in most crops ( munns , 2005 ) . one of the key physiological processes disrupted by na supply in this toxic range is the maintenance of cellular and whole - plant potassium homeostasis ( rains and epstein , 1967 ; flowers and luchli , 1983 ; watad et al . , 1991 ; gaxiola et al . , 1992 ; , 1998 ; santa - mara and epstein , 2001 ; peng et al . , 2004 ; cakmak , 2005 ; kader and lindberg , 2005 ; kronzucker et al . , 2006 the ratio of k to na is considered an excellent indicator of plant tolerance to salinity ; the higher the ratio , the higher the plant 's tolerance ( flowers and hajibagheri , 2001 ; cakmak , 2005 ; chen et al . as a result of this observation , selection or breeding cultivars that maintain high k : na ratios has emerged as an important strategy to counteract the detrimental effects of soil salinity ( deal et al . , 1999 ; a more precise proposal has been that a high k : na ratio specifically in the cytosolic compartment is critical to plant survival under sodium challenge , while a decrease in this ratio will predict the onset of salinity stress and growth decline ( hajibagheri et al . , 1987 , 1989 ; maathuis and amtmann , 1999 ; flowers and hajibagheri , 2001 ; carden et al . , 2003 ; peng et al . , 2004 ; kader et al . , 2006 ; james et al . , 2006 ; chen et al . , 2007a ; davenport et al . , 2007 ; obata et al . , 2007 ; takahashi et al . , 2007 this proposal has gained wide acceptance , even though cytosolic k : na ratios are , in fact , rarely measured . in a recent study in barley ( hordeum vulgare l. ) , it was shown that , at low to intermediate levels of external k supply ( [ k]ext=0.11.5 mm ) , and at varying salinity levels , the ratio did not in fact correlate with seedling growth in this major cereal ( kronzucker et al . , 2006 ) . on the contrary , no difference in growth was observed in the presence of a > 5-fold variation in the cytosolic k : na ratio . the study further demonstrated that na suppressed k influx across the plasma membrane to a similar extent at 0.1 and 1.5 mm [ k]ext , concentrations at which high - affinity transport systems for k predominate ( epstein et al . , 1963 ; kochian and lucas , 1982 ; szczerba et al . , 2006 ) , while na influxes and cytosolic pools were unaffected by k. however , a significant suppression of the cytosolic k pool by na was seen only at the higher [ k]ext , suggesting that different cellular responses may come into effect as high - affinity k transport gives way to low - affinity transport ( see szczerba et al . , 2006 ) . in the present study , to examine further the proposed pivotal role of k homeostasis in salinity stress and tolerance , the effects of k supply across the low - affinity transport range of k ( up to 40 mm [ k]ext ) upon the primary fluxes and cytosolic pools of na , and , conversely , the effects of na upon k fluxes and pools in this range were investigated . such an examination was particularly necessary in the light of recent disagreements in the literature pertaining to ( i ) the size of cytosolic na pools ( flowers and hajibagheri , 2001 ; carden et al . , 2003 ; james et al . ( ii ) the proposed , but as yet unresolved , roles of molecular candidates for toxic na influx into the plant ( tester and davenport , 2003 ; flowers , 2006 ; wang et al . , 2007 ) . the primary candidates proposed are k - specific channels ( wang et al . , 2007 ) , non - selective cation channels ( nsccs ; demidchik et al . , 2002 ) , hkt - type transporters ( rodriguez - navarro and rubio , 2006 ) , and the low - affinity cation transporter lct ( amtmann et al . , 2001 ) . indeed , were these candidates to catalyse toxicologically significant fluxes of sodium , they should be competitively influenced by the presence of potassium . for these reasons , a detailed study of the interactions between the two ions was carried out along a wide gradient of external k supply . klondike ) were surface - sterilized by immersing seeds in 1.0% sodium hypochlorite for 10 min . seeds were then washed under running tap water for 3 h , placed on discs of plastic mesh , and covered by 2 cm of moist sand . germination proceeded for the following 3 d in a walk - in growth chamber equipped with fluorescent lights ( philips econ - o - watt , f96t12 , with an irradiation of 200 mol photons m s at plant height , for 16 h d ) , and having a day / night temperature cycle of 20 c/15 c , and relative humidity of 70% . following germination , seedlings were transferred to opaque plastic 4 l hydroponic vessels , filled with modified quarter - strength johnson 's solution , consisting of : 5 mm ca(no3)2 , 0.5 mm nah2po4 , 0.25 mm mgso4 , 0.2 mm caso4 , 0.125 m na2moo4 , 20 m feedta , 25 m h3bo3 , 2 m znso4 , 0.5 m mnso4 , and 0.5 m cuso4 . control plants had no additional sodium , while salt - stressed plants were treated with 100 mm na ( as nacl ) . potassium concentrations were adjusted to treatments of 1.5 , 5 , 10 , 20 , and 40 mm by addition of k2so4 . solutions were replaced after 2 d. plants remained in hydroponic solutions for 4 d prior to experimentation . in select treatments ( 1.5 mm and 40 mm k , at low and high nacl ) , plants were also grown for 2 weeks ( 11 d in solution ; see fig . compartmental analysis by tracer efflux was conducted as described in detail elsewhere ( kronzucker et al . roots of intact seedlings were immersed for 1 h in a nutrient solution identical to the growth solution , except that it contained na or k in addition to non - radioactive na or k. roots were desorbed of radioactivity in tracer - free solutions for the monitoring of na or k efflux , by periodic washing with a timed series of non - radioactive aliquots of nutrient solution ( kronzucker et al . , 1999 , 2006 ; the time course of aliquots was as follows : 15 s ( 4 ) , 20 s ( 3 ) , 30 s ( 2 ) , 40 s ( 1 ) , 50 s ( 1 ) , 1 min ( 5 ) , 1.25 min ( 1 ) , 1.5 min ( 1 ) , 1.75 min ( 1 ) , and 2 min ( 8 ) . radioactivity was measured in aliquots and plant tissues by gamma counting , using a canberra - packard counter , quantum cobra series ii , model 5003 . unidirectional na or k fluxes were determined using standard analyses ( for further details , see kronzucker et al . efflux ( co ) was calculated from the initial rate of na or k release from the cytosol , divided by initial cytosolic specific activity ( sc ) of na or k in this compartment ; sc was estimated by using labelling time ( tl ) in the external medium of known specific activity ( so ) , and the kinetic constant k that describes the exponential rate of cytosolic tracer exchange , using the relationship sc = so(1e ) . 1).(ii ) net fluxes were determined from retention of tracer in root and shoot at the end of the desorption protocol , divided by so , while influx ( oc ) was calculated from the sum of co and the net flux.(iii ) cytosolic concentrations of na ( [ na]cyt ) or k ( [ k]cyt ) were determined by integrating rates of radioactivity release from this compartment ; in simplified form , this calculation is made using the equation [ na or k]cyt=oc / k , where is a proportionality constant accounting for the cytosolic compartment comprising 5% of tissue volume . activity coefficients ( ) for cytosolic concentrations were estimated using the debye hckel onsager equation , adapted for the monovalent cations na and k : log=(0.5i)/(1+i ) where i is ionic strength of the cytosol ( assuming that the dominant cations are na and k , and that these are charge - balanced by monovalent anions ; see jander and blasius , 1988 ) . ( i ) efflux ( co ) was calculated from the initial rate of na or k release from the cytosol , divided by initial cytosolic specific activity ( sc ) of na or k in this compartment ; sc was estimated by using labelling time ( tl ) in the external medium of known specific activity ( so ) , and the kinetic constant k that describes the exponential rate of cytosolic tracer exchange , using the relationship sc = so(1e ) . ( ii ) net fluxes were determined from retention of tracer in root and shoot at the end of the desorption protocol , divided by so , while influx ( oc ) was calculated from the sum of co and the net flux . ( iii ) cytosolic concentrations of na ( [ na]cyt ) or k ( [ k]cyt ) were determined by integrating rates of radioactivity release from this compartment ; in simplified form , this calculation is made using the equation [ na or k]cyt=oc / k , where is a proportionality constant accounting for the cytosolic compartment comprising 5% of tissue volume . activity coefficients ( ) for cytosolic concentrations were estimated using the debye hckel onsager equation , adapted for the monovalent cations na and k : log=(0.5i)/(1+i ) where i is ionic strength of the cytosol ( assuming that the dominant cations are na and k , and that these are charge - balanced by monovalent anions ; see jander and blasius , 1988 ) . representative plots of k and na ( inset ) efflux from roots of intact barley seedlings , under varying ionic conditions . roots had been preloaded for 60 min in radioactive solution , then eluted of radioactivity in a timed series of non - radioactive growth - solution aliquots . dashed lines represent the slowest exchanging compartment ( cytosolic ) , with a minimum of 12 time points used for linear regression . roots of barley seedlings were desorbed for 5 min in 10 mm caso4 to remove extracellular k and na . roots and shoots were then separated , weighed , and oven dried for a minimum of 72 h at 8085 c , then pulverized and digested with 30% hno3 for a minimum of 72 h. k and na concentration was determined using a single - channel flame photometer ( digital flame analyzer model 2655 - 00 ; cole - parmer , anjou , qubec ) . statistical analyses were conducted using one - way analysis of variance ( anova ) with the statistical package spss ( ver . klondike ) were surface - sterilized by immersing seeds in 1.0% sodium hypochlorite for 10 min . seeds were then washed under running tap water for 3 h , placed on discs of plastic mesh , and covered by 2 cm of moist sand . germination proceeded for the following 3 d in a walk - in growth chamber equipped with fluorescent lights ( philips econ - o - watt , f96t12 , with an irradiation of 200 mol photons m s at plant height , for 16 h d ) , and having a day / night temperature cycle of 20 c/15 c , and relative humidity of 70% . following germination , seedlings were transferred to opaque plastic 4 l hydroponic vessels , filled with modified quarter - strength johnson 's solution , consisting of : 5 mm ca(no3)2 , 0.5 mm nah2po4 , 0.25 mm mgso4 , 0.2 mm caso4 , 0.125 m na2moo4 , 20 m feedta , 25 m h3bo3 , 2 m znso4 , 0.5 m mnso4 , and 0.5 m cuso4 . control plants had no additional sodium , while salt - stressed plants were treated with 100 mm na ( as nacl ) . potassium concentrations were adjusted to treatments of 1.5 , 5 , 10 , 20 , and 40 mm by addition of k2so4 . solutions were replaced after 2 d. plants remained in hydroponic solutions for 4 d prior to experimentation . in select treatments ( 1.5 mm and 40 mm k , at low and high nacl ) , plants were also grown for 2 weeks ( 11 d in solution ; see fig . compartmental analysis by tracer efflux was conducted as described in detail elsewhere ( kronzucker et al . , 1999 , 2006 ; in brief , roots of intact seedlings were immersed for 1 h in a nutrient solution identical to the growth solution , except that it contained na or k in addition to non - radioactive na or k. roots were desorbed of radioactivity in tracer - free solutions for the monitoring of na or k efflux , by periodic washing with a timed series of non - radioactive aliquots of nutrient solution ( kronzucker et al . , 1999 , 2006 ; the time course of aliquots was as follows : 15 s ( 4 ) , 20 s ( 3 ) , 30 s ( 2 ) , 40 s ( 1 ) , 50 s ( 1 ) , 1 min ( 5 ) , 1.25 min ( 1 ) , 1.5 min ( 1 ) , 1.75 min ( 1 ) , and 2 min ( 8 ) . radioactivity was measured in aliquots and plant tissues by gamma counting , using a canberra - packard counter , quantum cobra series ii , model 5003 . unidirectional na or k fluxes were determined using standard analyses ( for further details , see kronzucker et al . , 1999 , 2003 , 2006 ; , 2001):(i ) efflux ( co ) was calculated from the initial rate of na or k release from the cytosol , divided by initial cytosolic specific activity ( sc ) of na or k in this compartment ; sc was estimated by using labelling time ( tl ) in the external medium of known specific activity ( so ) , and the kinetic constant k that describes the exponential rate of cytosolic tracer exchange , using the relationship sc = so(1e ) . 1).(ii ) net fluxes were determined from retention of tracer in root and shoot at the end of the desorption protocol , divided by so , while influx ( oc ) was calculated from the sum of co and the net flux.(iii ) cytosolic concentrations of na ( [ na]cyt ) or k ( [ k]cyt ) were determined by integrating rates of radioactivity release from this compartment ; in simplified form , this calculation is made using the equation [ na or k]cyt=oc / k , where is a proportionality constant accounting for the cytosolic compartment comprising 5% of tissue volume . activity coefficients ( ) for cytosolic concentrations were estimated using the debye hckel onsager equation , adapted for the monovalent cations na and k : log=(0.5i)/(1+i ) where i is ionic strength of the cytosol ( assuming that the dominant cations are na and k , and that these are charge - balanced by monovalent anions ; see jander and blasius , 1988 ) . ( i ) efflux ( co ) was calculated from the initial rate of na or k release from the cytosol , divided by initial cytosolic specific activity ( sc ) of na or k in this compartment ; sc was estimated by using labelling time ( tl ) in the external medium of known specific activity ( so ) , and the kinetic constant k that describes the exponential rate of cytosolic tracer exchange , using the relationship sc = so(1e ) . net fluxes were determined from retention of tracer in root and shoot at the end of the desorption protocol , divided by so , while influx ( oc ) was calculated from the sum of co and the net flux . ( iii ) cytosolic concentrations of na ( [ na]cyt ) or k ( [ k]cyt ) were determined by integrating rates of radioactivity release from this compartment ; in simplified form , this calculation is made using the equation [ na or k]cyt=oc / k , where is a proportionality constant accounting for the cytosolic compartment comprising 5% of tissue volume . activity coefficients ( ) for cytosolic concentrations were estimated using the debye hckel onsager equation , adapted for the monovalent cations na and k : log=(0.5i)/(1+i ) where i is ionic strength of the cytosol ( assuming that the dominant cations are na and k , and that these are charge - balanced by monovalent anions ; see jander and blasius , 1988 ) . representative plots of k and na ( inset ) efflux from roots of intact barley seedlings , under varying ionic conditions . roots had been preloaded for 60 min in radioactive solution , then eluted of radioactivity in a timed series of non - radioactive growth - solution aliquots . dashed lines represent the slowest exchanging compartment ( cytosolic ) , with a minimum of 12 time points used for linear regression . roots of barley seedlings were desorbed for 5 min in 10 mm caso4 to remove extracellular k and na . roots and shoots were then separated , weighed , and oven dried for a minimum of 72 h at 8085 c , then pulverized and digested with 30% hno3 for a minimum of 72 h. k and na concentration was determined using a single - channel flame photometer ( digital flame analyzer model 2655 - 00 ; cole - parmer , anjou , qubec ) . statistical analyses were conducted using one - way analysis of variance ( anova ) with the statistical package spss ( ver . figure 1 shows representative plots of k ( main plot ) and na ( inset ) release from roots of intact barley seedlings , previously labelled with respective tracers for 60 min . steady - state tracer efflux curves of this type were analysed under five external potassium conditions ( 1.5 , 5 , 10 , 20 , and 40 mm ) in control ( 1 mm nacl ) or salt - treated ( 100 mm nacl ) plants , to determine unidirectional fluxes of na and k across the plasma membrane of root cells , kinetic constants for cytosolic exchange of the two ions , and estimates of the ions ' cytosolic activities . in fig . 1 , the main plot shows the potent reduction , by elevated na provision , of the efflux of k from barley roots ( note that the y - axis is logarithmic ) , a phenomenon paralleled in reductions in the influx and net flux of k ( see fig . 2 ) . by contrast , the inset of this figure shows the lack of a reciprocal effect : a 27-fold difference in k supply had no influence on the efflux of na under saline conditions . components of unidirectional k influx in roots of intact barley seedlings , grown and monitored with or without 100 mm na . error bars refer to sem of 414 replicates , with asterisks indicating significant differences in influx values within a given k treatment ( p < 0.05 ) . where differences in influx between na treatments were observed , significant differences in efflux and net flux were also found ( p < 0.05 ) . figure 2 shows the steady - state unidirectional and net fluxes of k into barley roots , obtained using compartmental tracer analysis under the 10 conditions examined . over the range of k supply , the influx of potassium in salt - treated plants was lower than in controls ( by 2060% ) , significantly so in all cases except for 40 mm [ k]ext ( p < 0.05 ) . ( 1985 ) showed that application of 3 mm na resulted in a 50% suppression of k influx in maize , but this effect was limited to the low - affinity transport range for k , and was only seen in low - salt plants . this result contrasts with the present study and with previous work in barley , which showed the suppression , by na , of k influx at the high - affinity supply provision of 0.1 mm ( kronzucker et al . , 2006 ) , as well as in the low - affinity range in the high - salt plants ( grown at up to 40 mm k ) ; two possible explanations for this disagreement include the use of a much higher na provision ( 100 mm ) , and the examination of plants grown and tested under steady - state nutritional conditions . consistent with the present work , effects of na on k uptake have also been observed in both high- and low - affinity transport ranges in a wide range of other studies ( rains and epstein , 1967 ; santa - mara et al . , 1997 ; fu and luan , 1998 ; rubio et al . , 2000 , 2003 net fluxes and unidirectional effluxes of k were also reduced under elevated nacl , significantly so in all cases except for the 40 mm k treatment ( fig . the reduction of k efflux with high nacl provision ( figs 1 , 2 ) may appear , on the surface , to contradict the finding that na stimulated a net efflux of k from roots of several plant species ( see , for instance , chen et al . , 2005 ; cuin and shabala , 2007 ) , but it must be pointed out that such demonstrations of na - stimulated k efflux involve short - term changes following nacl shock , whereas the present study involves measurements made under steady - state conditions . influx and efflux of na at 100 mm [ na]ext were high under all k conditions ( fig . 3 ) , and indicated substantial futile cycling of the ion at the plasma membrane . rapid unidirectional influxes of na in glycophytes under salinity conditions have been observed by others ( essah et al . , 2003 ; wang et al . , 2006 ; davenport et al . , 2007 ; 2007 ) ; in particular , the influx values reported here are in excellent agreement with short - term na labelling data in a recent study by chen et al . . the high ratios of efflux to influx seen consistently throughout the present treatments are also supported by previous studies ( cheeseman , 1982 ; mills et al . , 1985 ; essah et al . , in sharp contrast to the suppression of unidirectional and net k fluxes by na , a reciprocal influence was not seen . at 100 mm [ na]ext , the 27-fold variation in k supply resulted in no significant differences in influx , efflux , or net flux of sodium . previous work on the interactions between these ions in the high - affinity k transport range ( kronzucker et al . , 2006 ) also showed no significant differences in na between 0.1 mm and 1.5 mm external [ k ] . the two studies together therefore show that a 400-fold range of k provision fails to modify na fluxes , while k fluxes across this range are generally suppressed by elevated sodium . this lack of reciprocity was further confirmed by experiments conducted in 2-week - old seedlings of barley , indicating that the pattern was not limited to one developmental stage ( data not shown ) . this conclusion , however , was not drawn in a classic study , also with barley , by rains and epstein ( 1967 ) : a strong reciprocal suppression of the flux of one ion ( k or na ) by the co - presence of the other was observed in the low - affinity mechanism 2 of alkali cation transport. again , however , it is important to note that the present study differed from that of rains and epstein ( 1967 ) in that the fluxes in the present study were measured under steady - state nutritional supply conditions , which may be more relevant to plant performance in the field than perturbational conditions might be . ( 2007 ) also showed that k supply had no effect on na uptake in the halophyte suaeda maritima , but only when the external [ na ] was below 75 mm . above that concentration , a suppression of na fluxes was found , indicating , in suaeda , the involvement of akt - type potassium channels in na influx under some conditions . components of unidirectional na influx in roots of intact barley seedlings , grown and monitored at 100 mm na and at varying external k provision . no significant differences between treatments were observed . in the present study , the steady - state , non - reciprocal effect of one ion on the fluxes of the other is borne out in the tissue accumulation patterns for k and na ( figs 4 , 5 ) . for potassium , there was a general reduction of tissue content with salt treatment , particularly in the shoot , where tissue k levels were seen to decline by as much as 50% ( fig . 5 ; as with na fluxes , the accumulation of this ion was not changed over the range of applied external [ k ] , either in the root or in the shoot . tissue content of k in roots and shoots of barley plants , grown with or without 100 mm na , and under varying external k provision . asterisks denote significant differences within a given k treatment and plant organ ( p < 0.05 ) . tissue content of na in roots and shoots of barley plants , grown at 100 mm na , and under varying external k provision . no significant differences between treatments were observed . on a finer scale of analysis , a steady - state , non - reciprocal interaction was also seen in the effects of ion supply on the activities of na and k in the cytosolic compartment of root cells ( ana+,cyt and ak+,cyt ; figs 6 , 7 ) , as estimated by compartmental analysis . under salinity conditions , ana+,cyt proved to be resistant to variations in k provision , showing no significant changes across the entire range of external k supply ( fig . 100 mm sodium provision resulted in severe drops in ak+,cyt , relative to low - sodium controls ( fig . this effect was somewhat alleviated with increasing k provision , but , even at the highest [ k]ext , the pool size of k was reduced by nearly one - half . again , this pattern of no reciprocal effect on cytosolic pool sizes of na and k was confirmed in 2-week - old barley seedlings ( not shown ) . cytosolic k activity in roots of barley seedlings grown under varying k provision , with or without 100 mm na . asterisks denote significant differences within a given k treatment ( p < 0.05 ) . cytosolic na activity in roots of barley seedlings grown under 100 mm na and varying k provision . other workers , using techniques of x - ray microanalysis , radiotracer analysis , subcellular fractionation , and ion - selective microelectrodes , have reported very similar values for cytosolic activities of sodium ( harvey and flowers , 1978 ; yeo , 1981 ; mills et al . , 1985 ; amtmann and gradmann , 1994 ; flowers and hajibagheri , 2001 ; james et al . , 2006 ) and potassium ( pitman and saddler , 1967 ; vorobiev , 1967 ; macklon , 1975 ; memon et al . , 1985 ; beilby and blatt , 1986 ; maathuis and sanders , 1993 ; walker et al . , 1996 ) . there have also been reports of suppressed cytosolic k pool by na ( jeschke and stelter , 1976 ; harvey et al . , 1981 ; mills et al . , 1985 ; hajibagheri et al . , 1987 , 1988 , 1989 ; flowers and hajibagheri , 2001 ; carden et al . a similar suppression of ak+,cyt has been observed to result from high ammonium ( nh4 ) provision in the same model system ( kronzucker et al . , 2003 ) , but , unlike the effect of na , this effect was only seen in the high - affinity k transport range ( 0.1 mm [ k]ext ) . by contrast , suppression of ak+,cyt by high na was not observed at this level of k supply in previous work ( kronzucker et al . , 2006 ) , suggesting that k homeostasis is more resilient to salt stress in the high - affinity range . the variability of the cytosolic k pool in response to both salinity and external k supply , and , by contrast , the uniformity of the na pool with changes in external k , together result in substantial ( greater than 4-fold ) changes in the ratio of the two cytosolic pools ( fig . this k : na ratio peaked at 40 mm [ k]ext , and was minimized at 5 mm [ k]ext , but did not correlate with the growth of the experimental plants ( fig . 9a ; also seen in 2-week - old plants , fig . 2006 ) , this lack of correlation calls into question the view that the cytosolic k : na ratio is a critical determinant of plant growth in response to salinity stress ( see introduction ) . on the other hand , the ratio of k to na on the coarser , tissue level , is rather uniform among salinity - treated plants ( fig . 8) , as is the growth of plants under these conditions , indicating that this measure is indeed more of a more accurate predictor of plant performance under salt stress ( see introduction ) . this conclusion is supported by previously observed correlations between salinity tolerance and the tissue k : na ratio in other cultivars of barley ( flowers and hajibagheri , 2001 ; chen et al . , 2007b ) . ( 2007 ) showed that even this measure did not correlate with growth in 21 cultivars of bread wheat . the authors explained this as possibly reflecting alternative strategies of salt tolerance in different cereal species . cytosolic and tissue k : na ratios in roots of intact barley seedlings , grown at 100 mm na and varying k provision . ( a ) fresh weights of barley seedlings ( roots+shoots ) , grown and monitored with or without 100 mm na and varying levels of k supply . asterisks denote significant differences within a given k treatment ( p < 0.05 ) . ( b ) fresh weights of barley seedlings ( roots+shoots ) , grown and monitored with or without 100 mm na and two different [ k]ext ( 1.5 mm and 40 mm ) for 2 weeks . error bars refer to sem of 582 replicates . in summary , neither na fluxes nor cytosolic na pools responded to k over a wide range of k supply [ 400-fold , when results from a previous work ( kronzucker et al . , 2006 ) are included ] , and elevated k provision was unable to rescue barley plants from the primary toxic entry of na , contrary to the rescue from nh4 toxicity that is effected by increasing external provision of k ( britto and kronzucker , 2002 ; kronzucker et al . , 2003 ; szczerba et al . , the non - reciprocal nature of potassium sodium interactions in barley provides insight into the mediation of primary na entry into plant roots under toxicity - inducing conditions , an unresolved issue under much current debate ( schachtman and liu , 1999 ; golldack et al . , 2003 ; tester and davenport , 2003 ; qi and spalding , 2004 ; fuchs et al . , 2005 ; kader and lindberg , 2005 ; flowers , 2006 ; davenport et al . , 2007 ; obata et al . , 2007 ; takahashi et al . , 2007 ; wang et al . , 2007 ) . the present results suggest that several ion transporters favoured as candidates in the recent literature should likely be discounted , at least in barley . these include , on the one hand , k channels , such as less selective members of the akt family of transporters , and , on the other , nsccs and the low - affinity cation transporter lct , which have been shown to allow the passage of a variety of cations under certain conditions ( amtmann and sanders , 1999 ; maathuis and amtmann , 1999 ; uozumi et al . , 2000 ; ; kader and lindberg , 2005 ; kader et al . , 2006 ; wang et al . , 2007 ) . were members of these families of transporters , or of high - affinity k transporters ( takahashi et al . , 2007 ) , critically involved in toxic na influx , increasing external k concentrations over an extensive range as employed in the present study would be expected to strongly reduce na influx by virtue of ion competition effects . while background levels of ca were high in the present study ( 5 mm ) , and it is recognized that this may have minimized to some extent na influx contribution from nsccs and lct transporters that are known to be ca - sensitive ( see rains and epstein , 1967 ; schachtman and liu , 1999 ; amtmann et al . , 2001 ; essah et al . , 2003 ; wang et al . , 2006 ; ) , it should be pointed out that ca levels in soils are also typically in excess of 1 mm ( reisenauer , 1966 ; hawkesford and miller , 2004 ) . thus , if a major contribution from nsccs or lct can not be realized or rationalized under such conditions , the contribution from these transporters may also not be particularly relevant in the field ( schachtman and liu , 1999 ; for a similar conclusion , also see wang et al . , 2007 ) . two recent studies have also suggested that k - specific shaker - type channels are unlikely candidates for na influx ; in one case , a knockout mutation of akt1 in arabidopsis thaliana resulted in reduced growth under na stress , showing that toxic entry of na can still proceed in the absence of akt1 ( qi and spalding , 2004 ) . in the other case , overexpression of kat1 in yeast cells resulted in lowered na accumulation ( obata et al . , 2007 ) . another group of transporters whose role in primary na influx has been rigorously discussed is the hkt family . it has been proposed that the most well - studied member of this group , hkt1;1 ( rubio et al . , 1995 ) is a major determinant of sodium influx in arabidopsis ( rus et al . , 2001 ) , but other reports suggest that its function is in long - distance recirculation of na ( berthomieu et al . , 2003 ; davenport et al . , 2007 ) . the strong suppression of hkt2-mediated na influx by even low amounts of external k in grasses ( laurie et al . , 2002 ; horie et al . , 2007 ) suggests that this transporter is not likely to play a significant role in na uptake under normal soil conditions or in the present study . however , other transporter types may emerge as mediators of toxic na entry into the plant ( horie et al . , 2001 ; , 2003 ; haro et al . , 2005 ; ren et al . , 2005 ; kader et al . , in particular , the involvement of sodium - specific influx transporters remains a possibility , even though such systems have not yet been identified through arabidopsis genomic analysis ( see hua et al . , 2003 ) . a transporter of this kind has recently been identified in arabidopsis ( colmenero - flores et al . , 2007 ) . clearly , however , a definitive answer has not been forthcoming , and the search for the primary na influx transporter mediating toxic plasma - membrane influx continues ( flowers , 2006 ) . the present study indicates that , in barley , this transporter ( or transport system ) appears to be k - independent , suggesting that attempts to improve salt tolerance in this species , via increased selectivity of k uptake pathways , are unlikely to be effective .

the interaction of sodium and potassium ions in the context of the primary entry of na+ into plant cells , and the subsequent development of sodium toxicity , has been the subject of much recent attention . in the present study , the technique of compartmental analysis with the radiotracers 42k+ and 24na+ was applied in intact seedlings of barley ( hordeum vulgare l. ) to test the hypothesis that elevated levels of k+ in the growth medium will reduce both rapid , futile na+ cycling at the plasma membrane , and na+ build - up in the cytosol of root cells , under saline conditions ( 100 mm nacl ) . we reject this hypothesis , showing that , over a wide ( 400-fold ) range of k+ supply , k+ neither reduces the primary fluxes of na+ at the root plasma membrane nor suppresses na+ accumulation in the cytosol . by contrast , 100 mm nacl suppressed the cytosolic k+ pool by 4773% , and also substantially decreased low - affinity k+ transport across the plasma membrane . we confirm that the cytosolic [ k+]:[na+ ] ratio is a poor predictor of growth performance under saline conditions , while a good correlation is seen between growth and the tissue ratios of the two ions . the data provide insight into the mechanisms that mediate the toxic influx of sodium across the root plasma membrane under salinity stress , demonstrating that , in the glycophyte barley , k+ and na+ are unlikely to share a common low - affinity pathway for entry into the plant cell .

Introduction Materials and methods Plant growth Flux analysis Tissue K Statistical analysis Results and discussion

, 2006 the ratio of k to na is considered an excellent indicator of plant tolerance to salinity ; the higher the ratio , the higher the plant 's tolerance ( flowers and hajibagheri , 2001 ; cakmak , 2005 ; chen et al . , 1999 ; a more precise proposal has been that a high k : na ratio specifically in the cytosolic compartment is critical to plant survival under sodium challenge , while a decrease in this ratio will predict the onset of salinity stress and growth decline ( hajibagheri et al . in a recent study in barley ( hordeum vulgare l. ) , it was shown that , at low to intermediate levels of external k supply ( [ k]ext=0.11.5 mm ) , and at varying salinity levels , the ratio did not in fact correlate with seedling growth in this major cereal ( kronzucker et al . on the contrary , no difference in growth was observed in the presence of a > 5-fold variation in the cytosolic k : na ratio . the study further demonstrated that na suppressed k influx across the plasma membrane to a similar extent at 0.1 and 1.5 mm [ k]ext , concentrations at which high - affinity transport systems for k predominate ( epstein et al . , 2006 ) , while na influxes and cytosolic pools were unaffected by k. however , a significant suppression of the cytosolic k pool by na was seen only at the higher [ k]ext , suggesting that different cellular responses may come into effect as high - affinity k transport gives way to low - affinity transport ( see szczerba et al . in the present study , to examine further the proposed pivotal role of k homeostasis in salinity stress and tolerance , the effects of k supply across the low - affinity transport range of k ( up to 40 mm [ k]ext ) upon the primary fluxes and cytosolic pools of na , and , conversely , the effects of na upon k fluxes and pools in this range were investigated . ( ii ) the proposed , but as yet unresolved , roles of molecular candidates for toxic na influx into the plant ( tester and davenport , 2003 ; flowers , 2006 ; wang et al . , 2002 ) , hkt - type transporters ( rodriguez - navarro and rubio , 2006 ) , and the low - affinity cation transporter lct ( amtmann et al . for these reasons , a detailed study of the interactions between the two ions was carried out along a wide gradient of external k supply . control plants had no additional sodium , while salt - stressed plants were treated with 100 mm na ( as nacl ) . efflux ( co ) was calculated from the initial rate of na or k release from the cytosol , divided by initial cytosolic specific activity ( sc ) of na or k in this compartment ; sc was estimated by using labelling time ( tl ) in the external medium of known specific activity ( so ) , and the kinetic constant k that describes the exponential rate of cytosolic tracer exchange , using the relationship sc = so(1e ) . (ii ) net fluxes were determined from retention of tracer in root and shoot at the end of the desorption protocol , divided by so , while influx ( oc ) was calculated from the sum of co and the net flux. activity coefficients ( ) for cytosolic concentrations were estimated using the debye hckel onsager equation , adapted for the monovalent cations na and k : log=(0.5i)/(1+i ) where i is ionic strength of the cytosol ( assuming that the dominant cations are na and k , and that these are charge - balanced by monovalent anions ; see jander and blasius , 1988 ) . ( i ) efflux ( co ) was calculated from the initial rate of na or k release from the cytosol , divided by initial cytosolic specific activity ( sc ) of na or k in this compartment ; sc was estimated by using labelling time ( tl ) in the external medium of known specific activity ( so ) , and the kinetic constant k that describes the exponential rate of cytosolic tracer exchange , using the relationship sc = so(1e ) . ( ii ) net fluxes were determined from retention of tracer in root and shoot at the end of the desorption protocol , divided by so , while influx ( oc ) was calculated from the sum of co and the net flux . ( iii ) cytosolic concentrations of na ( [ na]cyt ) or k ( [ k]cyt ) were determined by integrating rates of radioactivity release from this compartment ; in simplified form , this calculation is made using the equation [ na or k]cyt=oc / k , where is a proportionality constant accounting for the cytosolic compartment comprising 5% of tissue volume . activity coefficients ( ) for cytosolic concentrations were estimated using the debye hckel onsager equation , adapted for the monovalent cations na and k : log=(0.5i)/(1+i ) where i is ionic strength of the cytosol ( assuming that the dominant cations are na and k , and that these are charge - balanced by monovalent anions ; see jander and blasius , 1988 ) . control plants had no additional sodium , while salt - stressed plants were treated with 100 mm na ( as nacl ) . , 1999 , 2006 ; in brief , roots of intact seedlings were immersed for 1 h in a nutrient solution identical to the growth solution , except that it contained na or k in addition to non - radioactive na or k. roots were desorbed of radioactivity in tracer - free solutions for the monitoring of na or k efflux , by periodic washing with a timed series of non - radioactive aliquots of nutrient solution ( kronzucker et al . , 1999 , 2003 , 2006 ; , 2001):(i ) efflux ( co ) was calculated from the initial rate of na or k release from the cytosol , divided by initial cytosolic specific activity ( sc ) of na or k in this compartment ; sc was estimated by using labelling time ( tl ) in the external medium of known specific activity ( so ) , and the kinetic constant k that describes the exponential rate of cytosolic tracer exchange , using the relationship sc = so(1e ) . (ii ) net fluxes were determined from retention of tracer in root and shoot at the end of the desorption protocol , divided by so , while influx ( oc ) was calculated from the sum of co and the net flux. (iii ) cytosolic concentrations of na ( [ na]cyt ) or k ( [ k]cyt ) were determined by integrating rates of radioactivity release from this compartment ; in simplified form , this calculation is made using the equation [ na or k]cyt=oc / k , where is a proportionality constant accounting for the cytosolic compartment comprising 5% of tissue volume . activity coefficients ( ) for cytosolic concentrations were estimated using the debye hckel onsager equation , adapted for the monovalent cations na and k : log=(0.5i)/(1+i ) where i is ionic strength of the cytosol ( assuming that the dominant cations are na and k , and that these are charge - balanced by monovalent anions ; see jander and blasius , 1988 ) . ( i ) efflux ( co ) was calculated from the initial rate of na or k release from the cytosol , divided by initial cytosolic specific activity ( sc ) of na or k in this compartment ; sc was estimated by using labelling time ( tl ) in the external medium of known specific activity ( so ) , and the kinetic constant k that describes the exponential rate of cytosolic tracer exchange , using the relationship sc = so(1e ) . net fluxes were determined from retention of tracer in root and shoot at the end of the desorption protocol , divided by so , while influx ( oc ) was calculated from the sum of co and the net flux . ( iii ) cytosolic concentrations of na ( [ na]cyt ) or k ( [ k]cyt ) were determined by integrating rates of radioactivity release from this compartment ; in simplified form , this calculation is made using the equation [ na or k]cyt=oc / k , where is a proportionality constant accounting for the cytosolic compartment comprising 5% of tissue volume . activity coefficients ( ) for cytosolic concentrations were estimated using the debye hckel onsager equation , adapted for the monovalent cations na and k : log=(0.5i)/(1+i ) where i is ionic strength of the cytosol ( assuming that the dominant cations are na and k , and that these are charge - balanced by monovalent anions ; see jander and blasius , 1988 ) . steady - state tracer efflux curves of this type were analysed under five external potassium conditions ( 1.5 , 5 , 10 , 20 , and 40 mm ) in control ( 1 mm nacl ) or salt - treated ( 100 mm nacl ) plants , to determine unidirectional fluxes of na and k across the plasma membrane of root cells , kinetic constants for cytosolic exchange of the two ions , and estimates of the ions ' cytosolic activities . 1 , the main plot shows the potent reduction , by elevated na provision , of the efflux of k from barley roots ( note that the y - axis is logarithmic ) , a phenomenon paralleled in reductions in the influx and net flux of k ( see fig . by contrast , the inset of this figure shows the lack of a reciprocal effect : a 27-fold difference in k supply had no influence on the efflux of na under saline conditions . over the range of k supply , the influx of potassium in salt - treated plants was lower than in controls ( by 2060% ) , significantly so in all cases except for 40 mm [ k]ext ( p < 0.05 ) . ( 1985 ) showed that application of 3 mm na resulted in a 50% suppression of k influx in maize , but this effect was limited to the low - affinity transport range for k , and was only seen in low - salt plants . this result contrasts with the present study and with previous work in barley , which showed the suppression , by na , of k influx at the high - affinity supply provision of 0.1 mm ( kronzucker et al . , 2006 ) , as well as in the low - affinity range in the high - salt plants ( grown at up to 40 mm k ) ; two possible explanations for this disagreement include the use of a much higher na provision ( 100 mm ) , and the examination of plants grown and tested under steady - state nutritional conditions . consistent with the present work , effects of na on k uptake have also been observed in both high- and low - affinity transport ranges in a wide range of other studies ( rains and epstein , 1967 ; santa - mara et al . 3 ) , and indicated substantial futile cycling of the ion at the plasma membrane . at 100 mm [ na]ext , the 27-fold variation in k supply resulted in no significant differences in influx , efflux , or net flux of sodium . previous work on the interactions between these ions in the high - affinity k transport range ( kronzucker et al . the two studies together therefore show that a 400-fold range of k provision fails to modify na fluxes , while k fluxes across this range are generally suppressed by elevated sodium . this lack of reciprocity was further confirmed by experiments conducted in 2-week - old seedlings of barley , indicating that the pattern was not limited to one developmental stage ( data not shown ) . this conclusion , however , was not drawn in a classic study , also with barley , by rains and epstein ( 1967 ) : a strong reciprocal suppression of the flux of one ion ( k or na ) by the co - presence of the other was observed in the low - affinity mechanism 2 of alkali cation transport. again , however , it is important to note that the present study differed from that of rains and epstein ( 1967 ) in that the fluxes in the present study were measured under steady - state nutritional supply conditions , which may be more relevant to plant performance in the field than perturbational conditions might be . above that concentration , a suppression of na fluxes was found , indicating , in suaeda , the involvement of akt - type potassium channels in na influx under some conditions . in the present study , the steady - state , non - reciprocal effect of one ion on the fluxes of the other is borne out in the tissue accumulation patterns for k and na ( figs 4 , 5 ) . 5 ; as with na fluxes , the accumulation of this ion was not changed over the range of applied external [ k ] , either in the root or in the shoot . tissue content of k in roots and shoots of barley plants , grown with or without 100 mm na , and under varying external k provision . tissue content of na in roots and shoots of barley plants , grown at 100 mm na , and under varying external k provision . on a finer scale of analysis , a steady - state , non - reciprocal interaction was also seen in the effects of ion supply on the activities of na and k in the cytosolic compartment of root cells ( ana+,cyt and ak+,cyt ; figs 6 , 7 ) , as estimated by compartmental analysis . under salinity conditions , ana+,cyt proved to be resistant to variations in k provision , showing no significant changes across the entire range of external k supply ( fig . this effect was somewhat alleviated with increasing k provision , but , even at the highest [ k]ext , the pool size of k was reduced by nearly one - half . cytosolic k activity in roots of barley seedlings grown under varying k provision , with or without 100 mm na . cytosolic na activity in roots of barley seedlings grown under 100 mm na and varying k provision . other workers , using techniques of x - ray microanalysis , radiotracer analysis , subcellular fractionation , and ion - selective microelectrodes , have reported very similar values for cytosolic activities of sodium ( harvey and flowers , 1978 ; yeo , 1981 ; mills et al . , 2003 ) , but , unlike the effect of na , this effect was only seen in the high - affinity k transport range ( 0.1 mm [ k]ext ) . by contrast , suppression of ak+,cyt by high na was not observed at this level of k supply in previous work ( kronzucker et al . , 2006 ) , suggesting that k homeostasis is more resilient to salt stress in the high - affinity range . the variability of the cytosolic k pool in response to both salinity and external k supply , and , by contrast , the uniformity of the na pool with changes in external k , together result in substantial ( greater than 4-fold ) changes in the ratio of the two cytosolic pools ( fig . this k : na ratio peaked at 40 mm [ k]ext , and was minimized at 5 mm [ k]ext , but did not correlate with the growth of the experimental plants ( fig . 2006 ) , this lack of correlation calls into question the view that the cytosolic k : na ratio is a critical determinant of plant growth in response to salinity stress ( see introduction ) . 8) , as is the growth of plants under these conditions , indicating that this measure is indeed more of a more accurate predictor of plant performance under salt stress ( see introduction ) . this conclusion is supported by previously observed correlations between salinity tolerance and the tissue k : na ratio in other cultivars of barley ( flowers and hajibagheri , 2001 ; chen et al . ( a ) fresh weights of barley seedlings ( roots+shoots ) , grown and monitored with or without 100 mm na and varying levels of k supply . in summary , neither na fluxes nor cytosolic na pools responded to k over a wide range of k supply [ 400-fold , when results from a previous work ( kronzucker et al . , 2006 ) are included ] , and elevated k provision was unable to rescue barley plants from the primary toxic entry of na , contrary to the rescue from nh4 toxicity that is effected by increasing external provision of k ( britto and kronzucker , 2002 ; kronzucker et al . , the non - reciprocal nature of potassium sodium interactions in barley provides insight into the mediation of primary na entry into plant roots under toxicity - inducing conditions , an unresolved issue under much current debate ( schachtman and liu , 1999 ; golldack et al . the present results suggest that several ion transporters favoured as candidates in the recent literature should likely be discounted , at least in barley . these include , on the one hand , k channels , such as less selective members of the akt family of transporters , and , on the other , nsccs and the low - affinity cation transporter lct , which have been shown to allow the passage of a variety of cations under certain conditions ( amtmann and sanders , 1999 ; maathuis and amtmann , 1999 ; uozumi et al . , 2007 ) , critically involved in toxic na influx , increasing external k concentrations over an extensive range as employed in the present study would be expected to strongly reduce na influx by virtue of ion competition effects . while background levels of ca were high in the present study ( 5 mm ) , and it is recognized that this may have minimized to some extent na influx contribution from nsccs and lct transporters that are known to be ca - sensitive ( see rains and epstein , 1967 ; schachtman and liu , 1999 ; amtmann et al . thus , if a major contribution from nsccs or lct can not be realized or rationalized under such conditions , the contribution from these transporters may also not be particularly relevant in the field ( schachtman and liu , 1999 ; for a similar conclusion , also see wang et al . two recent studies have also suggested that k - specific shaker - type channels are unlikely candidates for na influx ; in one case , a knockout mutation of akt1 in arabidopsis thaliana resulted in reduced growth under na stress , showing that toxic entry of na can still proceed in the absence of akt1 ( qi and spalding , 2004 ) . , 2007 ) suggests that this transporter is not likely to play a significant role in na uptake under normal soil conditions or in the present study . however , other transporter types may emerge as mediators of toxic na entry into the plant ( horie et al . , in particular , the involvement of sodium - specific influx transporters remains a possibility , even though such systems have not yet been identified through arabidopsis genomic analysis ( see hua et al . clearly , however , a definitive answer has not been forthcoming , and the search for the primary na influx transporter mediating toxic plasma - membrane influx continues ( flowers , 2006 ) . the present study indicates that , in barley , this transporter ( or transport system ) appears to be k - independent , suggesting that attempts to improve salt tolerance in this species , via increased selectivity of k uptake pathways , are unlikely to be effective .

water plays a fundamental role in a wide range of fields of study , including geology , atmospheric sciences , chemistry , biology , and physics . because of its relevance and ubiquity , water is the most common solvent in experimental and computational studies of biological systems . the hydrophobic effect , the segregation of molecules based on relative interactions with water , governs membrane self - assembly , protein folding , and many other biological processes . despite its seemingly simple structure , the water molecule forms intricate and dynamic networks of hydrogen bonds that give it unique bulk and interfacial properties . the wealth of anomalous characteristics that water exhibits relative to most other liquids underscore its uniqueness . in part because of its enigmatic character , it has been the subject of many theoretical and modeling efforts in the past few decades . reproducing these anomalous characteristics poses a serious challenge for any effort to attain a detailed understanding of water dynamics and structure . quantum mechanical models of water based on first principles ( ab initio ) precipitate the evolution of the electronic structure along with nuclear coordinates over time . these ab initio models produce radial distribution functions ( rdfs ) and rotational / vibrational spectra that strongly agree with experimental data . the computational complexity of quantum models restricts systems to a range too limited to determine most statistical bulk quantities . these properties , such as the diffusion coefficient and thermodynamic variables , are more accurately modeled by semiempirical approaches . water models with atomic - level detail are the most common for simulation of biomolecules and their aggregates . to reproduce a wide range of complex water behavior , several models that consist of four ( bernal fowler , tip4p , and tips2 ) and five ( tip5p ) interaction sites have been developed . in the interest of reducing computational cost , most simulations use models ( spc , spc / e , and tip3p ) that are composed of three partially charged atoms connected with rigid bonds and a lennard - jones ( 6 - 12 ) intermolecular potential between oxygen atoms . each model is reasonably accurate within its domain of applicability , but none reproduce a large portion of the anomalous properties of water . various models include flexible bonds that allow the water dipole moment to change with the surrounding environment . coarse - grained ( cg ) models are able to achieve longer time scales by averaging over rapid inter- and intramolecular motions , thus permitting a larger time step . cg models are able to effectively represent a larger number of molecules by reducing the number of interaction sites considered . because of the unusual properties of water , the development of a cg model that reliably describes the bulk and medium - range properties is a challenging project . the development of models of molecular assemblies with lower complexity that reproduce important qualities of water is ongoing . for an extensive review , johnson et al . have identified limitations of cg models by demonstrating the lack of transferability of a cg pair potential across different states . the work also shows that the pair potential can not simultaneously resolve all the properties of the reference system for a given state . despite these limitations , one common cg protocol is to group a number of water molecules into a single bead with its center as the interaction site . electrostatic interactions are implicitly incorporated into the effective pairwise interaction potential which is either a lennard - jones ( lj ) or morse - like function . the free parameters in pair potentials are trained against thermodynamic properties such as density , surface tension , and solvation free energy . molinero and moore built a coarse - grained water model around the tetrahedral properties shared by water , silicon , and carbon by adapting the stillinger the model maps one water molecule onto one interacting bead and describes important properties of water across a wide range of temperatures . using a clustering algorithm , have large isothermal compressibility . using a lj 12 - 4 potential , shinoda et al . have designed a cg water that has density and surface tension comparable to those of water . electrostatic interactions can be introduced into cg water models with multiple charged sites per bead . these models can describe the dielectric properties of water . despite the incorporation of electrostatic interactions , both the bmw and polarizable martini models predict density different from experimental measurements . the parameter set of the wt4 model is tuned to match the experimental density . on the other hand , the isothermal compressibility and surface tension are modeled less accurately . the recent gromos cg is a 5-to-1 mapped water model with two electrostatic interaction sites . it delivers good results for density , surface tension , and dielectric constant as compared to those of real water but yields a coefficient of thermal expansion 1 order of magnitude higher than the experimental value . adaptive multiscale models allow transfer between simulation domains with differing granularity , from the quantum level up to cg . adaptive methods face unique challenges because of the variability in the degrees of freedom over the span of a simulation . in continuation of our previous cg development based on the morse potential , we upgrade our previous cg water model ( termed csj ) with polarizability and a morse - like potential with more flexibility in the landscape of pairwise interaction parameters . we will refer to the new model as modified morse coarse - grain ( mmcg ) . a cg mapping of four water molecules to one cg water bead was used for the current model . for interactions between water beads , we have further refined our previous model , which used the standard morse potential , to meet two goals : ( 1 ) to improve compressibility without sacrificing other features of the potential and ( 2 ) to build into the interaction potential a smoothing function that permits fairly accurate simulations using a shorter cutoff . the choice of pairwise potential gives flexibility in the functional form without introducing any extra simulation costs . the coarse - grained potential must model the average of several different interatomic and intermolecular forces . therefore , the functional form is solely based on a phenomenological understanding of the system rather than a first - principle derivation , such as the r behavior of the dispersion force included in the lennard - jones potential . a modified morse potential ( eq 1 ) was used to describe interactions between water bead centers.wherewhere r and de are the potential well location and depth , respectively . compared to the morse potential , the mmcg potential replaces , the scaling parameter in the exponent , with two separate distance - dependent variables , (r ) and (r ) . this form decouples the repulsive ( eq 1a ) and attractive ( eq 1b ) parts of the potential . the shape of the repulsive term as r tends to 0 is determined by 0 . the exponential factor tends toward l as r approaches r. parameters r and c provide similar behavior for the attractive part of the potential . beyond the cutoff distance rc , the potential , we have not conducted an exhaustive search for the value for rc that optimally combines accuracy and efficiency , but the conventional value of 1.6 nm gives good results with reasonable efficiency . comparing systems representing equal numbers of water molecules , the mmcg model achieves a > 1 order of magnitude improvement in performance over an atomistic system . the polarizability of cg water beads is modeled like that of the polarizable martini water model , through the addition of a harmonic angle potential term , with spring constant k0 and equilibrium angle 0 , among three charged points . this model of polarizability allows for not only dipole but also higher moments of the electrostatic energy . the central point , which acts as the interaction site for the modified morse potential , carries negative charge q , while the two outer points each carry charge q/2 . a mass equal to that of four water molecules is distributed evenly among the three points . the distance between the outer points and the central point is a tunable parameter i. the change in the angle ( ) between the points represents the polarizable nature of the bead . the set of potential parameters { de , r,0,l,r,c,0,k0,q , i } determines the space on which optimization will be performed . parameter optimization is exploration of a parameter space to determine extreme values of a target function . in the case of molecular dynamics force field optimization , the set of potential parameters defines the space and comparison of model predictions with either ab initio data or experimental measurements defines the target function to be minimized . we have developed a new software package , parameter optimizer ( paropt ) , for general optimization problems ( paropt is available for download under the gnu public license at https://csmlabfs1.cas.usf.edu/sites ) . the software provides various optimization algorithms , including an orthogonal steepest descent and the nelder mead simplex - based method . sources of difficulty include high dimensionality , discontinuities , possible stochasticity due to random numerical error and finite sample size effects , and difficulty in defining a derivative . thus , in the context of molecular dynamics parameter optimization methods such as simplex - based , approaches with the least demanding conditions on the target function are the most suitable . mead , a commonly used simplex - based optimization algorithm , iteratively evolves m + 1 points on the m - dimensional space using four basic moves . the central site with charge q provides the interaction site for the modified morse potential . outer charge sites ( with charge q/2 ) are connected to the central site by a fixed bond of length i. the central angle is a determined by a harmonic potential with equilibrium angle 0 . paropt provides an environment in which different optimization methods can be accessed by defining the generation of the target function . replace the highest point with a point reflected about the centroid of the remaining points.2 if the reflected point is lower in value than all points in the centroid , consider a point further from the centroid.3 if the reflected point would remain the highest , consider a point nearer to the simplex.4 move all points nearer to the lowest point.5the m + 1 individual points in the m - dimensional space are denoted by pi , while subscripts h and l denote the lowest and highest points , respectively . the centroid is defined by6values used for optimization of water parameters are those suggested by nelder mead : = 1.0 , = 2.0 , = 0.5 , and = 0.5 . arrows denote points that will be replaced and the new point to be considered : ( a ) the reflection of the highest point about the centroid of the remaining points , ( b ) the further expansion of the reflection move , ( c ) the contraction along the line joining the centroid and the highest point , and ( d ) the shrinking of the simplex by shifting all points nearer to the lowest point . the nelder mead algorithm replaces the highest point in the simplex with one with a lower target function value by applying these moves . a nelder mead step begins with calculation of a reflect move . if the target function value for this new reflected point is lower than all points in the simplex , an expansion move is attempted . if this expanded point is lower than all points in the simplex , it is accepted . if the reflected point is not higher than the highest point in the new simplex , it is accepted and replaces the highest point in the simplex . if the newly accepted reflected point is still the highest point in the simplex , a contraction step is attempted . if the contracted point is higher than the highest point in the simplex , then a shrink move is applied , replacing all points except the lowest . if the contracted point is lower than at least one point , it is accepted into the simplex . this process is repeated until the stopping criterion is met . the stopping criterion for the nelder mead method is a cutoff on the root - mean - square target function value over the entire simplex . the number of occurrences of each move in an optimization depends on the characteristics of both the simplex and the target function and is therefore problem specific . minimal and maximal values ( see table 1 ) for each parameter were chosen heuristically to restrict target function evaluations to reasonable combinations of parameters . the original description of the nelder mead algorithm has no set method for boundary conditions on the parameter space , though suggestions are made for modification of functional forms or assignment of large penalty we chose to replace points outside the boundary with the nearest point within the allowable space . for optimization of the mmcg water model , the target function was defined by the weighted percent error in comparison of cg simulation results with four experimental quantities : density , dielectric constant , diffusion coefficient , and surface tension . to ensure that all comparison data contribute to the target function and thus the evolution of the simplex , percent errors are assigned scalar weights to yield similar orders of magnitude among all quantities . with this goal in mind , density was weighted 100 times higher than surface tension , while permittivity and diffusion coefficient were weighted 10 times higher . density was computed by taking the ratio of the mass of the system and the ensemble average volume . the standard practice in comparing diffusion coefficients between cg models and experiment is to assume that diffusion for n - sized clusters scales as 1/n ; i.e. , dn = d1/n , where dn is the diffusion coefficient for an n - sized cluster . the gromos cg work compares the cg bead diffusion coefficient with the diffusion coefficient for the center of mass of clusters in the spc water model . in that work , static clusters in the atomistic representation were produced by adding distance restraints between oxygen atoms , which produces diffusion coefficient scaling close to the inverse behavior usually assumed . in the work presented here , clusters are constructed similarly , though unrestrained . to calculate diffusion coefficient scaling , clusters were constructed from each molecule and its nearest n 1 neighbors . the msd for such clusters was calculated using a window averaging method with eq 7 . though the method presented here may not be generalizable to all types of clusters , it provides an improvement over current assumptions for homogeneous systems . the plot for atomistic water shows significant deviation from the scaling behavior of uncorrelated clusters , especially at larger values of n. therefore , approximations leading to 1/n scaling are more appropriate at smaller cluster sizes . at a bead size of four water molecules , , we use a scale factor ( s ) of 3.16 to compare the cg diffusion coefficient with experiment , instead of the usual factor of 4.7where x(t ) is the position of the central bead at time t and the broken brackets denote an ensemble average . the line shows the usual practice of taking the diffusion of an n cluster beginning n times slower than the single molecule . spc / e was determined by simulation of spc / e at 303 k. clusters were chosen by the n 1 nearest neighbors for each water molecule , and the center of mass diffusion was calculated . the relative permittivity ( ) was calculated using a clausius mossotti - like equation with a reaction field with an infinite dielectric constant ( conducting boundary conditions).8where m is the total system dipole , 0 is the permittivity of free space , v is the ensemble average system volume , t is the target temperature for the thermostat , and 0 is the permittivity of the vacuum . the surface tension was taken from the gromacs internal calculation:9where the z - axis is normal to the interface , pnn terms are the pressure tensor diagonal elements , ns is the number of interfaces , and lz is the size of the system along the z - axis . these evaluations , at a point in parameter space , involve the execution of two md simulations . this places practical limits on the size and length of simulations used for optimization . therefore , systems used for determination of the diffusion coefficient , density , and electric field permittivity contained 512 cg beads , corresponding to 2048 water molecules . because of large fluctuations , the calculation of surface tension requires a larger system : 4096 cg beads , implying 16384 water molecules ( system s2 ) . system s2 was constructed , at every evaluation of the target function , with a slab near the average density computed from s1 in contact with a vacuum . system s2 was simulated under the constant number , volume , and temperature ( nvt ) ensemble . more details on simulation methods are found in appendix a. the first step in optimization is the construction of an initial simplex . in this work , optimization started with a simplex consisting of random points within the domain of parameter space that satisfied constraints . to further improve the accuracy of the final point , the optimization was restarted with an initial simplex consisting of the converged point and random points around it . this procedure was then iterated two more times , with the final optimization resulting in the point presented in table 1 . figure 5 shows the mean and root - mean - square ( rms ) target function value versus the nelder mead step for the final optimization iteration . because of the roughness of the hypersurface generated by the target function , the simplex may occasionally include a vertex with an abnormally high value , as observed in the mean and rms of the second shrink step of figure 5 . figure 6 shows the evolution of parameter values over the final run , while figure 7 shows the change in computed physical properties . further , figure 6 demonstrates that although parameters did encounter the boundaries over the course of the optimization , the final simplex converged away from the boundary . optimization results . the mean error , rms error , and rms cutoff as a function of nelder reflection ( ) , contraction ( ) , and shrink ( ) steps are labeled at the corresponding mean error point . variation in parameters as a function of nelder mead step . dashed lines indicate minimal and maximal values . training data [ density ( ) , electric field permittivity ( ) , diffusion coefficient ( d ) , and surface tension ( ) ] ( ) as a function of nelder mead step compared with target values ( --- ) . the final set of parameters was chosen as the point in the final converged simplex with the lowest target function value ( see table 1 ) . the decoupling of repulsive and attractive parameters , the added distance dependence of the exponential factors , and the added electrostatic interaction have allowed for a decrease in the equilibrium distance , while maintaining the experimental density . the large differences between exponential parameters in the new and original models hint that the extensions have added important flexibility . the simulation results corresponding to the final set of parameters are listed in table 2 . when validating the results of an optimization procedure , one can not consider the training data alone . the suitability of the force field will depend on matching data that the model was not explicitly trained to reproduce . therefore , validation simulations were performed to verify the reliability of the optimized parameters . because validation is not performed iteratively , all systems for validation consisted of 110592 cg beads , which corresponds to 442368 water molecules . the density , diffusion coefficient , relative permittivity , and surface tension were determined from the larger validation systems using the same methods that were used during optimization [ see section 3.1 ; in addition , bulk thermodynamic quantities were also calculated ( see table 3 ) ] . the coefficient of thermal expansion , , was calculated using the finite - difference method.10two constant number , pressure , and temperature ( npt ) ensemble simulations at temperatures of 303.15 k ( t1 ) and 308.0 k ( t2 ) were performed . the ensemble averages of the resulting densities were used for 1 and 2 , respectively . isothermal compressibility t was calculated using two methods : ( i ) a finite difference method for nvt simulations at different densities ( i)11and ( ii ) volume fluctuations in an npt ensemble simulation12where pi is the pressure on system i and v is the variance of the volume . for eq 11 , the following densities were used : 1 = 0.993 g cm , and 2 = 0.963 g cm . the enthalpy of vaporization was calculated from the interaction energy between cg beads ( vinter ) and the intermolecular interactions that occur within the bead vs of 128.5 kj mol determined from quantum mechanical calculations of the water tetramer binding energy.13where the factor of 4 is due to the level of cg . the average dipole moment of an individual bead was calculated for the mmcg water model . because a cg bead represents four water molecules , the total dipole moment for an atomistically detailed spc / e water molecule along with its three nearest neighbors the mmcg model yields an average dipole moment of 4.1 0.25 d , whereas the four - water cluster dipole moment from the spc / e water model was 5.3 1.0 d ; density function theory ( dft ) calculations for an isolated cluster produce no dipole moment . the effective polarizability ( 0 ) for a cg bead was calculated using eq 14 . the mmcg model yields a value of 63 au compared with a value of 39.33 au given by dft for an isolated four - water cluster . the disparity in these values is most likely a result of differences between clusters in the bulk and in isolation.14 quantities computed are density , diffusion coefficient d , dielectric permittivity , surface tension , coefficient of thermal expansion , isothermal compressibility t , and enthalpy of vaporization hvap . values computed both during optimization and for validation differ because of the size differences between simulated systems . to automate force field optimization , we have developed a general optimization framework paropt . the software utilizes various methods to locate minima of the defined target function on parameter space . we have applied this general framework to optimize the parameters of a polarizable cg water model with nonbonded interactions given by a modified morse potential via the nelder mead algorithm . the model has more flexibility in the functional form than most cg models , allowing it to match a wide range of experimental properties ( see table 3 ) . any model in a classical molecular dynamics framework is a simplification of a physical system and therefore involves a reduction in the degrees of freedom in the description of the interactions . though the set of parameters that govern the model are mathematically independent , their effects on observable data and therefore the target function are not necessarily uncorrelated . therefore , because of this interdependence of the parameters , the optimization is not necessarily underdetermined despite an apparent abundance of free parameters to match the target data . the question of uniqueness is nontrivial to answer and is in fact a matter of ongoing research ( e.g. , in the context of economic models ) . for complex systems in general , there is no straightforward , computationally tractable , and general solution to this problem ; rather , heuristic approaches must be developed for the particular system being studied . in light of these factors , it may be the case that our parameter set presented in this paper is one of many equally good sets . the point we have chosen was a suitable point that matched the training data well . additionally , it can be seen from the good agreement with the validation data , which is independent of the training data , that the resulting force field parameters are well - tuned . an artifact common to most cg models is the long - range correlation between cg water beads not seen in experiment or atomistic models . . likely as a result of the high degree of interbead correlation at long distances , the model has the propensity to spontaneously solidify at room temperature , a weakness found in other cg water models . this phenomenon would not be expected in a heterogeneous system , because long - range correlations would be broken by the presence of other interactions . additionally , spontaneous freezing can be avoided by periodically assigning random velocities chosen from a maxwell distribution , a method similar to one suggested by harvey et al . to prevent the overpopulation of low - frequency modes of a molecular dynamics system . because dynamic and structural properties of water clusters vary with temperature , the internal states of cg beads would , as well . thus , cg models can not necessarily be assumed to transfer to different temperatures . we believe that the interaction parameters for cg models would be more accurately treated as being temperature - dependent . the set of potential parameters has been tuned for one temperature and is not expected to accurately simulate behavior far from the target temperature or in thermodynamic ensembles without constant temperature control . rdf . comparison between g(r ) for the atomistic spc / e water model ( ) and the mmcg water model ( --- ) . the predictions of the model agree well with experiment for structural , dynamic , and bulk properties . the dielectric constant indicates the validity of the parameters that determine electrostatics and polarizability . accurate surface tension indicates an accurate representation of the strength of the interaction between beads . the thermodynamic quantities predicted by the model are in good agreement with those of experiment , validating the fitness of the parameters .

we have developed an automated parameter optimization software framework ( paropt ) that implements the nelder mead simplex algorithm and applied it to a coarse - grained polarizable water model . the model employs a tabulated , modified morse potential with decoupled short- and long - range interactions incorporating four water molecules per interaction site . polarizability is introduced by the addition of a harmonic angle term defined among three charged points within each bead . the target function for parameter optimization was based on the experimental density , surface tension , electric field permittivity , and diffusion coefficient . the model was validated by comparison of statistical quantities with experimental observation . we found very good performance of the optimization procedure and good agreement of the model with experiment .

Introduction Model Methods and Results Conclusion

water plays a fundamental role in a wide range of fields of study , including geology , atmospheric sciences , chemistry , biology , and physics . the hydrophobic effect , the segregation of molecules based on relative interactions with water , governs membrane self - assembly , protein folding , and many other biological processes . quantum mechanical models of water based on first principles ( ab initio ) precipitate the evolution of the electronic structure along with nuclear coordinates over time . the computational complexity of quantum models restricts systems to a range too limited to determine most statistical bulk quantities . these properties , such as the diffusion coefficient and thermodynamic variables , are more accurately modeled by semiempirical approaches . to reproduce a wide range of complex water behavior , several models that consist of four ( bernal fowler , tip4p , and tips2 ) and five ( tip5p ) interaction sites have been developed . in the interest of reducing computational cost , most simulations use models ( spc , spc / e , and tip3p ) that are composed of three partially charged atoms connected with rigid bonds and a lennard - jones ( 6 - 12 ) intermolecular potential between oxygen atoms . coarse - grained ( cg ) models are able to achieve longer time scales by averaging over rapid inter- and intramolecular motions , thus permitting a larger time step . because of the unusual properties of water , the development of a cg model that reliably describes the bulk and medium - range properties is a challenging project . have identified limitations of cg models by demonstrating the lack of transferability of a cg pair potential across different states . the work also shows that the pair potential can not simultaneously resolve all the properties of the reference system for a given state . despite these limitations , one common cg protocol is to group a number of water molecules into a single bead with its center as the interaction site . the free parameters in pair potentials are trained against thermodynamic properties such as density , surface tension , and solvation free energy . molinero and moore built a coarse - grained water model around the tetrahedral properties shared by water , silicon , and carbon by adapting the stillinger the model maps one water molecule onto one interacting bead and describes important properties of water across a wide range of temperatures . have designed a cg water that has density and surface tension comparable to those of water . the parameter set of the wt4 model is tuned to match the experimental density . on the other hand , the isothermal compressibility and surface tension are modeled less accurately . the recent gromos cg is a 5-to-1 mapped water model with two electrostatic interaction sites . it delivers good results for density , surface tension , and dielectric constant as compared to those of real water but yields a coefficient of thermal expansion 1 order of magnitude higher than the experimental value . adaptive methods face unique challenges because of the variability in the degrees of freedom over the span of a simulation . in continuation of our previous cg development based on the morse potential , we upgrade our previous cg water model ( termed csj ) with polarizability and a morse - like potential with more flexibility in the landscape of pairwise interaction parameters . we will refer to the new model as modified morse coarse - grain ( mmcg ) . a cg mapping of four water molecules to one cg water bead was used for the current model . for interactions between water beads , we have further refined our previous model , which used the standard morse potential , to meet two goals : ( 1 ) to improve compressibility without sacrificing other features of the potential and ( 2 ) to build into the interaction potential a smoothing function that permits fairly accurate simulations using a shorter cutoff . the coarse - grained potential must model the average of several different interatomic and intermolecular forces . therefore , the functional form is solely based on a phenomenological understanding of the system rather than a first - principle derivation , such as the r behavior of the dispersion force included in the lennard - jones potential . a modified morse potential ( eq 1 ) was used to describe interactions between water bead centers.wherewhere r and de are the potential well location and depth , respectively . compared to the morse potential , the mmcg potential replaces , the scaling parameter in the exponent , with two separate distance - dependent variables , (r ) and (r ) . this form decouples the repulsive ( eq 1a ) and attractive ( eq 1b ) parts of the potential . the shape of the repulsive term as r tends to 0 is determined by 0 . comparing systems representing equal numbers of water molecules , the mmcg model achieves a > 1 order of magnitude improvement in performance over an atomistic system . the polarizability of cg water beads is modeled like that of the polarizable martini water model , through the addition of a harmonic angle potential term , with spring constant k0 and equilibrium angle 0 , among three charged points . this model of polarizability allows for not only dipole but also higher moments of the electrostatic energy . the central point , which acts as the interaction site for the modified morse potential , carries negative charge q , while the two outer points each carry charge q/2 . a mass equal to that of four water molecules is distributed evenly among the three points . the distance between the outer points and the central point is a tunable parameter i. the change in the angle ( ) between the points represents the polarizable nature of the bead . parameter optimization is exploration of a parameter space to determine extreme values of a target function . in the case of molecular dynamics force field optimization , the set of potential parameters defines the space and comparison of model predictions with either ab initio data or experimental measurements defines the target function to be minimized . we have developed a new software package , parameter optimizer ( paropt ) , for general optimization problems ( paropt is available for download under the gnu public license at https://csmlabfs1.cas.usf.edu/sites ) . the software provides various optimization algorithms , including an orthogonal steepest descent and the nelder mead simplex - based method . thus , in the context of molecular dynamics parameter optimization methods such as simplex - based , approaches with the least demanding conditions on the target function are the most suitable . mead , a commonly used simplex - based optimization algorithm , iteratively evolves m + 1 points on the m - dimensional space using four basic moves . the central site with charge q provides the interaction site for the modified morse potential . outer charge sites ( with charge q/2 ) are connected to the central site by a fixed bond of length i. the central angle is a determined by a harmonic potential with equilibrium angle 0 . paropt provides an environment in which different optimization methods can be accessed by defining the generation of the target function . replace the highest point with a point reflected about the centroid of the remaining points.2 if the reflected point is lower in value than all points in the centroid , consider a point further from the centroid.3 if the reflected point would remain the highest , consider a point nearer to the simplex.4 move all points nearer to the lowest point.5the m + 1 individual points in the m - dimensional space are denoted by pi , while subscripts h and l denote the lowest and highest points , respectively . the centroid is defined by6values used for optimization of water parameters are those suggested by nelder mead : = 1.0 , = 2.0 , = 0.5 , and = 0.5 . arrows denote points that will be replaced and the new point to be considered : ( a ) the reflection of the highest point about the centroid of the remaining points , ( b ) the further expansion of the reflection move , ( c ) the contraction along the line joining the centroid and the highest point , and ( d ) the shrinking of the simplex by shifting all points nearer to the lowest point . the nelder mead algorithm replaces the highest point in the simplex with one with a lower target function value by applying these moves . a nelder mead step begins with calculation of a reflect move . if the target function value for this new reflected point is lower than all points in the simplex , an expansion move is attempted . the stopping criterion for the nelder mead method is a cutoff on the root - mean - square target function value over the entire simplex . the number of occurrences of each move in an optimization depends on the characteristics of both the simplex and the target function and is therefore problem specific . the original description of the nelder mead algorithm has no set method for boundary conditions on the parameter space , though suggestions are made for modification of functional forms or assignment of large penalty we chose to replace points outside the boundary with the nearest point within the allowable space . for optimization of the mmcg water model , the target function was defined by the weighted percent error in comparison of cg simulation results with four experimental quantities : density , dielectric constant , diffusion coefficient , and surface tension . to ensure that all comparison data contribute to the target function and thus the evolution of the simplex , percent errors are assigned scalar weights to yield similar orders of magnitude among all quantities . with this goal in mind , density was weighted 100 times higher than surface tension , while permittivity and diffusion coefficient were weighted 10 times higher . , dn = d1/n , where dn is the diffusion coefficient for an n - sized cluster . the gromos cg work compares the cg bead diffusion coefficient with the diffusion coefficient for the center of mass of clusters in the spc water model . in that work , static clusters in the atomistic representation were produced by adding distance restraints between oxygen atoms , which produces diffusion coefficient scaling close to the inverse behavior usually assumed . at a bead size of four water molecules , , we use a scale factor ( s ) of 3.16 to compare the cg diffusion coefficient with experiment , instead of the usual factor of 4.7where x(t ) is the position of the central bead at time t and the broken brackets denote an ensemble average . spc / e was determined by simulation of spc / e at 303 k. clusters were chosen by the n 1 nearest neighbors for each water molecule , and the center of mass diffusion was calculated . the relative permittivity ( ) was calculated using a clausius mossotti - like equation with a reaction field with an infinite dielectric constant ( conducting boundary conditions).8where m is the total system dipole , 0 is the permittivity of free space , v is the ensemble average system volume , t is the target temperature for the thermostat , and 0 is the permittivity of the vacuum . the surface tension was taken from the gromacs internal calculation:9where the z - axis is normal to the interface , pnn terms are the pressure tensor diagonal elements , ns is the number of interfaces , and lz is the size of the system along the z - axis . therefore , systems used for determination of the diffusion coefficient , density , and electric field permittivity contained 512 cg beads , corresponding to 2048 water molecules . because of large fluctuations , the calculation of surface tension requires a larger system : 4096 cg beads , implying 16384 water molecules ( system s2 ) . system s2 was constructed , at every evaluation of the target function , with a slab near the average density computed from s1 in contact with a vacuum . to further improve the accuracy of the final point , the optimization was restarted with an initial simplex consisting of the converged point and random points around it . figure 5 shows the mean and root - mean - square ( rms ) target function value versus the nelder mead step for the final optimization iteration . because of the roughness of the hypersurface generated by the target function , the simplex may occasionally include a vertex with an abnormally high value , as observed in the mean and rms of the second shrink step of figure 5 . further , figure 6 demonstrates that although parameters did encounter the boundaries over the course of the optimization , the final simplex converged away from the boundary . variation in parameters as a function of nelder mead step . training data [ density ( ) , electric field permittivity ( ) , diffusion coefficient ( d ) , and surface tension ( ) ] ( ) as a function of nelder mead step compared with target values ( --- ) . the final set of parameters was chosen as the point in the final converged simplex with the lowest target function value ( see table 1 ) . the decoupling of repulsive and attractive parameters , the added distance dependence of the exponential factors , and the added electrostatic interaction have allowed for a decrease in the equilibrium distance , while maintaining the experimental density . when validating the results of an optimization procedure , one can not consider the training data alone . the suitability of the force field will depend on matching data that the model was not explicitly trained to reproduce . because validation is not performed iteratively , all systems for validation consisted of 110592 cg beads , which corresponds to 442368 water molecules . the density , diffusion coefficient , relative permittivity , and surface tension were determined from the larger validation systems using the same methods that were used during optimization [ see section 3.1 ; in addition , bulk thermodynamic quantities were also calculated ( see table 3 ) ] . the coefficient of thermal expansion , , was calculated using the finite - difference method.10two constant number , pressure , and temperature ( npt ) ensemble simulations at temperatures of 303.15 k ( t1 ) and 308.0 k ( t2 ) were performed . the ensemble averages of the resulting densities were used for 1 and 2 , respectively . for eq 11 , the following densities were used : 1 = 0.993 g cm , and 2 = 0.963 g cm . the enthalpy of vaporization was calculated from the interaction energy between cg beads ( vinter ) and the intermolecular interactions that occur within the bead vs of 128.5 kj mol determined from quantum mechanical calculations of the water tetramer binding energy.13where the factor of 4 is due to the level of cg . the average dipole moment of an individual bead was calculated for the mmcg water model . because a cg bead represents four water molecules , the total dipole moment for an atomistically detailed spc / e water molecule along with its three nearest neighbors the mmcg model yields an average dipole moment of 4.1 0.25 d , whereas the four - water cluster dipole moment from the spc / e water model was 5.3 1.0 d ; density function theory ( dft ) calculations for an isolated cluster produce no dipole moment . the disparity in these values is most likely a result of differences between clusters in the bulk and in isolation.14 quantities computed are density , diffusion coefficient d , dielectric permittivity , surface tension , coefficient of thermal expansion , isothermal compressibility t , and enthalpy of vaporization hvap . values computed both during optimization and for validation differ because of the size differences between simulated systems . to automate force field optimization , we have developed a general optimization framework paropt . the software utilizes various methods to locate minima of the defined target function on parameter space . we have applied this general framework to optimize the parameters of a polarizable cg water model with nonbonded interactions given by a modified morse potential via the nelder mead algorithm . the model has more flexibility in the functional form than most cg models , allowing it to match a wide range of experimental properties ( see table 3 ) . any model in a classical molecular dynamics framework is a simplification of a physical system and therefore involves a reduction in the degrees of freedom in the description of the interactions . though the set of parameters that govern the model are mathematically independent , their effects on observable data and therefore the target function are not necessarily uncorrelated . therefore , because of this interdependence of the parameters , the optimization is not necessarily underdetermined despite an apparent abundance of free parameters to match the target data . additionally , it can be seen from the good agreement with the validation data , which is independent of the training data , that the resulting force field parameters are well - tuned . an artifact common to most cg models is the long - range correlation between cg water beads not seen in experiment or atomistic models . likely as a result of the high degree of interbead correlation at long distances , the model has the propensity to spontaneously solidify at room temperature , a weakness found in other cg water models . this phenomenon would not be expected in a heterogeneous system , because long - range correlations would be broken by the presence of other interactions . to prevent the overpopulation of low - frequency modes of a molecular dynamics system . the set of potential parameters has been tuned for one temperature and is not expected to accurately simulate behavior far from the target temperature or in thermodynamic ensembles without constant temperature control . comparison between g(r ) for the atomistic spc / e water model ( ) and the mmcg water model ( --- ) . the predictions of the model agree well with experiment for structural , dynamic , and bulk properties . accurate surface tension indicates an accurate representation of the strength of the interaction between beads . the thermodynamic quantities predicted by the model are in good agreement with those of experiment , validating the fitness of the parameters .

in the last decade , the field of health informatics ( hi ) has grown worldwide . the institute of medicine in the united states has declared that information technology ( it ) is an essential tool to improve healthcare costs , patient safety and the quality and equity of care [ 2 - 4 ] . these benefits make hi particularly relevant for developing countries , where common difficulties faced by most of them , such as poor economics , political uncertainty , and the lack of cutting edge infrastructure , have hampered the quality of healthcare . it has been shown that it is feasible to implement a broad spectrum of it solutions in developing countries . although success has been achieved in implementing pilots or isolated programs ( mostly in mobile health or local electronic medical records ) , large sustainable implementations at a country or regional level are less common [ 7 - 10 ] . this lack of sustainability and scalability prevents improvements in quality , efficiency , and equity outcomes . thus , the potential benefits and the ultimate goal to achieve quality of care improvements through it are not met , making sustainability a primary factor for its success . according to madani and aronsky , when addressing the application of it in healthcare , sustainability can be analyzed through the following factors : 1 ) effectiveness , measured by outcome variables , such as mortality , morbidity , safety , or quality of medical decision - making ; 2 ) efficiency , including factors that affect resource allocation for the development and maintenance of systems , such as user training ; 3 ) financial viability , including cost - effectiveness of applications and return - on - investment in the long - term ; 4 ) reproducibility , such as integration and application in a variety of different settings ; and 5 ) portability , measured by the ease of implementing and adapting concepts and approaches to other environments . the benefits of achieving major sustainable implementations in hi have been hampered by several challenges common to most developing countries . as the field matures and health information systems ( his ) become more relevant for healthcare , a review of the various factors affecting sustainability is needed . based on this reality , we conducted a non - systematic review of the literature . articles were searched using the keywords medical informatics , developing countries , implementation and challenges in pubmed , lilacs , cinahl , scopus , and embase . the retrieved articles were classified by consensus rounds into six broad categories related to the challenges in achieving sustainable implementations in hi : 1 ) resource and infrastructure limitations ; 2 ) development of health it agendas ; 3 ) overcoming uncertainty , ethics , and legal considerations ; 4 ) lack of use of common interoperability standards ; 5 ) lack of a skillfully trained workforce ; and 6 ) regional integration . this review article describes experiences published on the challenges and barriers faced by developing countries with possible ways to address them , in the hope of assisting policy makers , healthcare managers , and project leaders to anticipate the challenges they will face and help them overcome them early in their implementations . there are many financial and structural hurdles that developing countries face when implementing sustainable e - health programs . examples of these difficulties are the lack of reliable electricity and low - quality , expensive internet access . disparities between different regions of the same country are common , and infrastructure can be very different when comparing private or public initiatives . these problems are most relevant in rural areas . developing countries have considerable structural deficits in their physical networks , due to high costs , geographic dispersion , and high percentages of the people living in rural areas . the possibility of using wireless networks and the widespread adoption of mobile phones help to ameliorate this issue . the penetration rates of mobile phones are increasing year after year in developing countries , providing opportunities to implement systems that require less resources in new and imaginative ways . for these reasons , mobile health ( part of a broader field known as telemedicine ) is proving to be useful to avoid the lack of an adequate infrastructure . nevertheless , these solutions create new problems , such as fragmented information , and difficulties with project scalability . luckily , given the accuracy of moore 's law prediction , hardware costs are constantly falling ; giving developing countries access to technologies that were previously unattainable . in addition , some governments are implementing initiatives intended to reduce the digital divide , through the distribution of low - cost , portable computers to children . examples of these initiatives are the " ceibal " program in uruguay , which has recently distributed its millionth computer ; and the " conectar igualdad " program in argentina , with more than 3,500,000 netbooks already distributed . in africa , rwanda is the country with the largest number of laptops distributed to children , with 210,000 computers distributed by the end of 2012 . in terms of software , the rise of the open - source movement is helping resource limited countries to implement his . two well - known examples are postgresql , a powerful open - source object - relational database system , with more than 15 years of experience , and a strong reputation for reliability , and openmrs , a software platform based on the data model of the regenstrief institute , which enables the design of customized electronic health records ( ehrs ) with no programming experience . openmrs has been implemented in many developing countries in africa , asia , and central and latin america . it is noteworthy that the direct costs of hi implementations can be large , which makes long - term commitments necessary to maintain them . it is common for these programs to rely on donor funding for the pilot stages , and when looking for opportunities to scale up , alternative sources of financing are needed . these can be difficult to obtain in resource constrained countries , where an e - health agenda has to compete with more basic needs , like food , healthcare and education . these infrastructure limitations in networks , hardware , and software must be considered before planning an e - health project in developing countries . a comprehensive nationwide e - health agenda that contemplates most of the challenges we address in this review is vital . the need for health it application frameworks to better develop and sustain it projects has been advocated by the united nations , the world health organization ( who ) , and other international organizations . worldwide , large amounts of health data are electronically collected , but information is scattered and is not useful for high - quality decision - making . further development in hi will require the implementation of clear data standards in order to be of optimal value , and this situation demands a clear framework for understanding and moving forward on e - health . many developed countries have advanced in the development of e - health agendas : canada , australia , and denmark among others [ 30 - 32 ] . in 2010 , the economic commission for latin america and the caribbean ( eclac ) , described the advances made and the difficulties encountered by five developed countries ( belgium , sweden , spain , united kingdom , and denmark ) while implementing e - health actions . e - health agendas have suffered from a lack of sufficient focus and targeted priorities in developing countries . few of them have sufficiently strong and effective his to meet all their diverse needs . however , since 2008 , more than 20 developing countries have been working on health information strategic plans , supported by the who 's health metrics network , a global partnership created in 2005 , dedicated to assessing and strengthening national his . in this sense , private initiatives could generate a positive impact in the disclosure of these programs . international societies could advance the generation of regional or national agendas that enable or facilitate the implementation of hi programs through economic incentives and professional training programs . overcoming uncertainty represents a challenge in every new implementation . in the process of implementing an ehr , patients enrolled in healthcare systems must trust those invested with the responsibility to safeguard their personal information . these challenges are similar worldwide , but their consequences are less serious in developed countries , because their legal frameworks have better support for digital agendas . furthermore , security issues and legal accountability might represent a significant obstacle in the implementation process . the high levels of legal uncertainty present in most developing countries could act in two antagonistic modes . on one side , the lack of legalization in the field could be an incentive to work more freely . on the other side , the lack of needed laws could delay the start of implementations , while countries wait for a framework to organize such programs . still , in one way or another , the number of initiatives is lower than expected when compared to developed countries . to manage these challenges , local health personnel need to acquire knowledge of legal frameworks and medical ethics . there is an urgent need to enhance the teaching of the discipline at both the undergraduate and postgraduate levels . it is common for his to be fragmented , incomplete , inaccurate , and isolated , and this problem is even more explicit in developing countries . this leads to information silos , and the information contained inside them can not be used for patient care or data analysis . to overcome this problem , the ability to exchange and use information between different systems ( interoperability ) is a fundamental requirement to accomplish healthcare goals ; the most important consequence of the lack of interoperability is the loss of the continuity of care among practitioners . the who asked its member countries to adopt standards for effective information exchange between healthcare actors and e - health implementations through a resolution in its 66th world assembly . some of the aspects that need to be addressed to achieve effective interoperability are the correct and unambiguous identification of patients , improved cooperation among stakeholders to ensure the consistent application of standards across different domains , the use of data interchange standards to ensure syntactic interoperability , the application of semantic interoperability with the use of standard terminologies , and the use of quality measures to assure that data is accurate and relevant . most of the standards needed to create interoperable systems exist today , and are the same as those required in developed countries . governments and stakeholders must be aware of these issues and advance methods of reaching consensus on the common and consistent use of standards . international initiatives showing the benefits of defined standards could improve and accelerate the process to define these standards in developing regions . in the same way , international standard societies should help these countries in the process of implementing these interoperability programs by disseminating its standards and training staff . there is widespread agreement that an appropriately trained workforce is a critical dimension if sustained progress is to be achieved . there are too few well - trained medical informaticians , and they have an inadequate geographic distribution to meet the needs and expertise necessary for health it implementations . the general model to train the workforce needed emerged from the e - capacity meeting in bellagio in 2008 , during which components of the educational strategy to train clinical informaticians and improve the level of informatics knowledge , skills , and attitudes in both formally and informally trained health workers were described . one example is the american medical informatics association 's ( amia 's ) 10 10 initiative , a program that aimed to train 10,000 professionals in hi by 2010 . since its introduction , amia has been working alongside local institutions in developing countries , such as argentina and singapore , to create an international version adapted to local needs . other initiatives include the informatics training for global health program of the fogarty international center , us national institutes of health , that maintains eight partnerships between united states and international academic programs to expand informatics training in india , latin america , and africa as well as the amia 's global partnership program , funded by the bill and melinda gates foundation , with the goals of promoting project - centric approaches to training in developing countries to expand the local capacity to continue programs in the future . another approach to this problem is the use of telemedicine and mobile devices to connect trained resources with the population in need . this is especially useful in rural areas located far from urban centers where the specialized workforce tends to live . one example is the experience in india of using mobile tools for the screening of retinopathy . all of these actions are recognized as essential for advancing educational programs and implementing systems in ways that are compatible with local culture and healthcare needs , and should be coordinated with current efforts being made by the international medical informatics association ( imia ) through education working groups . the us office of the national coordinator for health information technology and the institute of medicine , recognizing the importance of integrating and sharing experiences , are committed to helping organizations share approaches to improve their likelihood of success . the flow of information needs to be nurtured to allow for the dissemination of potential patient safety issues , such as those involved in the implementation of his . the communication of existing projects , as well as of past experiences has proven to be a challenge in developing countries . critical to the transition from a pilot project to a sustainable implementation is the sharing of resources , experiences , and lessons learned from other projects . traditionally , the communication of medical knowledge , research , and advances has been achieved by the publication of articles in scientific journals . yet , this task also represents an obstacle in developing countries , and hi is not excluded . publication and retrieval of hi scientific material can be facilitated quickly with relatively little expenditure when compared with other investments needed . for example , the open - access approach to publishing helps authors in underdeveloped regions share information . the imia , through its working group for development , has focused on this challenge , creating a global repository for all ongoing projects related to health it , with the hope that it will fulfill the final goal of connecting all the actors working in hi solutions for developing countries . the site is still relatively new as it was introduced in july 2013 , and managing the input of data by project leaders has proven difficult mostly due to lack of knowledge of the tool . efforts to communicate successful programs in the region and to share experiences in implementing programs and teaching resources there are many financial and structural hurdles that developing countries face when implementing sustainable e - health programs . examples of these difficulties are the lack of reliable electricity and low - quality , expensive internet access . disparities between different regions of the same country are common , and infrastructure can be very different when comparing private or public initiatives . these problems are most relevant in rural areas . developing countries have considerable structural deficits in their physical networks , due to high costs , geographic dispersion , and high percentages of the people living in rural areas . the possibility of using wireless networks and the widespread adoption of mobile phones help to ameliorate this issue . the penetration rates of mobile phones are increasing year after year in developing countries , providing opportunities to implement systems that require less resources in new and imaginative ways . for these reasons , mobile health ( part of a broader field known as telemedicine ) is proving to be useful to avoid the lack of an adequate infrastructure . nevertheless , these solutions create new problems , such as fragmented information , and difficulties with project scalability . luckily , given the accuracy of moore 's law prediction , hardware costs are constantly falling ; giving developing countries access to technologies that were previously unattainable . in addition , some governments are implementing initiatives intended to reduce the digital divide , through the distribution of low - cost , portable computers to children . examples of these initiatives are the " ceibal " program in uruguay , which has recently distributed its millionth computer ; and the " conectar igualdad " program in argentina , with more than 3,500,000 netbooks already distributed . in africa , rwanda is the country with the largest number of laptops distributed to children , with 210,000 computers distributed by the end of 2012 . in terms of software , the rise of the open - source movement is helping resource limited countries to implement his . two well - known examples are postgresql , a powerful open - source object - relational database system , with more than 15 years of experience , and a strong reputation for reliability , and openmrs , a software platform based on the data model of the regenstrief institute , which enables the design of customized electronic health records ( ehrs ) with no programming experience . openmrs has been implemented in many developing countries in africa , asia , and central and latin america . it is noteworthy that the direct costs of hi implementations can be large , which makes long - term commitments necessary to maintain them . it is common for these programs to rely on donor funding for the pilot stages , and when looking for opportunities to scale up , alternative sources of financing are needed . these can be difficult to obtain in resource constrained countries , where an e - health agenda has to compete with more basic needs , like food , healthcare and education . these infrastructure limitations in networks , hardware , and software must be considered before planning an e - health project in developing countries . a comprehensive nationwide e - health agenda that contemplates most of the challenges we address in this review is vital . the need for health it application frameworks to better develop and sustain it projects has been advocated by the united nations , the world health organization ( who ) , and other international organizations . worldwide , large amounts of health data are electronically collected , but information is scattered and is not useful for high - quality decision - making . further development in hi will require the implementation of clear data standards in order to be of optimal value , and this situation demands a clear framework for understanding and moving forward on e - health . many developed countries have advanced in the development of e - health agendas : canada , australia , and denmark among others [ 30 - 32 ] . in 2010 , the economic commission for latin america and the caribbean ( eclac ) , described the advances made and the difficulties encountered by five developed countries ( belgium , sweden , spain , united kingdom , and denmark ) while implementing e - health actions . e - health agendas have suffered from a lack of sufficient focus and targeted priorities in developing countries . few of them have sufficiently strong and effective his to meet all their diverse needs . however , since 2008 , more than 20 developing countries have been working on health information strategic plans , supported by the who 's health metrics network , a global partnership created in 2005 , dedicated to assessing and strengthening national his . in this sense , international societies could advance the generation of regional or national agendas that enable or facilitate the implementation of hi programs through economic incentives and professional training programs . overcoming uncertainty represents a challenge in every new implementation . in the process of implementing an ehr , patients enrolled in healthcare systems must trust those invested with the responsibility to safeguard their personal information . these challenges are similar worldwide , but their consequences are less serious in developed countries , because their legal frameworks have better support for digital agendas . furthermore , security issues and legal accountability might represent a significant obstacle in the implementation process . the high levels of legal uncertainty present in most developing countries could act in two antagonistic modes . on one side , the lack of legalization in the field could be an incentive to work more freely . on the other side , the lack of needed laws could delay the start of implementations , while countries wait for a framework to organize such programs . still , in one way or another , the number of initiatives is lower than expected when compared to developed countries . to manage these challenges , local health personnel need to acquire knowledge of legal frameworks and medical ethics . there is an urgent need to enhance the teaching of the discipline at both the undergraduate and postgraduate levels . it is common for his to be fragmented , incomplete , inaccurate , and isolated , and this problem is even more explicit in developing countries . this leads to information silos , and the information contained inside them can not be used for patient care or data analysis . to overcome this problem , the ability to exchange and use information between different systems ( interoperability ) is a fundamental requirement to accomplish healthcare goals ; the most important consequence of the lack of interoperability is the loss of the continuity of care among practitioners . the who asked its member countries to adopt standards for effective information exchange between healthcare actors and e - health implementations through a resolution in its 66th world assembly . some of the aspects that need to be addressed to achieve effective interoperability are the correct and unambiguous identification of patients , improved cooperation among stakeholders to ensure the consistent application of standards across different domains , the use of data interchange standards to ensure syntactic interoperability , the application of semantic interoperability with the use of standard terminologies , and the use of quality measures to assure that data is accurate and relevant . most of the standards needed to create interoperable systems exist today , and are the same as those required in developed countries . governments and stakeholders must be aware of these issues and advance methods of reaching consensus on the common and consistent use of standards . international initiatives showing the benefits of defined standards could improve and accelerate the process to define these standards in developing regions . in the same way , international standard societies should help these countries in the process of implementing these interoperability programs by disseminating its standards and training staff . there is widespread agreement that an appropriately trained workforce is a critical dimension if sustained progress is to be achieved . there are too few well - trained medical informaticians , and they have an inadequate geographic distribution to meet the needs and expertise necessary for health it implementations . the general model to train the workforce needed emerged from the e - capacity meeting in bellagio in 2008 , during which components of the educational strategy to train clinical informaticians and improve the level of informatics knowledge , skills , and attitudes in both formally and informally trained health workers were described . one example is the american medical informatics association 's ( amia 's ) 10 10 initiative , a program that aimed to train 10,000 professionals in hi by 2010 . since its introduction , amia has been working alongside local institutions in developing countries , such as argentina and singapore , to create an international version adapted to local needs . other initiatives include the informatics training for global health program of the fogarty international center , us national institutes of health , that maintains eight partnerships between united states and international academic programs to expand informatics training in india , latin america , and africa as well as the amia 's global partnership program , funded by the bill and melinda gates foundation , with the goals of promoting project - centric approaches to training in developing countries to expand the local capacity to continue programs in the future . another approach to this problem is the use of telemedicine and mobile devices to connect trained resources with the population in need . this is especially useful in rural areas located far from urban centers where the specialized workforce tends to live . one example is the experience in india of using mobile tools for the screening of retinopathy . all of these actions are recognized as essential for advancing educational programs and implementing systems in ways that are compatible with local culture and healthcare needs , and should be coordinated with current efforts being made by the international medical informatics association ( imia ) through education working groups . the us office of the national coordinator for health information technology and the institute of medicine , recognizing the importance of integrating and sharing experiences , are committed to helping organizations share approaches to improve their likelihood of success . the flow of information needs to be nurtured to allow for the dissemination of potential patient safety issues , such as those involved in the implementation of his . the communication of existing projects , as well as of past experiences has proven to be a challenge in developing countries . critical to the transition from a pilot project to a sustainable implementation is the sharing of resources , experiences , and lessons learned from other projects . traditionally , the communication of medical knowledge , research , and advances has been achieved by the publication of articles in scientific journals . yet , this task also represents an obstacle in developing countries , and hi is not excluded . publication and retrieval of hi scientific material can be facilitated quickly with relatively little expenditure when compared with other investments needed . for example , the open - access approach to publishing helps authors in underdeveloped regions share information . the imia , through its working group for development , has focused on this challenge , creating a global repository for all ongoing projects related to health it , with the hope that it will fulfill the final goal of connecting all the actors working in hi solutions for developing countries . the site is still relatively new as it was introduced in july 2013 , and managing the input of data by project leaders has proven difficult mostly due to lack of knowledge of the tool . efforts to communicate successful programs in the region and to share experiences in implementing programs and teaching resources knowing the challenges to be faced is important for the success of any implementation in hi . we have reviewed the problems faced by developing countries arising from the lack of adequate infrastructure , and the ways these problems can be bypassed . the issues that must be addressed include : the fundamental need to develop nationwide e - health agendas to achieve sustainable implementations ; ways to overcome public uncertainty with respect to privacy and security ; the difficulties shared with developed countries to achieve interoperability ; the need for a trained workforce in hi and existing initiatives for its development ; and strategies for achieving regional integration ( see table 1 for a summary of recommendations ) . even while this review has some limitations , such as the fact that it is not a systematic review , and difficulty in finding published papers related to the topic for all the countries in this group ; the challenges and barriers seem to be common to most of them . more resources towards integration of the region may be necessary as well as programs to help incipient implementations through the sharing of experiences and workforce . although the challenges described in this article could be common to all the countries in this group , moreover , these challenges are discussed in relation to developing countries , where implementers must be aware of common barriers and difficulties that can strain efforts in all it implementations . we hope this article will assist policy makers , healthcare managers , and project leaders to successfully plan their implementations and make them sustainable , to avoid unexpected barriers as much as possible , and to make better use of their resources .

objectivesinformation technology is an essential tool to improve patient safety and the quality of care , and to reduce healthcare costs . there is a scarcity of large sustainable implementations in developing countries . the objective of this paper is to review the challenges faced by developing countries to achieve sustainable implementations in health informatics and possible ways to address them.methodsin this non - systematic review of the literature , articles were searched using the keywords medical informatics , developing countries , implementation , and challenges in pubmed , lilacs , cinahl , scopus , and embase . the authors , after reading the literature , reached a consensus to classify the challenges into six broad categories.resultsthe authors describe the problems faced by developing countries arising from the lack of adequate infrastructure and the ways these can be bypassed ; the fundamental need to develop nationwide e - health agendas to achieve sustainable implementations ; ways to overcome public uncertainty with respect to privacy and security ; the difficulties shared with developed countries in achieving interoperability ; the need for a trained workforce in health informatics and existing initiatives for its development ; and strategies to achieve regional integration.conclusionscentral to the success of any implementation in health informatics is knowledge of the challenges to be faced . this is even more important in developing countries , where uncertainty and instability are common . the authors hope this article will assist policy makers , healthcare managers , and project leaders to successfully plan their implementations and make them sustainable , avoiding unexpected barriers and making better use of their resources .

I. Introduction II. Methods III. Results 1. Resource and Infrastructure Limitations 2. Development of Health IT Agendas 3. Overcoming Uncertainty, Ethics and Legal Considerations 4. Lack of Use of Common Interoperability Standards 5. Lack of a Trained Workforce 6. Regional Integration IV. Conclusions

the institute of medicine in the united states has declared that information technology ( it ) is an essential tool to improve healthcare costs , patient safety and the quality and equity of care [ 2 - 4 ] . these benefits make hi particularly relevant for developing countries , where common difficulties faced by most of them , such as poor economics , political uncertainty , and the lack of cutting edge infrastructure , have hampered the quality of healthcare . it has been shown that it is feasible to implement a broad spectrum of it solutions in developing countries . although success has been achieved in implementing pilots or isolated programs ( mostly in mobile health or local electronic medical records ) , large sustainable implementations at a country or regional level are less common [ 7 - 10 ] . this lack of sustainability and scalability prevents improvements in quality , efficiency , and equity outcomes . thus , the potential benefits and the ultimate goal to achieve quality of care improvements through it are not met , making sustainability a primary factor for its success . according to madani and aronsky , when addressing the application of it in healthcare , sustainability can be analyzed through the following factors : 1 ) effectiveness , measured by outcome variables , such as mortality , morbidity , safety , or quality of medical decision - making ; 2 ) efficiency , including factors that affect resource allocation for the development and maintenance of systems , such as user training ; 3 ) financial viability , including cost - effectiveness of applications and return - on - investment in the long - term ; 4 ) reproducibility , such as integration and application in a variety of different settings ; and 5 ) portability , measured by the ease of implementing and adapting concepts and approaches to other environments . the benefits of achieving major sustainable implementations in hi have been hampered by several challenges common to most developing countries . as the field matures and health information systems ( his ) become more relevant for healthcare , a review of the various factors affecting sustainability is needed . based on this reality , we conducted a non - systematic review of the literature . articles were searched using the keywords medical informatics , developing countries , implementation and challenges in pubmed , lilacs , cinahl , scopus , and embase . the retrieved articles were classified by consensus rounds into six broad categories related to the challenges in achieving sustainable implementations in hi : 1 ) resource and infrastructure limitations ; 2 ) development of health it agendas ; 3 ) overcoming uncertainty , ethics , and legal considerations ; 4 ) lack of use of common interoperability standards ; 5 ) lack of a skillfully trained workforce ; and 6 ) regional integration . this review article describes experiences published on the challenges and barriers faced by developing countries with possible ways to address them , in the hope of assisting policy makers , healthcare managers , and project leaders to anticipate the challenges they will face and help them overcome them early in their implementations . there are many financial and structural hurdles that developing countries face when implementing sustainable e - health programs . examples of these difficulties are the lack of reliable electricity and low - quality , expensive internet access . disparities between different regions of the same country are common , and infrastructure can be very different when comparing private or public initiatives . developing countries have considerable structural deficits in their physical networks , due to high costs , geographic dispersion , and high percentages of the people living in rural areas . the possibility of using wireless networks and the widespread adoption of mobile phones help to ameliorate this issue . the penetration rates of mobile phones are increasing year after year in developing countries , providing opportunities to implement systems that require less resources in new and imaginative ways . for these reasons , mobile health ( part of a broader field known as telemedicine ) is proving to be useful to avoid the lack of an adequate infrastructure . nevertheless , these solutions create new problems , such as fragmented information , and difficulties with project scalability . in addition , some governments are implementing initiatives intended to reduce the digital divide , through the distribution of low - cost , portable computers to children . examples of these initiatives are the " ceibal " program in uruguay , which has recently distributed its millionth computer ; and the " conectar igualdad " program in argentina , with more than 3,500,000 netbooks already distributed . in terms of software , the rise of the open - source movement is helping resource limited countries to implement his . two well - known examples are postgresql , a powerful open - source object - relational database system , with more than 15 years of experience , and a strong reputation for reliability , and openmrs , a software platform based on the data model of the regenstrief institute , which enables the design of customized electronic health records ( ehrs ) with no programming experience . openmrs has been implemented in many developing countries in africa , asia , and central and latin america . these can be difficult to obtain in resource constrained countries , where an e - health agenda has to compete with more basic needs , like food , healthcare and education . these infrastructure limitations in networks , hardware , and software must be considered before planning an e - health project in developing countries . a comprehensive nationwide e - health agenda that contemplates most of the challenges we address in this review is vital . the need for health it application frameworks to better develop and sustain it projects has been advocated by the united nations , the world health organization ( who ) , and other international organizations . further development in hi will require the implementation of clear data standards in order to be of optimal value , and this situation demands a clear framework for understanding and moving forward on e - health . many developed countries have advanced in the development of e - health agendas : canada , australia , and denmark among others [ 30 - 32 ] . in 2010 , the economic commission for latin america and the caribbean ( eclac ) , described the advances made and the difficulties encountered by five developed countries ( belgium , sweden , spain , united kingdom , and denmark ) while implementing e - health actions . e - health agendas have suffered from a lack of sufficient focus and targeted priorities in developing countries . these challenges are similar worldwide , but their consequences are less serious in developed countries , because their legal frameworks have better support for digital agendas . on one side , the lack of legalization in the field could be an incentive to work more freely . on the other side , the lack of needed laws could delay the start of implementations , while countries wait for a framework to organize such programs . to manage these challenges , local health personnel need to acquire knowledge of legal frameworks and medical ethics . there is an urgent need to enhance the teaching of the discipline at both the undergraduate and postgraduate levels . it is common for his to be fragmented , incomplete , inaccurate , and isolated , and this problem is even more explicit in developing countries . this leads to information silos , and the information contained inside them can not be used for patient care or data analysis . to overcome this problem , the ability to exchange and use information between different systems ( interoperability ) is a fundamental requirement to accomplish healthcare goals ; the most important consequence of the lack of interoperability is the loss of the continuity of care among practitioners . the who asked its member countries to adopt standards for effective information exchange between healthcare actors and e - health implementations through a resolution in its 66th world assembly . some of the aspects that need to be addressed to achieve effective interoperability are the correct and unambiguous identification of patients , improved cooperation among stakeholders to ensure the consistent application of standards across different domains , the use of data interchange standards to ensure syntactic interoperability , the application of semantic interoperability with the use of standard terminologies , and the use of quality measures to assure that data is accurate and relevant . most of the standards needed to create interoperable systems exist today , and are the same as those required in developed countries . governments and stakeholders must be aware of these issues and advance methods of reaching consensus on the common and consistent use of standards . international initiatives showing the benefits of defined standards could improve and accelerate the process to define these standards in developing regions . in the same way , international standard societies should help these countries in the process of implementing these interoperability programs by disseminating its standards and training staff . there is widespread agreement that an appropriately trained workforce is a critical dimension if sustained progress is to be achieved . there are too few well - trained medical informaticians , and they have an inadequate geographic distribution to meet the needs and expertise necessary for health it implementations . the general model to train the workforce needed emerged from the e - capacity meeting in bellagio in 2008 , during which components of the educational strategy to train clinical informaticians and improve the level of informatics knowledge , skills , and attitudes in both formally and informally trained health workers were described . one example is the american medical informatics association 's ( amia 's ) 10 10 initiative , a program that aimed to train 10,000 professionals in hi by 2010 . since its introduction , amia has been working alongside local institutions in developing countries , such as argentina and singapore , to create an international version adapted to local needs . other initiatives include the informatics training for global health program of the fogarty international center , us national institutes of health , that maintains eight partnerships between united states and international academic programs to expand informatics training in india , latin america , and africa as well as the amia 's global partnership program , funded by the bill and melinda gates foundation , with the goals of promoting project - centric approaches to training in developing countries to expand the local capacity to continue programs in the future . another approach to this problem is the use of telemedicine and mobile devices to connect trained resources with the population in need . all of these actions are recognized as essential for advancing educational programs and implementing systems in ways that are compatible with local culture and healthcare needs , and should be coordinated with current efforts being made by the international medical informatics association ( imia ) through education working groups . the us office of the national coordinator for health information technology and the institute of medicine , recognizing the importance of integrating and sharing experiences , are committed to helping organizations share approaches to improve their likelihood of success . the flow of information needs to be nurtured to allow for the dissemination of potential patient safety issues , such as those involved in the implementation of his . the communication of existing projects , as well as of past experiences has proven to be a challenge in developing countries . critical to the transition from a pilot project to a sustainable implementation is the sharing of resources , experiences , and lessons learned from other projects . yet , this task also represents an obstacle in developing countries , and hi is not excluded . publication and retrieval of hi scientific material can be facilitated quickly with relatively little expenditure when compared with other investments needed . the site is still relatively new as it was introduced in july 2013 , and managing the input of data by project leaders has proven difficult mostly due to lack of knowledge of the tool . efforts to communicate successful programs in the region and to share experiences in implementing programs and teaching resources there are many financial and structural hurdles that developing countries face when implementing sustainable e - health programs . examples of these difficulties are the lack of reliable electricity and low - quality , expensive internet access . disparities between different regions of the same country are common , and infrastructure can be very different when comparing private or public initiatives . developing countries have considerable structural deficits in their physical networks , due to high costs , geographic dispersion , and high percentages of the people living in rural areas . the penetration rates of mobile phones are increasing year after year in developing countries , providing opportunities to implement systems that require less resources in new and imaginative ways . for these reasons , mobile health ( part of a broader field known as telemedicine ) is proving to be useful to avoid the lack of an adequate infrastructure . luckily , given the accuracy of moore 's law prediction , hardware costs are constantly falling ; giving developing countries access to technologies that were previously unattainable . examples of these initiatives are the " ceibal " program in uruguay , which has recently distributed its millionth computer ; and the " conectar igualdad " program in argentina , with more than 3,500,000 netbooks already distributed . in terms of software , the rise of the open - source movement is helping resource limited countries to implement his . two well - known examples are postgresql , a powerful open - source object - relational database system , with more than 15 years of experience , and a strong reputation for reliability , and openmrs , a software platform based on the data model of the regenstrief institute , which enables the design of customized electronic health records ( ehrs ) with no programming experience . openmrs has been implemented in many developing countries in africa , asia , and central and latin america . it is noteworthy that the direct costs of hi implementations can be large , which makes long - term commitments necessary to maintain them . it is common for these programs to rely on donor funding for the pilot stages , and when looking for opportunities to scale up , alternative sources of financing are needed . these can be difficult to obtain in resource constrained countries , where an e - health agenda has to compete with more basic needs , like food , healthcare and education . these infrastructure limitations in networks , hardware , and software must be considered before planning an e - health project in developing countries . a comprehensive nationwide e - health agenda that contemplates most of the challenges we address in this review is vital . the need for health it application frameworks to better develop and sustain it projects has been advocated by the united nations , the world health organization ( who ) , and other international organizations . further development in hi will require the implementation of clear data standards in order to be of optimal value , and this situation demands a clear framework for understanding and moving forward on e - health . many developed countries have advanced in the development of e - health agendas : canada , australia , and denmark among others [ 30 - 32 ] . in 2010 , the economic commission for latin america and the caribbean ( eclac ) , described the advances made and the difficulties encountered by five developed countries ( belgium , sweden , spain , united kingdom , and denmark ) while implementing e - health actions . e - health agendas have suffered from a lack of sufficient focus and targeted priorities in developing countries . these challenges are similar worldwide , but their consequences are less serious in developed countries , because their legal frameworks have better support for digital agendas . on one side , the lack of legalization in the field could be an incentive to work more freely . on the other side , the lack of needed laws could delay the start of implementations , while countries wait for a framework to organize such programs . to manage these challenges , local health personnel need to acquire knowledge of legal frameworks and medical ethics . there is an urgent need to enhance the teaching of the discipline at both the undergraduate and postgraduate levels . it is common for his to be fragmented , incomplete , inaccurate , and isolated , and this problem is even more explicit in developing countries . this leads to information silos , and the information contained inside them can not be used for patient care or data analysis . to overcome this problem , the ability to exchange and use information between different systems ( interoperability ) is a fundamental requirement to accomplish healthcare goals ; the most important consequence of the lack of interoperability is the loss of the continuity of care among practitioners . the who asked its member countries to adopt standards for effective information exchange between healthcare actors and e - health implementations through a resolution in its 66th world assembly . some of the aspects that need to be addressed to achieve effective interoperability are the correct and unambiguous identification of patients , improved cooperation among stakeholders to ensure the consistent application of standards across different domains , the use of data interchange standards to ensure syntactic interoperability , the application of semantic interoperability with the use of standard terminologies , and the use of quality measures to assure that data is accurate and relevant . most of the standards needed to create interoperable systems exist today , and are the same as those required in developed countries . in the same way , international standard societies should help these countries in the process of implementing these interoperability programs by disseminating its standards and training staff . there is widespread agreement that an appropriately trained workforce is a critical dimension if sustained progress is to be achieved . the general model to train the workforce needed emerged from the e - capacity meeting in bellagio in 2008 , during which components of the educational strategy to train clinical informaticians and improve the level of informatics knowledge , skills , and attitudes in both formally and informally trained health workers were described . since its introduction , amia has been working alongside local institutions in developing countries , such as argentina and singapore , to create an international version adapted to local needs . other initiatives include the informatics training for global health program of the fogarty international center , us national institutes of health , that maintains eight partnerships between united states and international academic programs to expand informatics training in india , latin america , and africa as well as the amia 's global partnership program , funded by the bill and melinda gates foundation , with the goals of promoting project - centric approaches to training in developing countries to expand the local capacity to continue programs in the future . all of these actions are recognized as essential for advancing educational programs and implementing systems in ways that are compatible with local culture and healthcare needs , and should be coordinated with current efforts being made by the international medical informatics association ( imia ) through education working groups . the us office of the national coordinator for health information technology and the institute of medicine , recognizing the importance of integrating and sharing experiences , are committed to helping organizations share approaches to improve their likelihood of success . the flow of information needs to be nurtured to allow for the dissemination of potential patient safety issues , such as those involved in the implementation of his . the communication of existing projects , as well as of past experiences has proven to be a challenge in developing countries . critical to the transition from a pilot project to a sustainable implementation is the sharing of resources , experiences , and lessons learned from other projects . yet , this task also represents an obstacle in developing countries , and hi is not excluded . the imia , through its working group for development , has focused on this challenge , creating a global repository for all ongoing projects related to health it , with the hope that it will fulfill the final goal of connecting all the actors working in hi solutions for developing countries . the site is still relatively new as it was introduced in july 2013 , and managing the input of data by project leaders has proven difficult mostly due to lack of knowledge of the tool . efforts to communicate successful programs in the region and to share experiences in implementing programs and teaching resources knowing the challenges to be faced is important for the success of any implementation in hi . we have reviewed the problems faced by developing countries arising from the lack of adequate infrastructure , and the ways these problems can be bypassed . the issues that must be addressed include : the fundamental need to develop nationwide e - health agendas to achieve sustainable implementations ; ways to overcome public uncertainty with respect to privacy and security ; the difficulties shared with developed countries to achieve interoperability ; the need for a trained workforce in hi and existing initiatives for its development ; and strategies for achieving regional integration ( see table 1 for a summary of recommendations ) . even while this review has some limitations , such as the fact that it is not a systematic review , and difficulty in finding published papers related to the topic for all the countries in this group ; the challenges and barriers seem to be common to most of them . although the challenges described in this article could be common to all the countries in this group , moreover , these challenges are discussed in relation to developing countries , where implementers must be aware of common barriers and difficulties that can strain efforts in all it implementations . we hope this article will assist policy makers , healthcare managers , and project leaders to successfully plan their implementations and make them sustainable , to avoid unexpected barriers as much as possible , and to make better use of their resources .

an essential step in the procedure for sentinel lymph node ( sln ) biopsy is to locate the first - echelon node of the drainage basin . lymphoscintigraphy is a minimally invasive diagnostic tool for mapping the sln . after injecting the patient with sulfur colloid radiolabeled with tc in locations proximal to a tumor site and waiting for lymphatic drainage to occur , the lymphatic drainage pathway from the primary injection site may be imaged with a gamma camera . interpretation of lymphoscintigrams is hindered by the absence of anatomical landmarks in the scintigraphy image . to partly cope with this limitation , it is customary to acquire additional transmission images via a backlighting co sheet source in order to facilitate the anatomic localization of the nodes seen on the scintigraphy image . while the backlighting imaging technique creates an outline of the patient 's body , it depicts no internal landmarks . the introduction of hybrid single photon emission computed tomography with computed tomography ( spect / ct ) systems into clinical practice presents an opportunity to improve the spatial localization in lymphoscintigraphy . improvement in the localization technique could potentially help the surgeon prepare for sentinel node biopsy ( snb ) operations , for example , when slns lie in the internal mammary chain where bony anatomy necessitates pinpointing the location of a node before incision . single photon emission computed tomography and computed tomography image fusion ( known as spect / ct ) provides accurate three - dimensional ( 3d ) localization of lymph nodes at the cost of a significant increase in the absorbed dose to the patient for the overall lymphoscintigraphy and ct procedure . at m.d . anderson cancer center ( mdacc ) , spect / ct is routinely performed for lymphoscintigraphy of the head and neck because of the intricate anatomy . for those situations in which planar scintigrams would suffice , we have developed a technique for fusing the ct topograms ( also known as the ct scout or pilot image ) with planar lymphoscintigrams in order to improve the localization of slns with better anatomical information than would be offered by co backlighting , but with less absorbed dose to the patient than would be imparted by ct localization . conventional method for node localization co backlighting : the most common and traditional localization method for breast lymphoscintigraphy is the use of a low - activity co sheet source to backlight the patient ( figure 1 ) [ 1 , 3 , 4 ] . following the acquisition of a ten - minute static anterior emission image , a co sheet source is placed below the patient on the frame of the posterior camera of a dual - head gamma camera . a three - minute static anterior patient silhouette ( or backlit ) image is acquired that allows the physician to locate the node in relation to the outline of the patient . such an approach aids in assessing whether lymphatic vessels from the tumor site in the breast are draining to the internal mammary nodal basin or to the axillary nodal basin . a lateral emission image is used to estimate the anterior depth of the node . one approach is to use a chair positioned adjacent to the bed to prop the sheet source vertically next to the patient [ 1 , 57 ] . in another case , the source is placed on top of the detector of the gamma camera below the patient for both the anterior and lateral images because the lateral images are acquired by rotating the patient to a lateral decubitus position while leaving the gamma camera gantry in the zero - degree orientation . the absorbed dose to the patient is kept low by placing a co sheet source of relatively low activity , 74185 mbq ( 25 mci ) , on the distal side of the patient for a maximum of two to three minutes , which provides sufficient photon fluence to obtain an adequate outline of the patient . clarke , notghi , and harding reported that co backlighting adds approximately 25% to the absorbed dose to the patient from a tc - nanocolloid injection . spect / ct for lymphoscintigraphy : the great promise of hybrid spect / ct and pet / ct cameras is the mechanical registration of emission data with a highly detailed x - ray computed tomogram , thereby combining the sensitivity and specificity of a radiopharmaceutical study with the high resolution morphological information of a ct scan . the value of spect / ct for sln identification and localization has been described in several reports [ 1114 ] . in special instances , contamination , nodes close to the injection site , and overweight patients are three noted instances in which sln identification and localization are better with spect than with standard planar methods [ 15 , 16 ] . overall , the detectability studies comparing planar imaging and spect for the general population do not indicate a compelling reason to migrate to spect for breast lymphoscintigraphy studies . this is in contrast to protocols such as head and neck lymphoscintigraphy that nearly always greatly benefit from using spect / ct for slns identification and localization , for example , because the anatomy in that area is much more complex than that of the breast . the development of our topogram fusion technique began with a retrospective chart review that was used as a guide toward the design and implementation of a phantom that would realistically mimic a patient undergoing a breast lymphoscintigraphic exam . topograms of a simple alignment phantom were analyzed to assess the spatial distortion effects of the fan - beam projection geometry . a protocol to acquire and process topograms and scintigrams in anteroposterior and a computer program was written to process and prepare both topogram and scintigram prior to their fusion . phantom data were acquired , prepared , and fused for the characterization and validation of our approach . in addition , data were acquired and doses calculated to estimate the absorbed doses to patients from the co - backlighting , topogram fusion , and spect / ct approaches , respectively . a retrospective review of lymphoscintigrams of 200 patients was approved by the institutional review board ( irb ) at u.t . although physiological processes vary from individual to individual ( affecting the drainage rate and amount of tc-99 m sulfur colloid collected in an sln ) , the apparent activity of the node ( an ) in a subset of the 200 patients reviewed was calculated using the total counts in the injection site ( nis ) , the total counts in the sln ( nn ) , and the known amount of injected activity ( ais ) . calculations made from regions of interest around slns in the clinical images estimated that the radioactivity in a typical lymph node was 1.11.8 mbq ( 3050 ci ) of tc-99 m . fusion of a lymphoscintigram that is , a projection along parallel ray paths with a ct to pogram that is , a projection with fan - beam geometry is complicated by the inherent misregistration between the two projection geometries . virtual detector lies at a plane through isocenter for the acquisition of topograms . as in projection radiography , the fan - beam geometry causes objects closer to the x - ray source than the gantry isocenter to appear magnified laterally in the detected image . unlike radiographs , where objects closest to the detector appear less distorted in the image than objects closer to the x - ray source , objects in topogram acquisitions that are closer to the x - ray detectors than the gantry isocenter will appear smaller in the image due to reconstruction software for topograms . the spatial distortion due to the geometry of the fan - beam affects the location of objects in the topogram in both the anteroposterior and lateral directions . there is no distortion along the long axis of the patient because the ct topogram beam is tightly collimated so the edges are nearly parallel in the cranio - caudal direction . as a special case , small objects at the isocenter of the gantry remain at the center of the imaging plane and suffer minimal magnification and geometric distortion in contrast to objects located away from the gantry isocenter . the amount of magnification in the lateral direction varies as a function of the distance of the coronal plane ( of a supine patient ) to the gantry isocenter axis . for vertical planes perpendicular to the patient couch ( sagittal planes of a supine patient ) , the lateral distortion is the difference between the actual lateral distance of an object relative to the gantry isocenter and the measured lateral distance of that object to the central axis of the image . the degree of magnification and geometric distortion of that object varies as a function of the distance of the sagittal plane to the gantry isocenter axis . to compute the magnitude of the distortion of objects at different coronal and lateral distances ( assuming a supine patient orientation ) , six 1.5 mm diameter skin markers used for mammography , consisting of a metal bead with adhesive tab , were positioned along one surface at 2.5 cm , 5.0 cm , 10 cm , 15 cm , and 20 cm from the center of a fillable sheet source phantom . the central axis of the rigid phantom was aligned with the gantry lasers of the spect / ct scanner and the height of the table was initially adjusted so that the center of the beads was at the isocenter . topograms of the phantom were acquired at different table heights between 6.7 cm and 14.0 cm , including the table height position corresponding to the beads at isocenter ( table position = 0 cm ) . the spatial distortion of point objects at different anteroposterior and lateral positions was quantified by using the digital ruler tool in the gamma camera workstation software ( e.soft , siemens medical solutions usa , hoffman estates , il ) to measure the lateral distance of the bead from the gantry isocenter axis in the topogram image . the lateral distortion ( or offset ) for each point was computed as the difference between the measured distance and the known distance from the isocenter axis . the camera is configured with the detectors in a 180 degree , opposed - view orientation and static images are acquired in spect mode at zero and 90 degree gantry positions . the resulting four images ( anterior , posterior , left lateral , and right lateral ) are saved as separate static dicom images in the same series . the emission images are acquired in a 256 256 matrix for ten - minutes per view at each camera position . posterior / anterior ( pa ) and ipsilateral topograms are acquired using a 110 kvp , 20 ma beam . as patients on a spect / ct scanner would not move for the lateral images , the position and shape of the breast tissue are more similar to the configuration that the breasts will have at the time of surgery than if the patient were to rotate to the decubitus position for the lateral views . following image acquisition , the original scintigrams are adjusted for single - point registration and fusion to the topogram of the sln as described below . the lateral magnification factor is applied to the scintigram to match the geometry of the topogram so that the fan - beam geometry of the topogram will remain consistent regardless of the location of an sln . the original topogram is output as a new image series with an nm modality tag ( rather than a ct modality tag ) in the dicom header , since the image fusion software we used would not permit fusing planar nm data with ct datasets . a computer program was written in the interactive data language ( idl , itt visual solutions , boulder , co ) to automate the process of adjusting the lateral magnification of each scintigram . the program was integrated into the image processing application environment of the gamma camera workstation ( e.soft , siemens medical solutions usa , hoffman estates , il ) . the program accepts as inputs four dicom images : anterior and ipsilateral planar lymphoscintigrams , and posterior / anterior and ipsilateral ct topograms . assuming that the central pixel of the image corresponds to the isocenter of the gantry , the distance from the central pixel to the sln in both the anterior and lateral images can be used to laterally magnify or minify the scintigrams to match the geometries of the topograms and scintigrams for a particular node location . the operator manually marks a desired feature such as an sln on each of the two orthogonal lymphoscintigrams . the location of the sln relative to the isocenter in the lateral lymphoscintigram is then used to apply the appropriate lateral magnification factor to the anterior lymphoscintigram . this is because the degree of distortion of objects in the posterior / anterior topogram is dependent upon the coronal plane in which the object lies . the depth of the sln in the lateral lymphoscintigram gives the distance of the coronal plane from the isocenter and the appropriate amount of distortion correction can then be applied . the location of the node in the anterior lymphoscintigram provides the offset of the sagittal plane containing the node relative to the gantry isocenter . after the magnification factor has been applied , the program generates two new image series : a series of laterally stretched lymphoscintigrams and a series of ct topograms with nm modality tags . in cases where several nodes are visible and widely scattered , the scintigram can be adjusted differently and a separate fused image created for each one of the nodes we chose to modify the lymphoscintigram rather than the topogram so that the geometry of the morphological information will retain a consistent fan - beam presentation . a rigid - plane phantom was placed on the patient bed of the hybrid spect / ct system with three co button sources positioned at various lateral distances from the central axis of the scanner . static emission images and ct topograms were acquired in both the anterior and lateral views at several bed height positions to take into account different sln anterior - posterior positions . the images were processed using the geometric compensation program and analyzed to compute the residual misregistration of each point source between the scintigram and the topogram . in order to have a reproducible test object , a nuclear medicine anthropomorphic phantom ( radiology support devices , inc , long beach , ca ) was augmented in order to simulate a breast lymphoscintigraphy injection site and sentinel lymph nodes ( figure 3 ) . fillable spheres ( data spectrum corporation , chapel hill , nc ) , such as those used in the nema iec body phantom , were used to mimic the node and injection sites . therefore , fillable spheres of inner diameter 4.95 mm and volume 0.063 ml were used to represent slns in the phantom . initial images of the phantom indicated that the previously estimated 1.11.8 mbq ( 3050 ci ) nodal activity , determined by the chart review , was too high . a total activity in the node between 37 kbq and 370 kbq ( 1 and 10 ci ) was used , leading to a range of activity concentrations from 0.595.9 mbq per ml ( 16 to 160 ci per ml ) . a fillable sphere of inner diameter 19.79 mm and volume 4.0 ml was used for the injection site . the sphere used for the injection site was filled with an absolute activity of 92.5 mbq ( 2.5 mci ) ; the volume concentration for the 4.0 ml sphere is thus 23.1 mbq per ml ( 0.625 mci per ml ) . generally , the distance between the anterior surface of the breast and the injection site ranges from a few millimeters for a subcutaneous injection to about 3 cm for a perilesional injection . the distance between the injection site and the visualized node in patient studies is highly dependent on the location of both the tumor and the node , but generally the lymph node is at the junction of the fatty breast tissue and chest wall . superflab ( wfr - aquaplast , wyckoff , ny ) is a tissue - equivalent material used in radiation therapy . because the material is flexible and its characteristics mimic the scattering properties of tissue , it was used to simulate superficial tissues in the construction of the phantom . the setup for the thorax phantom study consisted of securing the small spheres directly to the thorax phantom in the anterior axillary region to represent axillary slns and near the mediastinum to represent internal mammary slns . a 0.5 cm layer of superflab was placed over the small sphere to cushion the objects when the chest overlay was placed onto the thorax phantom . a cavity just big enough to accommodate the larger sphere ( representing the injection site ) was formed in a layer of 1.0 cm thick superflab in order to provide a scatter medium for imaging . an additional 1 - 2 cm of superflab was added as needed to provide scatter similar to that seen in the patient studies of the retrospective chart review for both the anterior and lateral emission images . static emission images and ct topograms were acquired for both the anterior and lateral views of the thorax phantom . the images were processed using the geometry compensation program and registered to validate the processing protocol for phantom images that mimic those of patients . the photon flux of co sheet sources is too low for measurement with routinely used radiographic ion chambers such as a triad or radcal because of the insensitivity of the chambers ( chamber volumes are not sufficient to detect the low exposure rate of the sheet source ) . the absorbed dose from a 111 mbq ( 3 mci ) co sheet source was therefore estimated by measuring the exposure rate using a dose - equivalent ion chamber ( inovision 451b - de - si , fluke , everett , wa ) . with the gamma camera configured for opposed - head imaging at a gantry angle of zero ( i.e. , to produce anterior and posterior views of a supine patient ) , the sheet source was placed on the face of the collimator of the posterior ( lower ) detector . the ion chamber was placed on the patient couch and centered above the sheet source . acrylic blocks were placed around the chamber to simulate the scatter in the human torso . the meter readings , in sv per hour , represent the dose - equivalent rate at that location . ( the 451b - de - si conversion factor varies between 0.9 and 1.0 over the range of photon energies present in this setup ) . the total absorbed dose for unilateral and bilateral breast lymphoscintigraphy studies was calculated by accounting for each view of the study . unilateral studies had two transmission images acquired whereas bilateral studies had three transmission images acquired . it is most important that the images provide anatomical landmarks by depicting bony anatomy and providing some soft tissue information . topograms of the anthropomorphic phantom were acquired at 80 , 110 , and 130 kvp for various ma settings . the images were presented to two physicians who assessed image quality with respect to the ability to adequately localize the hot spheres when fused to the lymphoscintigrams . anterior and lateral topograms acquired at 110 kvp and 20 ma were deemed to be of sufficient quality for sln localization . the dosimetry for the topogram localization technique was estimated using a 6 cubic centimeter cylindrical ionization chamber ( model 10x5 - 6 ) and electrometer ( model 9010 ) ( radcal corporation , monrovia , ca ) as described by o'daniel et al . . the chamber was centered in the gantry of the ct scanner with the stem of the ion chamber ( the long axis of the cylinder ) parallel to the long axis of the couch . in - air exposure measurements were recorded for both the anterior / posterior ( ap ) and posterior / anterior ( pa ) tube positions . the entrance skin exposure was calculated for an average size patient ( 23 cm anterior - posterior thickness ) for each tube position by correcting the isocenter exposure measurement using the inverse - square method . the skin dose was calculated using the roentgen - to - rad conversion ( 0.87 rad = 1 r and for si units 1 rad = 10 mgy ) and back - scatter factor ( assumed to be 1.35 ) . in order to properly convert the skin dose measurements to effective dose , a conversion factor of 0.1 msv per mgy was obtained from the national radiological protection board report w4 . if ct tomographic images are to be used only to localize slns , the ct need not be of diagnostic quality and should be acquired at a relatively low dose ( i.e. , low mas ) . for the purposes of this study , five different scan parameter combinations were tested to assess the range of absorbed doses that might be delivered to the patient : 130 kvp , 90 mas ; 130 kvp , 20 mas ; 110 kvp , 20 mas ; 110 kvp , 10 mas ; and 80 kvp , 20 mas . the ct dose index ( ctdi ) dosimetry method employs a ctdi body phantom ( model 20ct14 ) and a 10 cm long , 3 cubic centimeter pencil ionization chamber ( model 10x5 - 3ct ) ( radcal corporation , monrovia , ca ) . in - phantom measurements of exposure ( mr ) were recorded for the central chamber position and four peripheral positions . then , ctdi100 , ctdiw , ctdivol , and dose - length product ( dlp ) values were calculated according to the definitions in american association of physicists in medicine ( aapm ) report number 96 , using a detector configuration of 6 2 mm and pitch of 1.2 . the dlp ( based on 40 cm scan length ) for a given ct scan was converted to effective dose using the conversion factors in the european guidelines on quality criteria for computed tomography . since the localization technique for the breast lymphoscintigraphy protocol has a scan extent throughout the chest region , the -factor used for this estimation of effective dose was 0.017 msv per mgy - cm . the measured spatial mismatch between uncompensated scintigrams and topograms increased linearly with distance from the isocenter in the anterior - posterior and lateral directions ( figure 4 ) . the measured distance ( x ) of the point sources from the long axis of the topogram when the point source is in a given coronal plane ( ) can be approximated by ( 2 ) . the actual lateral distance of the point source from the long axis of the hybrid scanner is denoted by ( 2)x[cm]=(0.08+4.98)(5 cm ) . the measured spatial mismatch is calculated by the difference of the measured position of the point source , x , and the actual lateral position of the point source from the central axis of the scanner , . the spatial mismatch is estimated by ( 3)x[cm][cm]=(0.0160.004)( ) . the misregistration between nuclear medicine scintigrams and ct topograms was reduced to within 3.4 mm in the plane of a particular co button source . the residual misregistration is less than the intrinsic resolution of the gamma camera ( 3.23.4 mm ) and much less than the overall system resolution of the images . fused images of the registered , geometrically compensated scintigrams , and topograms of the point source phantom are shown in figure 5 . as the lateral magnification of the topogram is processed separately for each individual object of interest , only the indicated point source is correctly registered since each source is in a different sagittal plane . in addition to measuring the spatial distortion of topograms by using a point - source phantom , the anthropomorphic thorax phantom was used for qualitative comparison of various lymphoscintigraphic imaging methods . the phantom studies were performed to visually compare our proposed method of localization to the conventional method ( figure 6 ) . the direct comparison of the two techniques illustrates how much additional information is provided by our approach with respect to localizing an sln relative to bony anatomy . the measured dose rate for a 111 mbq ( 3 mci ) sheet source was 87 usv per hour . if the co backlit images were acquired for 3 minutes per view , then the estimated effective dose for unilateral and bilateral breast lymphoscintigraphy studies localized with the backlighting technique would be 9 sv and 13 sv , respectively . the measured in - air exposure at gantry isocenter for a 110 kvp , 20 ma pa topogram was 18 mr , so the exposure at the skin surface would be 19 mr using an inverse - square correction . the estimated skin dose for the ct topogram is 0.23 mgy . using the 0.1 msv per mgy conversion from skin dose to effective dose , the estimated effective dose for the 110 kvp , 20 ma topogram would be 22.5 sv . nrpb - w4 does not provide an msv per mgy conversion from skin dose to effective dose for lateral exams . however , based on several other known acquisition techniques and configurations , it is reasonable to expect that the effective dose for the lateral topogram would be only one order of magnitude greater than the estimated dose using co backlighting ( table 1 ) . the effective dose to the chest for a ct scan ranged from 0.308 msv for the lowest possible dose setting ( 80 kvp , 20 mas ) to 5.265 msv for the default clinical ct scan setting ( 130 kvp , 90 mas ) . the calculations for each dose index , the dlp , and the effective dose for each of the ct scan parameter combinations are compiled in table 2 . the fusion of a geometry - compensated lymphoscintigram with a ct topogram is a new method of anatomical localization for lymphoscintigraphy . when compared to the time - honored means of localization using backlighting with a co sheet source , it offers a substantial increase in both the number and detail of anatomical references at the relative detriment of an order of magnitude increase in the still quite modest absorbed dose to the patient . furthermore , the gantry of the spect / ct camera can be rotated to acquire the lateral topogram and the lateral scintigrams while the patient does not move . thus all of the views may be acquired without moving the patient from a position close to that in which subsequent biopsy or surgery would be performed . while a full ct exam would avoid the need for geometrical compensation , it would impose a further increase of one to two orders of magnitude over our proposed approach in absorbed dose to the patient . the software program used to adjust the scintigrams was written in idl in such a fashion that it functions stand - alone or it can be incorporated into a vendor image processing workstation using workflow broker activities . the image registration and fusion of the stretched scintigrams with nm dicom tags and ct topograms with nm dicom tags can be performed with any vendor software program that registers and fuses two planar nuclear medicine ( nm ) images . although the idl program was integrated into the siemens e.soft image processing workstation for this project , the images were also fused successfully using osirix ( pixmeo , switzerland ) . the most fruitful cases in which to conduct the first clinical evaluations of this technique would be in regions of the body of moderate complexity such as for drainage to the internal mammary lymphatic chain , where the surgeon must know which rib to remove in order to access a given node . extremely complex anatomy , such as the head and neck , calls for a full spect / ct study , while in cases of drainage to the axillary lymphatic chain , the plasticity of the tissues and the absence of skeletal interference to the surgical approach make the improvement of topogram fusion over sheet source backlighting of less value . gamma camera images of the phantom described herein are realistic - looking surrogates for actual patient lymphoscintigrams . the phantom meets the need for a reproducible object in which the true location and activity of a simulated lymph node is known . we demonstrated that fusing lymphoscintigrams to ct topograms is superior to co backlighting for producing detailed anatomic localization images . through our approach , simulated lymph nodes in anterior and lateral scintigrams can be localized to within 3.4 mm between the fused scintigram and topogram . this misregistration error is less than the intrinsic resolution , and much less than the system compound resolution , of the gamma camera for lymphoscintigraphic imaging . moreover , for many clinical applications , fusion of a planar scintigram with a ct topogram may offer the best compromise between absorbed dose to the patient and critical anatomic detail .

lymphoscintigraphy is a nuclear medicine procedure that is used to detect sentinel lymph nodes ( slns ) . this project sought to investigate fusion of planar scintigrams with ct topograms as a means of improving the anatomic reference for the sln localization . heretofore , the most common lymphoscintigraphy localization method has been backlighting with a 57co sheet source . currently , the most precise method of localization through hybrid spect / ct increases the patient absorbed dose by a factor of 34 to 585 ( depending on the specific ct technique factors ) over the conventional 57co backlighting . the new approach described herein also uses a spect / ct scanner , which provides mechanically aligned planar scintigram and ct topogram data sets , but only increases the dose by a factor of two over that from 57co backlighting . planar nuclear medicine image fusion with ct topograms has been proven feasible and offers a clinically suitable compromise between improved anatomic details and minimally increased radiation dose .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

an essential step in the procedure for sentinel lymph node ( sln ) biopsy is to locate the first - echelon node of the drainage basin . lymphoscintigraphy is a minimally invasive diagnostic tool for mapping the sln . after injecting the patient with sulfur colloid radiolabeled with tc in locations proximal to a tumor site and waiting for lymphatic drainage to occur , the lymphatic drainage pathway from the primary injection site may be imaged with a gamma camera . to partly cope with this limitation , it is customary to acquire additional transmission images via a backlighting co sheet source in order to facilitate the anatomic localization of the nodes seen on the scintigraphy image . while the backlighting imaging technique creates an outline of the patient 's body , it depicts no internal landmarks . the introduction of hybrid single photon emission computed tomography with computed tomography ( spect / ct ) systems into clinical practice presents an opportunity to improve the spatial localization in lymphoscintigraphy . single photon emission computed tomography and computed tomography image fusion ( known as spect / ct ) provides accurate three - dimensional ( 3d ) localization of lymph nodes at the cost of a significant increase in the absorbed dose to the patient for the overall lymphoscintigraphy and ct procedure . anderson cancer center ( mdacc ) , spect / ct is routinely performed for lymphoscintigraphy of the head and neck because of the intricate anatomy . for those situations in which planar scintigrams would suffice , we have developed a technique for fusing the ct topograms ( also known as the ct scout or pilot image ) with planar lymphoscintigrams in order to improve the localization of slns with better anatomical information than would be offered by co backlighting , but with less absorbed dose to the patient than would be imparted by ct localization . conventional method for node localization co backlighting : the most common and traditional localization method for breast lymphoscintigraphy is the use of a low - activity co sheet source to backlight the patient ( figure 1 ) [ 1 , 3 , 4 ] . following the acquisition of a ten - minute static anterior emission image , a co sheet source is placed below the patient on the frame of the posterior camera of a dual - head gamma camera . a three - minute static anterior patient silhouette ( or backlit ) image is acquired that allows the physician to locate the node in relation to the outline of the patient . a lateral emission image is used to estimate the anterior depth of the node . one approach is to use a chair positioned adjacent to the bed to prop the sheet source vertically next to the patient [ 1 , 57 ] . in another case , the source is placed on top of the detector of the gamma camera below the patient for both the anterior and lateral images because the lateral images are acquired by rotating the patient to a lateral decubitus position while leaving the gamma camera gantry in the zero - degree orientation . the absorbed dose to the patient is kept low by placing a co sheet source of relatively low activity , 74185 mbq ( 25 mci ) , on the distal side of the patient for a maximum of two to three minutes , which provides sufficient photon fluence to obtain an adequate outline of the patient . clarke , notghi , and harding reported that co backlighting adds approximately 25% to the absorbed dose to the patient from a tc - nanocolloid injection . spect / ct for lymphoscintigraphy : the great promise of hybrid spect / ct and pet / ct cameras is the mechanical registration of emission data with a highly detailed x - ray computed tomogram , thereby combining the sensitivity and specificity of a radiopharmaceutical study with the high resolution morphological information of a ct scan . the value of spect / ct for sln identification and localization has been described in several reports [ 1114 ] . overall , the detectability studies comparing planar imaging and spect for the general population do not indicate a compelling reason to migrate to spect for breast lymphoscintigraphy studies . this is in contrast to protocols such as head and neck lymphoscintigraphy that nearly always greatly benefit from using spect / ct for slns identification and localization , for example , because the anatomy in that area is much more complex than that of the breast . the development of our topogram fusion technique began with a retrospective chart review that was used as a guide toward the design and implementation of a phantom that would realistically mimic a patient undergoing a breast lymphoscintigraphic exam . phantom data were acquired , prepared , and fused for the characterization and validation of our approach . in addition , data were acquired and doses calculated to estimate the absorbed doses to patients from the co - backlighting , topogram fusion , and spect / ct approaches , respectively . although physiological processes vary from individual to individual ( affecting the drainage rate and amount of tc-99 m sulfur colloid collected in an sln ) , the apparent activity of the node ( an ) in a subset of the 200 patients reviewed was calculated using the total counts in the injection site ( nis ) , the total counts in the sln ( nn ) , and the known amount of injected activity ( ais ) . fusion of a lymphoscintigram that is , a projection along parallel ray paths with a ct to pogram that is , a projection with fan - beam geometry is complicated by the inherent misregistration between the two projection geometries . virtual detector lies at a plane through isocenter for the acquisition of topograms . as in projection radiography , the fan - beam geometry causes objects closer to the x - ray source than the gantry isocenter to appear magnified laterally in the detected image . there is no distortion along the long axis of the patient because the ct topogram beam is tightly collimated so the edges are nearly parallel in the cranio - caudal direction . as a special case , small objects at the isocenter of the gantry remain at the center of the imaging plane and suffer minimal magnification and geometric distortion in contrast to objects located away from the gantry isocenter . the amount of magnification in the lateral direction varies as a function of the distance of the coronal plane ( of a supine patient ) to the gantry isocenter axis . for vertical planes perpendicular to the patient couch ( sagittal planes of a supine patient ) , the lateral distortion is the difference between the actual lateral distance of an object relative to the gantry isocenter and the measured lateral distance of that object to the central axis of the image . the degree of magnification and geometric distortion of that object varies as a function of the distance of the sagittal plane to the gantry isocenter axis . to compute the magnitude of the distortion of objects at different coronal and lateral distances ( assuming a supine patient orientation ) , six 1.5 mm diameter skin markers used for mammography , consisting of a metal bead with adhesive tab , were positioned along one surface at 2.5 cm , 5.0 cm , 10 cm , 15 cm , and 20 cm from the center of a fillable sheet source phantom . the central axis of the rigid phantom was aligned with the gantry lasers of the spect / ct scanner and the height of the table was initially adjusted so that the center of the beads was at the isocenter . as patients on a spect / ct scanner would not move for the lateral images , the position and shape of the breast tissue are more similar to the configuration that the breasts will have at the time of surgery than if the patient were to rotate to the decubitus position for the lateral views . following image acquisition , the original scintigrams are adjusted for single - point registration and fusion to the topogram of the sln as described below . the original topogram is output as a new image series with an nm modality tag ( rather than a ct modality tag ) in the dicom header , since the image fusion software we used would not permit fusing planar nm data with ct datasets . the program accepts as inputs four dicom images : anterior and ipsilateral planar lymphoscintigrams , and posterior / anterior and ipsilateral ct topograms . assuming that the central pixel of the image corresponds to the isocenter of the gantry , the distance from the central pixel to the sln in both the anterior and lateral images can be used to laterally magnify or minify the scintigrams to match the geometries of the topograms and scintigrams for a particular node location . the location of the sln relative to the isocenter in the lateral lymphoscintigram is then used to apply the appropriate lateral magnification factor to the anterior lymphoscintigram . the depth of the sln in the lateral lymphoscintigram gives the distance of the coronal plane from the isocenter and the appropriate amount of distortion correction can then be applied . after the magnification factor has been applied , the program generates two new image series : a series of laterally stretched lymphoscintigrams and a series of ct topograms with nm modality tags . in cases where several nodes are visible and widely scattered , the scintigram can be adjusted differently and a separate fused image created for each one of the nodes we chose to modify the lymphoscintigram rather than the topogram so that the geometry of the morphological information will retain a consistent fan - beam presentation . a rigid - plane phantom was placed on the patient bed of the hybrid spect / ct system with three co button sources positioned at various lateral distances from the central axis of the scanner . static emission images and ct topograms were acquired in both the anterior and lateral views at several bed height positions to take into account different sln anterior - posterior positions . the images were processed using the geometric compensation program and analyzed to compute the residual misregistration of each point source between the scintigram and the topogram . in order to have a reproducible test object , a nuclear medicine anthropomorphic phantom ( radiology support devices , inc , long beach , ca ) was augmented in order to simulate a breast lymphoscintigraphy injection site and sentinel lymph nodes ( figure 3 ) . fillable spheres ( data spectrum corporation , chapel hill , nc ) , such as those used in the nema iec body phantom , were used to mimic the node and injection sites . therefore , fillable spheres of inner diameter 4.95 mm and volume 0.063 ml were used to represent slns in the phantom . a fillable sphere of inner diameter 19.79 mm and volume 4.0 ml was used for the injection site . the sphere used for the injection site was filled with an absolute activity of 92.5 mbq ( 2.5 mci ) ; the volume concentration for the 4.0 ml sphere is thus 23.1 mbq per ml ( 0.625 mci per ml ) . generally , the distance between the anterior surface of the breast and the injection site ranges from a few millimeters for a subcutaneous injection to about 3 cm for a perilesional injection . the distance between the injection site and the visualized node in patient studies is highly dependent on the location of both the tumor and the node , but generally the lymph node is at the junction of the fatty breast tissue and chest wall . superflab ( wfr - aquaplast , wyckoff , ny ) is a tissue - equivalent material used in radiation therapy . because the material is flexible and its characteristics mimic the scattering properties of tissue , it was used to simulate superficial tissues in the construction of the phantom . the setup for the thorax phantom study consisted of securing the small spheres directly to the thorax phantom in the anterior axillary region to represent axillary slns and near the mediastinum to represent internal mammary slns . a 0.5 cm layer of superflab was placed over the small sphere to cushion the objects when the chest overlay was placed onto the thorax phantom . an additional 1 - 2 cm of superflab was added as needed to provide scatter similar to that seen in the patient studies of the retrospective chart review for both the anterior and lateral emission images . static emission images and ct topograms were acquired for both the anterior and lateral views of the thorax phantom . the photon flux of co sheet sources is too low for measurement with routinely used radiographic ion chambers such as a triad or radcal because of the insensitivity of the chambers ( chamber volumes are not sufficient to detect the low exposure rate of the sheet source ) . the absorbed dose from a 111 mbq ( 3 mci ) co sheet source was therefore estimated by measuring the exposure rate using a dose - equivalent ion chamber ( inovision 451b - de - si , fluke , everett , wa ) . , to produce anterior and posterior views of a supine patient ) , the sheet source was placed on the face of the collimator of the posterior ( lower ) detector . the ion chamber was placed on the patient couch and centered above the sheet source . the meter readings , in sv per hour , represent the dose - equivalent rate at that location . ( the 451b - de - si conversion factor varies between 0.9 and 1.0 over the range of photon energies present in this setup ) . the total absorbed dose for unilateral and bilateral breast lymphoscintigraphy studies was calculated by accounting for each view of the study . anterior and lateral topograms acquired at 110 kvp and 20 ma were deemed to be of sufficient quality for sln localization . the dosimetry for the topogram localization technique was estimated using a 6 cubic centimeter cylindrical ionization chamber ( model 10x5 - 6 ) and electrometer ( model 9010 ) ( radcal corporation , monrovia , ca ) as described by o'daniel et al . the chamber was centered in the gantry of the ct scanner with the stem of the ion chamber ( the long axis of the cylinder ) parallel to the long axis of the couch . in order to properly convert the skin dose measurements to effective dose , a conversion factor of 0.1 msv per mgy was obtained from the national radiological protection board report w4 . if ct tomographic images are to be used only to localize slns , the ct need not be of diagnostic quality and should be acquired at a relatively low dose ( i.e. for the purposes of this study , five different scan parameter combinations were tested to assess the range of absorbed doses that might be delivered to the patient : 130 kvp , 90 mas ; 130 kvp , 20 mas ; 110 kvp , 20 mas ; 110 kvp , 10 mas ; and 80 kvp , 20 mas . in - phantom measurements of exposure ( mr ) were recorded for the central chamber position and four peripheral positions . since the localization technique for the breast lymphoscintigraphy protocol has a scan extent throughout the chest region , the -factor used for this estimation of effective dose was 0.017 msv per mgy - cm . the measured spatial mismatch is calculated by the difference of the measured position of the point source , x , and the actual lateral position of the point source from the central axis of the scanner , . the misregistration between nuclear medicine scintigrams and ct topograms was reduced to within 3.4 mm in the plane of a particular co button source . in addition to measuring the spatial distortion of topograms by using a point - source phantom , the anthropomorphic thorax phantom was used for qualitative comparison of various lymphoscintigraphic imaging methods . the phantom studies were performed to visually compare our proposed method of localization to the conventional method ( figure 6 ) . the measured dose rate for a 111 mbq ( 3 mci ) sheet source was 87 usv per hour . the estimated skin dose for the ct topogram is 0.23 mgy . using the 0.1 msv per mgy conversion from skin dose to effective dose , the estimated effective dose for the 110 kvp , 20 ma topogram would be 22.5 sv . however , based on several other known acquisition techniques and configurations , it is reasonable to expect that the effective dose for the lateral topogram would be only one order of magnitude greater than the estimated dose using co backlighting ( table 1 ) . the effective dose to the chest for a ct scan ranged from 0.308 msv for the lowest possible dose setting ( 80 kvp , 20 mas ) to 5.265 msv for the default clinical ct scan setting ( 130 kvp , 90 mas ) . the calculations for each dose index , the dlp , and the effective dose for each of the ct scan parameter combinations are compiled in table 2 . the fusion of a geometry - compensated lymphoscintigram with a ct topogram is a new method of anatomical localization for lymphoscintigraphy . when compared to the time - honored means of localization using backlighting with a co sheet source , it offers a substantial increase in both the number and detail of anatomical references at the relative detriment of an order of magnitude increase in the still quite modest absorbed dose to the patient . furthermore , the gantry of the spect / ct camera can be rotated to acquire the lateral topogram and the lateral scintigrams while the patient does not move . thus all of the views may be acquired without moving the patient from a position close to that in which subsequent biopsy or surgery would be performed . while a full ct exam would avoid the need for geometrical compensation , it would impose a further increase of one to two orders of magnitude over our proposed approach in absorbed dose to the patient . the software program used to adjust the scintigrams was written in idl in such a fashion that it functions stand - alone or it can be incorporated into a vendor image processing workstation using workflow broker activities . the image registration and fusion of the stretched scintigrams with nm dicom tags and ct topograms with nm dicom tags can be performed with any vendor software program that registers and fuses two planar nuclear medicine ( nm ) images . although the idl program was integrated into the siemens e.soft image processing workstation for this project , the images were also fused successfully using osirix ( pixmeo , switzerland ) . the most fruitful cases in which to conduct the first clinical evaluations of this technique would be in regions of the body of moderate complexity such as for drainage to the internal mammary lymphatic chain , where the surgeon must know which rib to remove in order to access a given node . extremely complex anatomy , such as the head and neck , calls for a full spect / ct study , while in cases of drainage to the axillary lymphatic chain , the plasticity of the tissues and the absence of skeletal interference to the surgical approach make the improvement of topogram fusion over sheet source backlighting of less value . gamma camera images of the phantom described herein are realistic - looking surrogates for actual patient lymphoscintigrams . we demonstrated that fusing lymphoscintigrams to ct topograms is superior to co backlighting for producing detailed anatomic localization images . through our approach , simulated lymph nodes in anterior and lateral scintigrams can be localized to within 3.4 mm between the fused scintigram and topogram . moreover , for many clinical applications , fusion of a planar scintigram with a ct topogram may offer the best compromise between absorbed dose to the patient and critical anatomic detail .

sufficient healing after open or arthroscopic rotator cuff repair still remains one of the challenges in shoulder surgery . despite an improvement in the surgical technique , suggested that the repair should provide high initial fixation strength , allowing for only minimal gap formation between the tendon and its insertion site . in addition , it is postulated that re - establishing the anatomical footprint of the tendon bone insertion is a key factor to facilitate healing of the tendon to the bone . the integrity of the repair site , in particular , has been shown to correlate with clinical improvement , particularly the return of strength [ 4 , 13 ] . therefore , the goal is to reconstruct the footprint configuration accompanied by the restoration of its mechanical performance in order to establish an adequate environment for optimal healing of the tendon to the bone . the native footprint area could be recreated more accurately with the use of the double - row technique when compared to a single - row of anchors [ 3 , 26 , 29 ] . additionally , previous studies were able to demonstrate a higher initial mechanical strength of the double - row repair by means of isometric cyclic loading at time zero [ 2 , 18 , 21 , 22 ] . nonetheless , recent trials have not been able to confirm these results for clinical applications [ 10 , 12 , 16 ] . recently , the suture - bridge technique was introduced to maximise the utility of a single - row construct by using the suture limbs from the medial mattress sutures to bridge and compress the repaired tendon . the suture - bridge technique is postulated to restore the footprint contact area and unite the advantages of repair with those of suture anchor and transosseous repair [ 11 , 24 ] . thus , there is still no consensus as to which procedure , i.e. suture anchors or suture - bridge fixation , will provide the optimal conditions for cell biological tendon - to - bone healing . given this current situation in the treatment of rotator cuff injury , the purpose of this study was to determine the initial mechanical properties in cyclic loading and load - to - failure as well as the initial contact pressure over a defined rotator cuff footprint for a modified suture - bridge technique , using a titanium anchor screw . the study was conducted with the hypothesis that the presented technique exceeds the clinically relevant 250-n load threshold and would resist an isometric load of 3,000 cycles . thirty sheep shoulders ( specimen age , 2 years ) were harvested fresh , wrapped in saline - soaked gauze and stored frozen at 20c [ 2 , 3 , 31 ] . shoulders were dissected from all of the soft tissues except for the infraspinatus muscle and tendon . the infraspinatus tendon was sharply detached from its insertion site to mimic a standardised full - thickness tear . right and left shoulders were randomly assigned among three investigation groups that were based on the use of a special titanium anchor screw ( lasa - dr - screw , knigsee implantate , aschau , germany ) : ( 1 ) cyclic loading , ( 2 ) load - to - failure testing and ( 3 ) tendon bone interface contact pressure measurement . all of the repairs were coupled with braided nonabsorbable polyethylene suture sized usp no . 2 ( hifi ; conmed linvatec , largo , fl , usa ) . the lasa - dr - screw ( fig . 1 ) consists of a pinpoint to fix the medial row of sutures , a threaded rod to assure anchorage of the screw to the bone , a thread - free neck and a flat head . using a freehand technique , a medial bone channel is made with a punch at the medial footprint border , with a lateral tilt of 7080 inclination towards the footprint area ( fig . after that a punch is inserted through the cortex of the lesser tuberosity in parallel direction to the footprint surface ( fig . . then the length of the screw is determined , according to the length of the footprint between the tuberosity and the medial footprint border . two sutures are then inserted around the tip of the screw using a guiding device ( fig . after perforating the sutures medially through the tendon , they were secured by arthroscopic mason - allen stitches . the sutures were then tensioned , crossed over the tendon s footprint , tied around the screw neck and secured by the flat head ( fig . stitches were first tied with the use of a sliding double half - hitch knot , and then secured by a series of four reversing half hitches on alternative posts . to standardise tension for the repair , no less than 4 kg of tensile force dissections , preparations , repairs and experimental testing were performed by one single - experienced shoulder surgeon after thawing the shoulders for 24 h at room temperature.fig . 1lasa - dr - screw ( knigsee implantate , aschau , germany ) demonstrating the pinpoint to fix the medial row of sutures , the threaded rod to assure anchorage of the screw to the bone , the thread - free neck and the flat headfig . 2a medial bone channel is made with a punch ( * ) at the medial footprint border ( a ) . a suturing instrument is equipped with a shuttle suture and brought into this bone channel . a punch ( # ) then , the screw is placed in parallel to the footprint , with two sutures inserted around the screw pinpoint ( b ) . the tendon is medially fixed by arthroscopic mason - allen stitches ( c e ) . sutures were tensioned over the tendon s footprint , then tied around the screw neck , and secured by the flat head ( f h ) lasa - dr - screw ( knigsee implantate , aschau , germany ) demonstrating the pinpoint to fix the medial row of sutures , the threaded rod to assure anchorage of the screw to the bone , the thread - free neck and the flat head a medial bone channel is made with a punch ( * ) at the medial footprint border ( a ) . a suturing instrument is equipped with a shuttle suture and brought into this bone channel . a punch ( # ) then , the screw is placed in parallel to the footprint , with two sutures inserted around the screw pinpoint ( b ) . the tendon is medially fixed by arthroscopic mason - allen stitches ( c e ) . sutures were tensioned over the tendon s footprint , then tied around the screw neck , and secured by the flat head ( f h ) examinations were performed using a material - testing machine [ zwick 1445 , zwick - roell , ulm , germany ] . the data were recorded with the dedicated software [ testxpert 12 , zwick - roell , ulm , germany ] and a load displacement curve . the proximal end of the infraspinatus tendon was set in a tendon clamp , leaving approximately 6 cm between the clamp and the site of repair . to prevent it from slipping out of the clamp during testing , cryo - jaws for soft tissue fixation were used [ 2 , 19 ] . the humerus was fixed in a custom rig designed to evenly distribute loads across the tendon . to simulate postoperative conditions , a cyclic loading shoulders were loaded in the physiologic direction of the rotator cuff tendon , perpendicular to the longitudinal axis of the humerus , as previously described [ 2 , 19 , 27 ] . the loading force was applied with the humerus maintained at a constant angle relative to the tendon , resulting in simulated isometric muscle contraction . after pretension to 10 n for 1 min , each construct was cyclically loaded to 3,000 cycles from 10 to 180 n with a 5-s cycle [ 2 , 7 , 8 , 19 ] . these parameters have been reported as the physiologic loads and speeds that occur in normal daily activity and were therefore considered the best manner to simulate the postoperative condition [ 8 , 27 ] . the tests were stopped when complete failure ( e.g. repair site gap 10 mm , defect of the tendon bone construct ) or a total of 3,000 cycles was attained . the number of cycles creating a 5-mm and 10-mm gap was recorded along with the mechanism of failure [ 2 , 19 , 27 ] . with use of a material - testing machine [ zwick 1445 , zwick - roell , ulm , germany ] , a pretension of 10 n was applied and the clamp and custom rig were checked for tightness before beginning the load - to - failure testing . the load [ n ] and the displacement [ mm ] were digitally recorded by a deformation curve , and the mode of failure was documented . tests were stopped when complete failure was attained ( e.g. repair site gap of 10 mm , defect of the tendon bone construct ) . a load of 250 n was chosen as the minimum clinically relevant ultimate failure load , based on previous studies of rotator cuff fixation techniques [ 6 , 7 , 15 ] . a pressure - sensitive film ( prescale film , super low pressure type , fuji photo film co ltd , tokyo , japan ) was used to measure the interface contact pattern and pressure between the infraspinatus tendon and the insertion site [ 3 , 24 , 29 ] . the pressure - sensitive film was cut in a standardised fashion for all specimens to conform to the 1 2 cm footprint defect . then , the film was placed under a prepared template so that we were able to prepare holes on the film in a uniform pattern . bone interfaces after insertion of the screws and sutures , while great care was taken to keep the film dry by constantly using gauze to absorb the moisture from the tendon to the bone [ 3 , 24 , 29 ] . the sutures connecting the tendon to the bone were carefully passed through the prepared holes . these measures were conformed so as to attain the best possible panoramic view of the contact pressure and pressure pattern . after the repair , the film was left in place for 2 min , as recommended by the manufacturer . sutures were then cut , the film was scanned into a fuji film prescale pressure densitometer ( fdp-305e , fuji photo film co ltd , tokyo , japan ) , and the average contact pressure and pressure pattern were detected . examinations were performed using a material - testing machine [ zwick 1445 , zwick - roell , ulm , germany ] . the data were recorded with the dedicated software [ testxpert 12 , zwick - roell , ulm , germany ] and a load displacement curve . the proximal end of the infraspinatus tendon was set in a tendon clamp , leaving approximately 6 cm between the clamp and the site of repair . to prevent it from slipping out of the clamp during testing , cryo - jaws for soft tissue fixation the humerus was fixed in a custom rig designed to evenly distribute loads across the tendon . to simulate postoperative conditions , a cyclic loading was performed , similar to previous studies [ 2 , 8 , 27 ] . shoulders were loaded in the physiologic direction of the rotator cuff tendon , perpendicular to the longitudinal axis of the humerus , as previously described [ 2 , 19 , 27 ] . the loading force was applied with the humerus maintained at a constant angle relative to the tendon , resulting in simulated isometric muscle contraction . after pretension to 10 n for 1 min , each construct was cyclically loaded to 3,000 cycles from 10 to 180 n with a 5-s cycle [ 2 , 7 , 8 , 19 ] . these parameters have been reported as the physiologic loads and speeds that occur in normal daily activity and were therefore considered the best manner to simulate the postoperative condition [ 8 , 27 ] . the tests were stopped when complete failure ( e.g. repair site gap 10 mm , defect of the tendon bone construct ) or a total of 3,000 cycles was attained . the number of cycles creating a 5-mm and 10-mm gap was recorded along with the mechanism of failure [ 2 , 19 , 27 ] . with use of a material - testing machine [ zwick 1445 , zwick - roell , ulm , germany ] , a pretension of 10 n was applied and the clamp and custom rig were checked for tightness before beginning the load - to - failure testing . the load [ n ] and the displacement [ mm ] were digitally recorded by a deformation curve , and the mode of failure was documented . tests were stopped when complete failure was attained ( e.g. repair site gap of 10 mm , defect of the tendon bone construct ) . a load of 250 n was chosen as the minimum clinically relevant ultimate failure load , based on previous studies of rotator cuff fixation techniques [ 6 , 7 , 15 ] . a pressure - sensitive film ( prescale film , super low pressure type , fuji photo film co ltd , tokyo , japan ) was used to measure the interface contact pattern and pressure between the infraspinatus tendon and the insertion site [ 3 , 24 , 29 ] . the pressure - sensitive film was cut in a standardised fashion for all specimens to conform to the 1 2 cm footprint defect . then , the film was placed under a prepared template so that we were able to prepare holes on the film in a uniform pattern . bone interfaces after insertion of the screws and sutures , while great care was taken to keep the film dry by constantly using gauze to absorb the moisture from the tendon to the bone [ 3 , 24 , 29 ] . the sutures connecting the tendon to the bone were carefully passed through the prepared holes . these measures were conformed so as to attain the best possible panoramic view of the contact pressure and pressure pattern . after the repair , the film was left in place for 2 min , as recommended by the manufacturer . sutures were then cut , the film was scanned into a fuji film prescale pressure densitometer ( fdp-305e , fuji photo film co ltd , tokyo , japan ) , and the average contact pressure and pressure pattern were detected . the number of cycles to 5-mm gap formation was 2,884.5 96.8 cycles . the ultimate tensile strength of the suture - bridge technique was 565.8 17.8 n and stiffness was 173.7 9.9 n / mm . the entire specimen presented a previously described unique mode of failure as it is well known in using high strength sutures by pulling them through the tendon [ 2 , 5 , 20 ] . there was no screw - related failure during load - to - failure testing as well . we detected a mean contact pressure of 1.19 0.03 mpa applied on the footprint area . however , the pressure around the insertion of the knots was higher than in the area between the knots . the most important findings of the study are excellent biomechanical and tendon bone - pressure characteristics of the presented modified suture - bridge technique . we confirmed our hypothesis , with the principle that this technique exceeds the clinically relevant 250-n load threshold as well as resisted against an isometric load of 3,000 cycles . additionally , the contact pressure over a defined footprint area is comparable to the results of a double - row technique [ 3 , 24 , 29 ] . several studies have demonstrated the mechanical characteristics of different techniques for rotator cuff repair , mainly dealing with the use of suture anchor systems . these studies have reported excellent mechanical behaviour for the double - row repair [ 2 , 18 , 21 , 22 ] . the double- and single - row techniques provided comparable clinical and mri arthrography short - term outcomes , especially in patients with small and medium rotator cuff tears ( <3 cm ) . only charousset et al . demonstrated significantly better tendon healing results for the double - row technique , in a computed tomographic arthrography follow - up . despite its widespread use , our review of the literature revealed only a few papers examining clinical use of the suture - bridge technique , in basic trials [ 6 , 9 , 24 ] or presented as a technical note [ 11 , 25 ] . in a recent study , frank et al . confirmed a healing rate of 88% with mri studies , as well as excellent clinical function after a mean follow - up of 14.6 months the authors concluded that the suture - bridge technique could protect the biological environment of tendon - to - bone healing over a short - term period . simulated a suture - bridge technique repair with a combination of transosseous sutures and suture anchor systems in cadaver shoulder specimen . they used a monotonic loading regime and found a mean failure of the repair at 404 n. in comparison with transosseous and single - row techniques , the repair failed at 5567% higher loads , respectively . a cyclic loading test was not performed ( table 1 ) . comparing the mean load - to - failure result of 565.81 n to a recent study evaluating the differences between single- and double - row techniques , we were able to confirm higher failure loads , as shown by burkhead et al . . certainly , comparing these results to the mentioned study , it has to be considered that we used an animal model and the infraspinatus tendon instead of the human supraspinatus tendon.table 1summary of the results of current studies investigating the suture - bridge or suture - bridge - like repair techniquesexperimental settingtested techniquefixation materialresultburkhead et al . load - to - failuretos versus sr versus sbtos : 3 ethibond suturesr : 3 metallic anchor systemssb : panalok anchor systemsb > tos > srbusfield et al . cyclic loadingload - to - failuredrsb1 versus drsb2dr - sb1 : 2 metallic anchor systems medial , 2 pushlocks lateraldr - sb2 : 2 metallic anchor systems medial , 2 pushlocks lateraldrsb2 > contact pressurecontact areadr versus sb2 versus sb4dr : 4 bio - corkscrew anchor systemssb2 : 1 bio - tenodesis screwsb4 : 2 bio - tenodesis screwsb4 > sb2 > drdr double - row , drsb1 double - row - suture - bridge group 1 ( without medial row knots ) , drsb2 double - row - suture - bridge group 2 ( with medial row knots ) , tos transosseous , sr single - row , sb suture bridge , sb2 suture bridge ( 2 suture bridges ) , sb4 suture bridge ( 4 suture bridges ) summary of the results of current studies investigating the suture - bridge or suture - bridge - like repair techniques dr double - row , drsb1 double - row - suture - bridge group 1 ( without medial row knots ) , drsb2 double - row - suture - bridge group 2 ( with medial row knots ) , tos transosseous , sr single - row , sb suture bridge , sb2 suture bridge ( 2 suture bridges ) , sb4 suture bridge ( 4 suture bridges ) busfield et al . conducted a biomechanical comparison of two suture - bridge repair techniques with and without medial row knots using metallic suture anchors and pushlocks ( arthrex , naples , fl , usa ) . the authors confirmed the necessity of medial row knots , in order to preserve the integrity of the repair construct and reach higher ultimate failure loads of up to 352.9 n ( table 1 ) . the average number of cycles to 5-mm gap formation in the study at hand was 2,884.5 cycles . the entire specimen resisted 3,000 cycles . comparing these results to studies using an analogue experimental set - up , it seems that the suture - bridge technique provides strength superior to that of a single - row , double - row and transosseous techniques [ 2 , 19 ] . the contact pressure at the tendon bone interface was the same when comparing this technique to a double - row technique using a combination of arthroscopic mason - allen stitches and horizontal mattress stitches . when using a combination of simple and horizontal mattress stitches the use of a suture - bridge technique maintained a homogenous pressure pattern at the tendon bone interface , as supported by the results of park et al . . the entire tested specimen in the current study failed by pulling the suture through the tendon when the maximum load was reached , as noticed in former investigations [ 2 , 5 , 20 ] . nevertheless , the maximum load was significantly higher compared to a double - row repair that was tested in a prior study , with the use of braided polyblend polyethylene sutures as well as with braided polyester sutures . the problem of early suture breakage seems to be solved by the improved material properties of the new high - performance suture material . however , it has to be noted that the more rigid characteristics of the new braided polyblend polyethylene suture material [ 20 , 32 ] can more easily cut in a parallel direction through the defective tendon , when used in healthy rotator cuff specimens [ 2 , 5 , 20 ] . this aspect is particularly important , because most of the torn human rotator cuff tendons are degenerated . tendons that fail a single - row repair are typically grade iii degenerate tendons , according to the classification of goutallier et al . . in the recent study , failure of the repair is indeed due to suture pulling through the tendon , resulting in a considerably higher failure force when compared to double - row repair . we could assume that this phenomenon is due to a steady and homogenous pressure distribution of the tendon the contact pattern between the fixation points could be increased by the suture - bridge repair , resulting in more footprint coverage than is observed for single - row techniques . interconnected repair , in contrast to the double - row technique that is based on separate fixation points . additionally , the suture - bridge technique provides a better compression vector when compared with suture anchor techniques . analysed the contact area of two different suture - bridge techniques in which a 4-suture - bridge technique , similar to that used in our evaluation , yielded a significantly higher contact area and interface pressure than double - row and 2-suture - bridge techniques ( table 1 ) . the authors conclude that this technique may lead to further improvement in the repair of rotator cuff tears , thus optimising the chances of healing the tendon to the bone . in our study , the mean contact pressure was 1.19 mpa , which is comparable to the results of a double - row technique . the sheep infraspinatus tendon was chosen because it has previously been demonstrated to be similar in size , shape and microstructure to the human supraspinatus tendon . but it has to be stated that it is different from the degenerated and thinner supraspinatus tendons seen in human shoulders with chronic rotator cuff tears , as already mentioned earlier . consequently , it remains an approximation to the human condition and clinically used rotator cuff repair techniques . nevertheless , this animal model has been used extensively to evaluate rotator cuff tendon repairs in previous studies [ 2 , 3 , 15 , 19 ] and allows interstudy comparison . furthermore , although great care was taken to minimise interference when preparing holes in the film , with regard to the pressure measurements , this may have yielded some artefacts . however , it would be impossible to obtain the entire contact pressure distribution and pressure pattern of the investigated techniques if pressure - sensitive film was only inserted between the tendon bone interfaces . additionally , using pressure - sensitive film that detects a pressure range between 0.50 and 2.50 mpa may have underestimated the contact area . the results of the current study provide information on the footprint coverage and mechanical strength of a suture - bridge technique during the immediate postoperative clinical situation . by passing the sutures medially , the repair takes advantage of healthier tendon tissue to improve fixation strength . due to the interconnected repair , one is able to achieve a homogenous tendon - to - bone pressure that has been shown to influence healing . beyond these considerations , the optimal pressure range for the tendon to heal to the bone is not known . bone interface , whereas high pressure may affect the vascularisation of the tendon and result in impaired tissue healing . thus , it should be noted that our evaluation does not address the influence of healing and/or remodelling on the strengths and footprint coverage of the repair . after comparing our results with recent reports in the literature [ 2 , 3 , 19 , 21 , 22 , 24 , 26 , 29 ] , the suture - bridge technique seems to be a further improvement in rotator cuff surgery [ 13 , 2426 ] . therefore , the presented technique may lead to clinical results superior to several double - row fixations , because it offers biomechanical advantages with low gap formation in cyclic loading and a uniform tendon - bone - contact pressure . in conclusion , the fundamental results of our study ( i.e. high mechanical load resistance , high contact pressure and contact area ) support the use of a suture - bridge technique for reconstruction of a torn rotator cuff tendon . nevertheless , an individual estimation has to be done when using the suture - bridge technique clinically as suggested by park et al . . a biomechanical comparison of a double - row versus the presented suture - bridge technique may be helpful but finally , in vivo trials are necessary to gain information on the cell biological healing process of suture - bridge techniques , to emphasise potential biological advantages and to accomplish more sufficient advancements in rotator cuff repair .

the aim of the study was to evaluate the time - zero mechanical and footprint properties of a suture - bridge technique for rotator cuff repair in an animal model . thirty fresh - frozen sheep shoulders were randomly assigned among three investigation groups : ( 1 ) cyclic loading , ( 2 ) load - to - failure testing , and ( 3 ) tendon bone interface contact pressure measurement . shoulders were cyclically loaded from 10 to 180 n and displacement to gap formation of 5- and 10-mm at the repair site . cycles to failure were determined . additionally , the ultimate tensile strength and stiffness were verified along with the mode of failure . the average contact pressure and pressure pattern were investigated using a pressure - sensitive film system . all of the specimens resisted against 3,000 cycles and none of them reached a gap formation of 10 mm . the number of cycles to 5-mm gap formation was 2,884.5 96.8 cycles . the ultimate tensile strength was 565.8 17.8 n and stiffness was 173.7 9.9 n / mm . the entire specimen presented a unique mode of failure as it is well known in using high strength sutures by pulling them through the tendon . we observed a mean contact pressure of 1.19 0.03 mpa , applied on the footprint area . the fundamental results of our study support the use of a suture - bridge technique for optimising the conditions of the healing biology of a reconstructed rotator cuff tendon . nevertheless , an individual estimation has to be done if using the suture - bridge technique clinically . further investigation is necessary to evaluate the cell biological healing process in order to achieve further sufficient advancements in rotator cuff repair .

Introduction Materials and methods Cyclic load testing Load-to-failure testing Contact pressure testing Results Discussion Conclusion

sufficient healing after open or arthroscopic rotator cuff repair still remains one of the challenges in shoulder surgery . despite an improvement in the surgical technique , suggested that the repair should provide high initial fixation strength , allowing for only minimal gap formation between the tendon and its insertion site . in addition , it is postulated that re - establishing the anatomical footprint of the tendon bone insertion is a key factor to facilitate healing of the tendon to the bone . the integrity of the repair site , in particular , has been shown to correlate with clinical improvement , particularly the return of strength [ 4 , 13 ] . therefore , the goal is to reconstruct the footprint configuration accompanied by the restoration of its mechanical performance in order to establish an adequate environment for optimal healing of the tendon to the bone . the native footprint area could be recreated more accurately with the use of the double - row technique when compared to a single - row of anchors [ 3 , 26 , 29 ] . additionally , previous studies were able to demonstrate a higher initial mechanical strength of the double - row repair by means of isometric cyclic loading at time zero [ 2 , 18 , 21 , 22 ] . recently , the suture - bridge technique was introduced to maximise the utility of a single - row construct by using the suture limbs from the medial mattress sutures to bridge and compress the repaired tendon . the suture - bridge technique is postulated to restore the footprint contact area and unite the advantages of repair with those of suture anchor and transosseous repair [ 11 , 24 ] . suture anchors or suture - bridge fixation , will provide the optimal conditions for cell biological tendon - to - bone healing . given this current situation in the treatment of rotator cuff injury , the purpose of this study was to determine the initial mechanical properties in cyclic loading and load - to - failure as well as the initial contact pressure over a defined rotator cuff footprint for a modified suture - bridge technique , using a titanium anchor screw . the study was conducted with the hypothesis that the presented technique exceeds the clinically relevant 250-n load threshold and would resist an isometric load of 3,000 cycles . shoulders were dissected from all of the soft tissues except for the infraspinatus muscle and tendon . right and left shoulders were randomly assigned among three investigation groups that were based on the use of a special titanium anchor screw ( lasa - dr - screw , knigsee implantate , aschau , germany ) : ( 1 ) cyclic loading , ( 2 ) load - to - failure testing and ( 3 ) tendon bone interface contact pressure measurement . 1 ) consists of a pinpoint to fix the medial row of sutures , a threaded rod to assure anchorage of the screw to the bone , a thread - free neck and a flat head . using a freehand technique , a medial bone channel is made with a punch at the medial footprint border , with a lateral tilt of 7080 inclination towards the footprint area ( fig . after that a punch is inserted through the cortex of the lesser tuberosity in parallel direction to the footprint surface ( fig . stitches were first tied with the use of a sliding double half - hitch knot , and then secured by a series of four reversing half hitches on alternative posts . a punch ( # ) then , the screw is placed in parallel to the footprint , with two sutures inserted around the screw pinpoint ( b ) . sutures were tensioned over the tendon s footprint , then tied around the screw neck , and secured by the flat head ( f h ) lasa - dr - screw ( knigsee implantate , aschau , germany ) demonstrating the pinpoint to fix the medial row of sutures , the threaded rod to assure anchorage of the screw to the bone , the thread - free neck and the flat head a medial bone channel is made with a punch ( * ) at the medial footprint border ( a ) . a punch ( # ) then , the screw is placed in parallel to the footprint , with two sutures inserted around the screw pinpoint ( b ) . sutures were tensioned over the tendon s footprint , then tied around the screw neck , and secured by the flat head ( f h ) examinations were performed using a material - testing machine [ zwick 1445 , zwick - roell , ulm , germany ] . to prevent it from slipping out of the clamp during testing , cryo - jaws for soft tissue fixation were used [ 2 , 19 ] . to simulate postoperative conditions , a cyclic loading shoulders were loaded in the physiologic direction of the rotator cuff tendon , perpendicular to the longitudinal axis of the humerus , as previously described [ 2 , 19 , 27 ] . after pretension to 10 n for 1 min , each construct was cyclically loaded to 3,000 cycles from 10 to 180 n with a 5-s cycle [ 2 , 7 , 8 , 19 ] . repair site gap 10 mm , defect of the tendon bone construct ) or a total of 3,000 cycles was attained . the number of cycles creating a 5-mm and 10-mm gap was recorded along with the mechanism of failure [ 2 , 19 , 27 ] . with use of a material - testing machine [ zwick 1445 , zwick - roell , ulm , germany ] , a pretension of 10 n was applied and the clamp and custom rig were checked for tightness before beginning the load - to - failure testing . the load [ n ] and the displacement [ mm ] were digitally recorded by a deformation curve , and the mode of failure was documented . repair site gap of 10 mm , defect of the tendon bone construct ) . a pressure - sensitive film ( prescale film , super low pressure type , fuji photo film co ltd , tokyo , japan ) was used to measure the interface contact pattern and pressure between the infraspinatus tendon and the insertion site [ 3 , 24 , 29 ] . the pressure - sensitive film was cut in a standardised fashion for all specimens to conform to the 1 2 cm footprint defect . then , the film was placed under a prepared template so that we were able to prepare holes on the film in a uniform pattern . bone interfaces after insertion of the screws and sutures , while great care was taken to keep the film dry by constantly using gauze to absorb the moisture from the tendon to the bone [ 3 , 24 , 29 ] . the sutures connecting the tendon to the bone were carefully passed through the prepared holes . these measures were conformed so as to attain the best possible panoramic view of the contact pressure and pressure pattern . sutures were then cut , the film was scanned into a fuji film prescale pressure densitometer ( fdp-305e , fuji photo film co ltd , tokyo , japan ) , and the average contact pressure and pressure pattern were detected . to prevent it from slipping out of the clamp during testing , cryo - jaws for soft tissue fixation the humerus was fixed in a custom rig designed to evenly distribute loads across the tendon . shoulders were loaded in the physiologic direction of the rotator cuff tendon , perpendicular to the longitudinal axis of the humerus , as previously described [ 2 , 19 , 27 ] . the loading force was applied with the humerus maintained at a constant angle relative to the tendon , resulting in simulated isometric muscle contraction . after pretension to 10 n for 1 min , each construct was cyclically loaded to 3,000 cycles from 10 to 180 n with a 5-s cycle [ 2 , 7 , 8 , 19 ] . repair site gap 10 mm , defect of the tendon bone construct ) or a total of 3,000 cycles was attained . the number of cycles creating a 5-mm and 10-mm gap was recorded along with the mechanism of failure [ 2 , 19 , 27 ] . with use of a material - testing machine [ zwick 1445 , zwick - roell , ulm , germany ] , a pretension of 10 n was applied and the clamp and custom rig were checked for tightness before beginning the load - to - failure testing . the load [ n ] and the displacement [ mm ] were digitally recorded by a deformation curve , and the mode of failure was documented . repair site gap of 10 mm , defect of the tendon bone construct ) . a pressure - sensitive film ( prescale film , super low pressure type , fuji photo film co ltd , tokyo , japan ) was used to measure the interface contact pattern and pressure between the infraspinatus tendon and the insertion site [ 3 , 24 , 29 ] . the pressure - sensitive film was cut in a standardised fashion for all specimens to conform to the 1 2 cm footprint defect . these measures were conformed so as to attain the best possible panoramic view of the contact pressure and pressure pattern . after the repair , the film was left in place for 2 min , as recommended by the manufacturer . sutures were then cut , the film was scanned into a fuji film prescale pressure densitometer ( fdp-305e , fuji photo film co ltd , tokyo , japan ) , and the average contact pressure and pressure pattern were detected . the number of cycles to 5-mm gap formation was 2,884.5 96.8 cycles . the ultimate tensile strength of the suture - bridge technique was 565.8 17.8 n and stiffness was 173.7 9.9 n / mm . the entire specimen presented a previously described unique mode of failure as it is well known in using high strength sutures by pulling them through the tendon [ 2 , 5 , 20 ] . there was no screw - related failure during load - to - failure testing as well . we detected a mean contact pressure of 1.19 0.03 mpa applied on the footprint area . however , the pressure around the insertion of the knots was higher than in the area between the knots . the most important findings of the study are excellent biomechanical and tendon bone - pressure characteristics of the presented modified suture - bridge technique . we confirmed our hypothesis , with the principle that this technique exceeds the clinically relevant 250-n load threshold as well as resisted against an isometric load of 3,000 cycles . additionally , the contact pressure over a defined footprint area is comparable to the results of a double - row technique [ 3 , 24 , 29 ] . several studies have demonstrated the mechanical characteristics of different techniques for rotator cuff repair , mainly dealing with the use of suture anchor systems . despite its widespread use , our review of the literature revealed only a few papers examining clinical use of the suture - bridge technique , in basic trials [ 6 , 9 , 24 ] or presented as a technical note [ 11 , 25 ] . confirmed a healing rate of 88% with mri studies , as well as excellent clinical function after a mean follow - up of 14.6 months the authors concluded that the suture - bridge technique could protect the biological environment of tendon - to - bone healing over a short - term period . simulated a suture - bridge technique repair with a combination of transosseous sutures and suture anchor systems in cadaver shoulder specimen . they used a monotonic loading regime and found a mean failure of the repair at 404 n. in comparison with transosseous and single - row techniques , the repair failed at 5567% higher loads , respectively . a cyclic loading test was not performed ( table 1 ) . comparing the mean load - to - failure result of 565.81 n to a recent study evaluating the differences between single- and double - row techniques , we were able to confirm higher failure loads , as shown by burkhead et al . certainly , comparing these results to the mentioned study , it has to be considered that we used an animal model and the infraspinatus tendon instead of the human supraspinatus tendon.table 1summary of the results of current studies investigating the suture - bridge or suture - bridge - like repair techniquesexperimental settingtested techniquefixation materialresultburkhead et al . load - to - failuretos versus sr versus sbtos : 3 ethibond suturesr : 3 metallic anchor systemssb : panalok anchor systemsb > tos > srbusfield et al . cyclic loadingload - to - failuredrsb1 versus drsb2dr - sb1 : 2 metallic anchor systems medial , 2 pushlocks lateraldr - sb2 : 2 metallic anchor systems medial , 2 pushlocks lateraldrsb2 > contact pressurecontact areadr versus sb2 versus sb4dr : 4 bio - corkscrew anchor systemssb2 : 1 bio - tenodesis screwsb4 : 2 bio - tenodesis screwsb4 > sb2 > drdr double - row , drsb1 double - row - suture - bridge group 1 ( without medial row knots ) , drsb2 double - row - suture - bridge group 2 ( with medial row knots ) , tos transosseous , sr single - row , sb suture bridge , sb2 suture bridge ( 2 suture bridges ) , sb4 suture bridge ( 4 suture bridges ) summary of the results of current studies investigating the suture - bridge or suture - bridge - like repair techniques dr double - row , drsb1 double - row - suture - bridge group 1 ( without medial row knots ) , drsb2 double - row - suture - bridge group 2 ( with medial row knots ) , tos transosseous , sr single - row , sb suture bridge , sb2 suture bridge ( 2 suture bridges ) , sb4 suture bridge ( 4 suture bridges ) busfield et al . the authors confirmed the necessity of medial row knots , in order to preserve the integrity of the repair construct and reach higher ultimate failure loads of up to 352.9 n ( table 1 ) . the average number of cycles to 5-mm gap formation in the study at hand was 2,884.5 cycles . the entire specimen resisted 3,000 cycles . comparing these results to studies using an analogue experimental set - up , it seems that the suture - bridge technique provides strength superior to that of a single - row , double - row and transosseous techniques [ 2 , 19 ] . the contact pressure at the tendon bone interface was the same when comparing this technique to a double - row technique using a combination of arthroscopic mason - allen stitches and horizontal mattress stitches . when using a combination of simple and horizontal mattress stitches the use of a suture - bridge technique maintained a homogenous pressure pattern at the tendon bone interface , as supported by the results of park et al . the entire tested specimen in the current study failed by pulling the suture through the tendon when the maximum load was reached , as noticed in former investigations [ 2 , 5 , 20 ] . nevertheless , the maximum load was significantly higher compared to a double - row repair that was tested in a prior study , with the use of braided polyblend polyethylene sutures as well as with braided polyester sutures . the problem of early suture breakage seems to be solved by the improved material properties of the new high - performance suture material . however , it has to be noted that the more rigid characteristics of the new braided polyblend polyethylene suture material [ 20 , 32 ] can more easily cut in a parallel direction through the defective tendon , when used in healthy rotator cuff specimens [ 2 , 5 , 20 ] . in the recent study , failure of the repair is indeed due to suture pulling through the tendon , resulting in a considerably higher failure force when compared to double - row repair . we could assume that this phenomenon is due to a steady and homogenous pressure distribution of the tendon the contact pattern between the fixation points could be increased by the suture - bridge repair , resulting in more footprint coverage than is observed for single - row techniques . additionally , the suture - bridge technique provides a better compression vector when compared with suture anchor techniques . analysed the contact area of two different suture - bridge techniques in which a 4-suture - bridge technique , similar to that used in our evaluation , yielded a significantly higher contact area and interface pressure than double - row and 2-suture - bridge techniques ( table 1 ) . the authors conclude that this technique may lead to further improvement in the repair of rotator cuff tears , thus optimising the chances of healing the tendon to the bone . in our study , the mean contact pressure was 1.19 mpa , which is comparable to the results of a double - row technique . but it has to be stated that it is different from the degenerated and thinner supraspinatus tendons seen in human shoulders with chronic rotator cuff tears , as already mentioned earlier . consequently , it remains an approximation to the human condition and clinically used rotator cuff repair techniques . nevertheless , this animal model has been used extensively to evaluate rotator cuff tendon repairs in previous studies [ 2 , 3 , 15 , 19 ] and allows interstudy comparison . however , it would be impossible to obtain the entire contact pressure distribution and pressure pattern of the investigated techniques if pressure - sensitive film was only inserted between the tendon bone interfaces . additionally , using pressure - sensitive film that detects a pressure range between 0.50 and 2.50 mpa may have underestimated the contact area . the results of the current study provide information on the footprint coverage and mechanical strength of a suture - bridge technique during the immediate postoperative clinical situation . by passing the sutures medially , the repair takes advantage of healthier tendon tissue to improve fixation strength . due to the interconnected repair , one is able to achieve a homogenous tendon - to - bone pressure that has been shown to influence healing . beyond these considerations , the optimal pressure range for the tendon to heal to the bone is not known . bone interface , whereas high pressure may affect the vascularisation of the tendon and result in impaired tissue healing . thus , it should be noted that our evaluation does not address the influence of healing and/or remodelling on the strengths and footprint coverage of the repair . after comparing our results with recent reports in the literature [ 2 , 3 , 19 , 21 , 22 , 24 , 26 , 29 ] , the suture - bridge technique seems to be a further improvement in rotator cuff surgery [ 13 , 2426 ] . therefore , the presented technique may lead to clinical results superior to several double - row fixations , because it offers biomechanical advantages with low gap formation in cyclic loading and a uniform tendon - bone - contact pressure . in conclusion , the fundamental results of our study ( i.e. high mechanical load resistance , high contact pressure and contact area ) support the use of a suture - bridge technique for reconstruction of a torn rotator cuff tendon . nevertheless , an individual estimation has to be done when using the suture - bridge technique clinically as suggested by park et al . a biomechanical comparison of a double - row versus the presented suture - bridge technique may be helpful but finally , in vivo trials are necessary to gain information on the cell biological healing process of suture - bridge techniques , to emphasise potential biological advantages and to accomplish more sufficient advancements in rotator cuff repair .

although the myocardial reperfusion following ischemia can produce the salvage of the ischemic tissue , it may also contribute not only to the endothelial dysfunction and the myocardial cellular injury , but it also may change the release of the biologically active substances , such as the endothelium - derived relaxing factor ( no ) . even though no obtained from l - arginine is a free radical per se , it also can quench other free radicals , including the superoxide radicals and it thereby protects both the endothelial cells and the cardiac myocytes [ 2 , 3 ] . in addition , no may stabilize mast cells and inhibit the release of histamine from the both isolated and the resident cardiac mast cells , thus influencing the postischemic myocardial injury [ 46 ] . thus , low doses of no appear to play a key protective role from the myocardial ischemia - reperfusion injury . however , high concentrations of no exert highly toxic and harmful effect during the ischemia - reperfusion injury [ 3 , 79 ] . beside the fact that no may stabilize the cardiac mast cells and it consequently decreases the histamine release [ 4 , 5 , 10 ] , earlier results showed a positive feedback relationship between no and histamine in the regulation of both coronary ischemic vasodilation and the coronary autoregulation . in our previous work , we evaluated both the dynamic response and the biomechanical properties of the isolated blood vessels in the presence of no precursor , l - arginine . our results demonstrated that some relaxation , such as the increase in the diameter of the blood vessel , was evident in the presence of l - arginine . at the same time , the development of the pressure was significantly faster compared to the group without the l - arginine . the analysis of the biomechanical parameters , such as the stress - strain and the shear stress , suggested that the isolated blood vessel became more rigid in the presence of l - arginine . the myocardial ischemia and the reperfusion have also been suggested to increase the histamine production . the histamine not only exists ( to a great extent ) within the mast cells and the basophilic leukocytes , but it also could be released by the endothelial cells , the aggregating platelets , lymphocytes and monocytes / macrophages [ 14 , 15 ] . high concentrations of the histamine are present in the cardiac tissue of most animal species , including humans provoking various cellular functions via the stimulation of four different g - protein - coupled receptors ( h1 , h2 , h3 , and h4 ) . the stimulation of the h2 receptors by the histamine induces both positive inotropic and positive chronotropic effects [ 16 , 17 ] . it has been shown that the blockage of the histamine h2 receptors improved the anaerobic metabolism of the myocardium in the ischemic heart disease that has been associated with the reduced myocardial oxygen consumption demonstrating the highly beneficial effect against the ischemia - reperfusion injury . moreover , the antagonists to the h2-histamine receptors in the mast cells of the heart may reduce the activity of the mast cells , thereby ; they may reduce the release of the mast cell 's mediators [ 13 , 18 , 19 ] . even though the blockage of the histamine h2 receptors limited infarct size , the inhibition of the histamine h1 receptors did not demonstrate this beneficial effect . in the heart , the stimulation of the h1 receptors with histamine impairs the atrioventricular ( av ) conduction , thus contributing to the development of the ventricular fibrillation in the myocardial ischemia - reperfusion injury [ 20 , 21 ] . furthermore , the mast cells play a role in the generation of the reactive oxygen species ( ros ) , such as superoxide anion and hydrogen peroxide , that are responsible for the further increased release of the histamine from the mast cells and vice versa . the histamine effects on the coronary arteries are the result of the multiple actions of this molecule on both smooth muscle cells and the endothelium . some reports showed different effects of the histamine including both the relaxation and the constriction of the coronary vessels depending on species , dose of the histamine , the diameter of the blood vessels and the initial vessel 's tone , as well as the relative location within the coronary circulation . the glucagon is an endogenous polypeptide hormone that exerts both positive inotropic and the chronotropic effects on the myocardial tissue [ 23 , 24 ] . the investigations of the glucagon influence on the transport of the radiolabeled histamine in the isolated guinea pig heart demonstrated the elevation of the histamine transport in the presence of glucagon [ 25 , 26 ] . previous findings showed that the cardiac anaphylactic crisis was markedly reduced in the presence of glucagon . furthermore , these data suggested that the antiarrhythmic action of glucagon during the cardiac anaphylaxis involved the inhibition of histamine release , the vasodilation of the coronary vessels , the increase in the automaticity of the sinoatrial node , and the enhancement of the atrioventricular conduction velocity . moreover , it has been demonstrated that the glucagon influenced the accumulation of histamine in the vesicles of enterochromaffin - like cells ; however , until now there have not yet been reported data explaining the glucagon - induced stimulation of the histamine storage in the heart cells . we postulated that glucagon can affect the changes of the coronary perfusion pressure during the ischemia - reperfusion , by altering the release of no and/or histamine in the heart . therefore , in the present study , we investigated the changes in the coronary perfusion pressure during the ischemia - reperfusion period , as well as the concentrations of nitric oxide , thiobarbituric acid reactive substances ( tbars ) , and histamine either in the presence or in the absence of glucagon in the coronary venous effluent of the isolated rat heart . here , we applied the experimental model and originally developed mathematical procedures ( 12 ) to describe the dynamic response of the coronary blood vessels . all experiments were performed according to eu ( 86/609/eec ) and the local ethical guidelines . wister rats of both sexes ( 200250 g body weight ) were killed by the cervical dislocation . the rat hearts were rapidly isolated and retrogradely perused via the aorta , according to the langendorff 's technique at a constant flow ( 7 - 8 ml / min / g wt ) of the perfusion buffer containing ( in mm ) : nacl 118.1 , kcl 4.7 , mgso4 1.66 , nahco3 24.88 , kh2po4 1.18 , glucose 5.5 , sodium pyruvate 20 , cacl2 2h2o 2.52 . the perfusate was continuously bubbled with 95% o2 and 5% co2 , with the ph adjusted to 7.4 at 37c . the coronary perfusion pressure was measured by a pressure transducer ( elunit , yugoslavia ) and recorded by a kipp & zonen chart recorder ( uk ) . the electrical pacing of the isolated hearts ( 3 v , 2 ms , 300 bpm ) was performed with the electrodes placed on the right auricles near sa node . after the equilibration period ( 20 minutes ) , the ischemic vasodilation was induced by the interruption of coronary inflow ( global ischemia ) for 60 seconds . two to three bouts of the ischemic vasodilation were induced in each heart , both without ( the control group , n = 10 ) and with glucagon solution ( the test group , n = 10 ) approximately 1520 minutes between the interruptions of the coronary inflow . glucagon ( sigma , usa ) was added in the physiological solution in the final concentration of 400 nm , at least 2 minutes before the occlusion , and the addition continued during the reperfusion , until 2 minutes after the reestablishment of the coronary pressure to the preischemic level . the samples of the venous effluent were continuously collected during the reperfusion period and prepared for further analysis : the determination of the histamine concentration , and the levels of no and tbars . after the coronary occlusion , the coronary pressure decreased near to zero . following the period of ischemia , the coronary flow was reestablished ( the reperfusion period ) and the changes in the coronary perfusion pressure ( cpp ) versus time the experimentally recorded dependence of cpp on time during the reperfusion period was fitted using an exponential mathematical function : ( 1)y = b1(1eb2x ) , where y was the cpp ( in mmhg ) , x was the time ( in s ) , and b1 and b2 were the coefficients of this relationship : b 1 has units of pressure , and b 2 has units of time . this function is shown in the figure 1 . as the cpp versus time curve . the constant b1 represents the maximum developed pressure , that is , the pressure corresponding to the alternate steady state . we introduced a dominant time constant ( t ) as the time value corresponding to the cross section point between the asymptote of the exponential curve and the tangent of the exponential curve at the zero point . we considered that the alternate steady state was reached for t = 5 t ( t = time ) , because in this case , e = e 0 , and y b1 . beside the time duration of the cpp restoration ( or the flow restoration ) , the area above the cpp or the flow curve had been commonly used as the parameter for the estimation of cpp or the flow restoration after the ischemic period , by means of the conventional mathematical methods . in this paper , the area above the experimental cpp curve was defined as eaiv ( area of ischemic vasodilation ) . the area above the fitted curve - faiv ( figure 2 ) can be defined by the following function ( 3 ) : ( 3)faiv=0[b1b1(1eb2x ) ] and calculated from the following function ( 4 ) : ( 4)faiv = b1b2 . nitric oxide was assessed as nitrite and quantified by the spectrophotometric method using the griess - reagent . 0.5 ml of the perfusate was precipitated with 200 l of 30% sulfosalicylic acid , vortexed for 30 minutes and centrifuged at 3000 g. the equal volumes of the supernatant and griess 's reagent , containing 1% sulfanilamide in 5% phosphoric acid/0.1% naphthalene ethylenediamine - dihydrochloride were added and incubated for 10 minutes in the dark and read at 543 the degree of lipid peroxidation in the coronary venous effluent was estimated by the measurement of thiobarbituric acid reactive substances ( tbars ) using 1% tba ( thiobarbituric acid ) in 0.05 naoh incubated with the coronary effluent at 100c for 15 minutes and read at 530 nm . statistical analysis was performed by using the multifactorial analysis of the variance for repeated measurements between the subject factors as well as bonferroni - test . also , the data were analyzed using student 's t - test , where p value of < .05 was considered as statistically significant . to investigate the effect of the glucagon on the concentration of the histamine in the coronary venous effluents , the levels of histamine were measured both in the control and in the test groups either in the absence ( n = 10 ) or in the presence ( n = 10 ) of the glucagon , respectively . our results demonstrated no statistically significant differences in the levels of the histamine concentration both in the control and the test groups , as shown in the table 1 . on the other hand , there was a significant increase of the histamine release both in the control ( 126% ) and the test groups ( 33% ) during the reperfusion period compared to the preischemic levels . the increase of the histamine concentrations achieved the maximal values within 30 to 90 s in the control group and within 30 to 60 s in the test group during the reperfusion period . in addition , our results indicated a significant decrease of the histamine release in the presence of glucagon during the reperfusion period compared to the control group . furthermore , we measured the levels of no in the coronary venous effluent , collected both during preischemic and the reperfusion periods , both in the control ( n = 10 ) and the test ( n = 10 ) groups , as presented in the figure 3 . the results showed the increase of no concentrations in both groups during the reperfusion period . also , the levels of no in the test group were significantly elevated ( 71% ) during the preischemic period in comparison to the control group . the elevated concentrations of no in the control group collected during the reperfusion period showed two peak values : the first peak was evident 30 seconds following the reperfusion period with the increase of 187% , and the second peak showed 90 seconds following the reperfusion period with the increase of 221% . on the other hand , the no level in the test group achieved one peak value within first 30 seconds of the reperfusion period . after reaching the peak value , the no level in the test group gradually decreased until the end of the reperfusion period . moreover , within first 30 seconds of the reperfusion period , the no levels in the test group were initially significantly higher ( 60% ) but eventually reached significantly lower levels ( 45% ) within first 90 seconds of the reperfusion period , compared to the corresponding no levels in the control group . next , we investigated the concentrations of tbars in the samples collected during both the preischemic and the reperfusion periods , both in the control and in the test groups . our results showed that the concentrations of tbars in the samples collected during the reperfusion period were greatly elevated in comparison to the preischemic levels , both in the control group ( 202.6% , n = 10 ) and in the test group ( 226.7% , n = 10 ) , as shown in the table 2 . the highest tbars concentrations were achieved within the first 30 seconds of the reperfusion period in both groups . in addition , our results showed no statistically significant differences in the tbars levels neither in the control nor in the test group both during the preischemic and the reperfusion periods . further , we analyzed the experimental cpp - time curves using a method as described in materials and methods . the values of 5 t , the b1 and b2 coefficients of the fitted experimental data ( coefficient of correlation was 0.998 0.002 ) both in the control ( n = 10 ) and in the test ( n = 1 ) groups were shown in the table 3 . our analyzed data indicated significant increase of the coefficient b2 in the test group compared to the control group . however , the 5 t value in the test group was significantly lower in comparison to the value of the control group . moreover , our results showed no great difference in b1 coefficients between the control and the test groups of the animals . when analyzing the faiv in both the control and the test groups , our results showed that faiv in the control group was 1373 33 , while in the presence of glucagon this area was significantly lower ( 940 28 ) . at the same time the values of eaiv both in the control group ( 1360 29 ) and the test group ( 951 30 ) were not significantly different in comparison to the corresponding faiv values . the restoration of the coronary perfusion pressure during the reperfusion period in our experiments had an exponential form rather than the step function , indicating that the ischemic vasodilation occurred within this period . in this study , we investigated the ischemic vasodilation of the isolated rat heart , as well as the no , tbars , and histamine concentrations in the coronary venous effluent either in the presence or in the absence of glucagon . we used a new approach in the ex vivo experiments considering not only the end points of the vessels response , but also the dynamics of this response . in order to elucidate the mentioned phenomena , we described the dynamic behavior of the coronary blood vessel during the ischemic vasodilation by proposing a mathematical relation where the parameters were evaluated through the relationships of the coronary pressure - time curves with the experimental data . the coronary pressure - time curves were fitted using the simple exponential mathematical function in the way that both b1 and b2 coefficients had the physical meaning . in this function , the first coefficient b1numerically described the maximal developed coronary pressure that corresponded to the pressure in the alternate steady state . the second coefficient b2was relevant for the description of the vessel transition response between the alternate steady states . in our previous work , we demonstrated that these coefficients were highly sensitive parameters to the conditions of the blood vessel dynamics . the mathematical analysis of the cpp dynamics showed no significant differences in the b1 coefficients ( table 3 ) both in the control and in the test groups of animals . this result revealed that the maximal coronary perfusion pressure during the ischemic vasodilation was similar in both groups of animals . on the contrary , the b2 coefficient in the test group was significantly higher compared to the b2 coefficient of the control group , indicating the alteration in the dynamic response during the ischemic vasodilation in the presence of the glucagon . in addition , the time interval for the development of the maximal cpp during the alternate steady state was 125 seconds in the control group and it was significantly higher compared to the maximal cpp developmental period of 82 seconds in the test group . therefore , these results demonstrated a faster increase of the coronary pressure in the glucagon - treated hearts ( table 3 ) . the values of faiv in the presence of the glucagon were significantly lower compared to the faiv values of the control group confirming the glucagon alteration of the ischemic vasodilation in our experimental design . on the other hand , the faiv values were not notably different from the eaiv values in both groups , demonstrating the logical consequence of the extremely high coefficient of the correlation ( r = 0.998 0.002 ) between the fitted and the experimental cpp curves ( figure 2 ) . in addition , we calculated the index of lipid peroxidation by measuring the levels of thiobarbituric acid reactive substances - tbars ( the known markers of the oxidative stress ) . our data demonstrated significantly the elevated levels of tbars within 2 minutes of the reperfusion both in the control and the test groups compared to the preischemic levels ( table 2 ) . these findings were in accordance with the previous reports [ 1 , 8 , 32 ] . also , our data demonstrated no apparent differences in the tbars levels between the control and the test groups , during both the preischemic and the ischemia - reperfusion periods . these results indicated that the glucagon administration did not affect the tbars levels in the isolated rat heart during both the preischemic and the ischemia - reperfusion periods in our experimental conditions . investigation of the no level both before and during the ischemic vasodilation in the presence and in the absence of the glucagon clearly demonstrated that : the no levels during the preischemic period were significantly elevated in the presence of glucagon compared to the control group ; the no level within the first 30 seconds of the reperfusion period was significantly higher , and after 90 seconds it was significantly lower in the test group , compared to the corresponding no levels in the control group . these findings were similar to the previous data reporting that the nitrite levels were higher under the glucagon treatment in the rat hepatocyte culture and that the no levels increased during the ischemia - reperfusion conditions , as well [ 7 , 9 , 34 , 35 ] . our experimental data also showed no apparent changes in the preischemic cpp values measured continuously during the glucagon administration . these results indicated that the elevated preischemic no level in the test group did not significantly affect the coronary vessel diameters in our experimental conditions . even though the elevated preischemic no level did not have the significant effects on the histamine release , its role in influencing the mast cells stabilization and consequently reducing the histamine release in the test group during ischemic vasodilation should not be neglected . our previous findings demonstrated the influence of l - arginine on the faster development of the perfusion pressure in the isolated blood vessels . although l - arginine in the previous investigation relaxed the smooth muscle in the blood vessel wall , the introduction of the hydrostatic pressure caused the blood vessel wall to become more rigid . these findings indicated that the increase of the no release might not only induce the vasodilation , but it also might affect the biomechanical properties of the blood vessel wall . even though the vasodilation occurred at the alternate steady states , the transient or the dynamic response of the blood vessel showed faster development of the pressure . it is well known that the histamine release is elevated during the ischemic vasodilation and its concentration is increased during the ischemia . also , our results showed that the histamine liberation during the ischemic vasodilation was significantly reduced in the presence of glucagon in the isolated rat heart . these data were in the accordance with the previous reports describing the influence of the glucagon on the inhibition of the histamine release during the anaphylactic response in a guinea - pig isolated heart and the protective effects of the glucagon pretreatment during cardiac anaphylaxis . changes in both no and histamine releases during both the ischemic vasodilation and the coronary autoregulation suggested a positive feedback between no and histamine . the initial increase of no within the first 30 seconds of the reperfusion period may lead to the increase of histamine release reaching the peak values between 3090 seconds . furthermore , the histamine stimulation of the endothelial histamine receptors produced the second peak of the no release , by positive feedback mechanism , after 90 seconds of the reperfusion . as mentioned earlier , the no level during the preischemic period was significantly elevated in the presence of glucagon . at the same time , there was neither a change in the cpp nor in the basal ( preischemic ) histamine concentrations . even though the preischemic no level was significantly elevated , the amount of no released seemed to be insufficient to dilate the coronary blood vessels , since there was no change in the cpp at that time . on the other hand , the increased amount of no might be sufficient to stabilize mast cells , therefore influencing the lack of the positive feedback relationship between no and the histamine in the presence of glucagon . this could be the cause of the significantly lower no release in the test group 90 seconds following the ischemic vasodilation . in the summary , the influence of the glucagon on the dynamic responses of the coronary vessels of the isolated rat heart was uncertain during the ischemic vasodilation . in this study , our results showed a faster restoration of the coronary perfusion pressure during the reperfusion period in the presence of glucagon compared to the control group . therefore , one of the possible postulated mechanism(s ) explaining this process could be as following : the glucagon increases no release both during the preischemic period and within the first 30 seconds of the ischemic vasodilation ; no stabilizes the mast cells and prevents the histamine release during the ischemic vasodilation ; further , the decreased histamine release then leads to the attenuated stimulation of the endothelial histamine receptor ( h1 ) and consequent decrease of the no release , thus switching off this vasodilatory mechanism ; simultaneously , the decreased histamine release leads to the attenuation of the h2/h3 mediated coronary vasodilation in the presence of glucagon ; as a result , the duration of the ischemic vasodilation is significantly shorter in the presence of glucagons ; moreover , the increased no release within the first 30 seconds of the ischemic vasodilation leads to the vasodilation in the presence of glucagon that in turn may also increase the rigidity of the coronary blood vessels , generating the faster development of cpp . finally , even though the mechanism(s ) of the glucagon influences on the coronary vessels during the ischemic vasodilation is still unclear , our results clearly indicate the key involvement of both histamine and no during this process .

the myocardial reperfusion following ischemia leads to the ischemic vasodilation by affecting the release of various vasoactive substances , such as free radicals , no , and histamine . in addition , some evidences suggest that glucagon itself may alter the release of those substances . in this study , we investigated the ischemic vasodilation of the isolated rat heart , as well as the concentrations of no , tbars , and histamine in the coronary venous effluent either in the presence or in the absence of glucagon . our results showed that in the presence of glucagon , there was a faster restoration of coronary perfusion pressure during ischemic vasodilation compared to the absence of glucagon ( 124 5.6 versus 81 5.2 s ) with no apparent changes in tbars concentration . the glucagon 's administration leads to the decreased release of histamine by approximately 35% . biphasic release of no in the presence of glucagon initially showed augmentation by 60% , followed by the significant attenuation of 45% .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusions

although the myocardial reperfusion following ischemia can produce the salvage of the ischemic tissue , it may also contribute not only to the endothelial dysfunction and the myocardial cellular injury , but it also may change the release of the biologically active substances , such as the endothelium - derived relaxing factor ( no ) . even though no obtained from l - arginine is a free radical per se , it also can quench other free radicals , including the superoxide radicals and it thereby protects both the endothelial cells and the cardiac myocytes [ 2 , 3 ] . in addition , no may stabilize mast cells and inhibit the release of histamine from the both isolated and the resident cardiac mast cells , thus influencing the postischemic myocardial injury [ 46 ] . thus , low doses of no appear to play a key protective role from the myocardial ischemia - reperfusion injury . beside the fact that no may stabilize the cardiac mast cells and it consequently decreases the histamine release [ 4 , 5 , 10 ] , earlier results showed a positive feedback relationship between no and histamine in the regulation of both coronary ischemic vasodilation and the coronary autoregulation . in our previous work , we evaluated both the dynamic response and the biomechanical properties of the isolated blood vessels in the presence of no precursor , l - arginine . our results demonstrated that some relaxation , such as the increase in the diameter of the blood vessel , was evident in the presence of l - arginine . at the same time , the development of the pressure was significantly faster compared to the group without the l - arginine . the analysis of the biomechanical parameters , such as the stress - strain and the shear stress , suggested that the isolated blood vessel became more rigid in the presence of l - arginine . high concentrations of the histamine are present in the cardiac tissue of most animal species , including humans provoking various cellular functions via the stimulation of four different g - protein - coupled receptors ( h1 , h2 , h3 , and h4 ) . the stimulation of the h2 receptors by the histamine induces both positive inotropic and positive chronotropic effects [ 16 , 17 ] . it has been shown that the blockage of the histamine h2 receptors improved the anaerobic metabolism of the myocardium in the ischemic heart disease that has been associated with the reduced myocardial oxygen consumption demonstrating the highly beneficial effect against the ischemia - reperfusion injury . moreover , the antagonists to the h2-histamine receptors in the mast cells of the heart may reduce the activity of the mast cells , thereby ; they may reduce the release of the mast cell 's mediators [ 13 , 18 , 19 ] . in the heart , the stimulation of the h1 receptors with histamine impairs the atrioventricular ( av ) conduction , thus contributing to the development of the ventricular fibrillation in the myocardial ischemia - reperfusion injury [ 20 , 21 ] . furthermore , the mast cells play a role in the generation of the reactive oxygen species ( ros ) , such as superoxide anion and hydrogen peroxide , that are responsible for the further increased release of the histamine from the mast cells and vice versa . the histamine effects on the coronary arteries are the result of the multiple actions of this molecule on both smooth muscle cells and the endothelium . some reports showed different effects of the histamine including both the relaxation and the constriction of the coronary vessels depending on species , dose of the histamine , the diameter of the blood vessels and the initial vessel 's tone , as well as the relative location within the coronary circulation . the investigations of the glucagon influence on the transport of the radiolabeled histamine in the isolated guinea pig heart demonstrated the elevation of the histamine transport in the presence of glucagon [ 25 , 26 ] . previous findings showed that the cardiac anaphylactic crisis was markedly reduced in the presence of glucagon . furthermore , these data suggested that the antiarrhythmic action of glucagon during the cardiac anaphylaxis involved the inhibition of histamine release , the vasodilation of the coronary vessels , the increase in the automaticity of the sinoatrial node , and the enhancement of the atrioventricular conduction velocity . moreover , it has been demonstrated that the glucagon influenced the accumulation of histamine in the vesicles of enterochromaffin - like cells ; however , until now there have not yet been reported data explaining the glucagon - induced stimulation of the histamine storage in the heart cells . we postulated that glucagon can affect the changes of the coronary perfusion pressure during the ischemia - reperfusion , by altering the release of no and/or histamine in the heart . therefore , in the present study , we investigated the changes in the coronary perfusion pressure during the ischemia - reperfusion period , as well as the concentrations of nitric oxide , thiobarbituric acid reactive substances ( tbars ) , and histamine either in the presence or in the absence of glucagon in the coronary venous effluent of the isolated rat heart . here , we applied the experimental model and originally developed mathematical procedures ( 12 ) to describe the dynamic response of the coronary blood vessels . the coronary perfusion pressure was measured by a pressure transducer ( elunit , yugoslavia ) and recorded by a kipp & zonen chart recorder ( uk ) . after the equilibration period ( 20 minutes ) , the ischemic vasodilation was induced by the interruption of coronary inflow ( global ischemia ) for 60 seconds . two to three bouts of the ischemic vasodilation were induced in each heart , both without ( the control group , n = 10 ) and with glucagon solution ( the test group , n = 10 ) approximately 1520 minutes between the interruptions of the coronary inflow . glucagon ( sigma , usa ) was added in the physiological solution in the final concentration of 400 nm , at least 2 minutes before the occlusion , and the addition continued during the reperfusion , until 2 minutes after the reestablishment of the coronary pressure to the preischemic level . the samples of the venous effluent were continuously collected during the reperfusion period and prepared for further analysis : the determination of the histamine concentration , and the levels of no and tbars . following the period of ischemia , the coronary flow was reestablished ( the reperfusion period ) and the changes in the coronary perfusion pressure ( cpp ) versus time the experimentally recorded dependence of cpp on time during the reperfusion period was fitted using an exponential mathematical function : ( 1)y = b1(1eb2x ) , where y was the cpp ( in mmhg ) , x was the time ( in s ) , and b1 and b2 were the coefficients of this relationship : b 1 has units of pressure , and b 2 has units of time . we introduced a dominant time constant ( t ) as the time value corresponding to the cross section point between the asymptote of the exponential curve and the tangent of the exponential curve at the zero point . beside the time duration of the cpp restoration ( or the flow restoration ) , the area above the cpp or the flow curve had been commonly used as the parameter for the estimation of cpp or the flow restoration after the ischemic period , by means of the conventional mathematical methods . 0.5 ml of the perfusate was precipitated with 200 l of 30% sulfosalicylic acid , vortexed for 30 minutes and centrifuged at 3000 g. the equal volumes of the supernatant and griess 's reagent , containing 1% sulfanilamide in 5% phosphoric acid/0.1% naphthalene ethylenediamine - dihydrochloride were added and incubated for 10 minutes in the dark and read at 543 the degree of lipid peroxidation in the coronary venous effluent was estimated by the measurement of thiobarbituric acid reactive substances ( tbars ) using 1% tba ( thiobarbituric acid ) in 0.05 naoh incubated with the coronary effluent at 100c for 15 minutes and read at 530 nm . statistical analysis was performed by using the multifactorial analysis of the variance for repeated measurements between the subject factors as well as bonferroni - test . to investigate the effect of the glucagon on the concentration of the histamine in the coronary venous effluents , the levels of histamine were measured both in the control and in the test groups either in the absence ( n = 10 ) or in the presence ( n = 10 ) of the glucagon , respectively . our results demonstrated no statistically significant differences in the levels of the histamine concentration both in the control and the test groups , as shown in the table 1 . on the other hand , there was a significant increase of the histamine release both in the control ( 126% ) and the test groups ( 33% ) during the reperfusion period compared to the preischemic levels . in addition , our results indicated a significant decrease of the histamine release in the presence of glucagon during the reperfusion period compared to the control group . furthermore , we measured the levels of no in the coronary venous effluent , collected both during preischemic and the reperfusion periods , both in the control ( n = 10 ) and the test ( n = 10 ) groups , as presented in the figure 3 . also , the levels of no in the test group were significantly elevated ( 71% ) during the preischemic period in comparison to the control group . the elevated concentrations of no in the control group collected during the reperfusion period showed two peak values : the first peak was evident 30 seconds following the reperfusion period with the increase of 187% , and the second peak showed 90 seconds following the reperfusion period with the increase of 221% . after reaching the peak value , the no level in the test group gradually decreased until the end of the reperfusion period . moreover , within first 30 seconds of the reperfusion period , the no levels in the test group were initially significantly higher ( 60% ) but eventually reached significantly lower levels ( 45% ) within first 90 seconds of the reperfusion period , compared to the corresponding no levels in the control group . next , we investigated the concentrations of tbars in the samples collected during both the preischemic and the reperfusion periods , both in the control and in the test groups . our results showed that the concentrations of tbars in the samples collected during the reperfusion period were greatly elevated in comparison to the preischemic levels , both in the control group ( 202.6% , n = 10 ) and in the test group ( 226.7% , n = 10 ) , as shown in the table 2 . in addition , our results showed no statistically significant differences in the tbars levels neither in the control nor in the test group both during the preischemic and the reperfusion periods . our analyzed data indicated significant increase of the coefficient b2 in the test group compared to the control group . however , the 5 t value in the test group was significantly lower in comparison to the value of the control group . moreover , our results showed no great difference in b1 coefficients between the control and the test groups of the animals . when analyzing the faiv in both the control and the test groups , our results showed that faiv in the control group was 1373 33 , while in the presence of glucagon this area was significantly lower ( 940 28 ) . at the same time the values of eaiv both in the control group ( 1360 29 ) and the test group ( 951 30 ) were not significantly different in comparison to the corresponding faiv values . the restoration of the coronary perfusion pressure during the reperfusion period in our experiments had an exponential form rather than the step function , indicating that the ischemic vasodilation occurred within this period . in this study , we investigated the ischemic vasodilation of the isolated rat heart , as well as the no , tbars , and histamine concentrations in the coronary venous effluent either in the presence or in the absence of glucagon . we used a new approach in the ex vivo experiments considering not only the end points of the vessels response , but also the dynamics of this response . in order to elucidate the mentioned phenomena , we described the dynamic behavior of the coronary blood vessel during the ischemic vasodilation by proposing a mathematical relation where the parameters were evaluated through the relationships of the coronary pressure - time curves with the experimental data . the coronary pressure - time curves were fitted using the simple exponential mathematical function in the way that both b1 and b2 coefficients had the physical meaning . in this function , the first coefficient b1numerically described the maximal developed coronary pressure that corresponded to the pressure in the alternate steady state . in our previous work , we demonstrated that these coefficients were highly sensitive parameters to the conditions of the blood vessel dynamics . the mathematical analysis of the cpp dynamics showed no significant differences in the b1 coefficients ( table 3 ) both in the control and in the test groups of animals . this result revealed that the maximal coronary perfusion pressure during the ischemic vasodilation was similar in both groups of animals . on the contrary , the b2 coefficient in the test group was significantly higher compared to the b2 coefficient of the control group , indicating the alteration in the dynamic response during the ischemic vasodilation in the presence of the glucagon . in addition , the time interval for the development of the maximal cpp during the alternate steady state was 125 seconds in the control group and it was significantly higher compared to the maximal cpp developmental period of 82 seconds in the test group . therefore , these results demonstrated a faster increase of the coronary pressure in the glucagon - treated hearts ( table 3 ) . the values of faiv in the presence of the glucagon were significantly lower compared to the faiv values of the control group confirming the glucagon alteration of the ischemic vasodilation in our experimental design . in addition , we calculated the index of lipid peroxidation by measuring the levels of thiobarbituric acid reactive substances - tbars ( the known markers of the oxidative stress ) . our data demonstrated significantly the elevated levels of tbars within 2 minutes of the reperfusion both in the control and the test groups compared to the preischemic levels ( table 2 ) . these results indicated that the glucagon administration did not affect the tbars levels in the isolated rat heart during both the preischemic and the ischemia - reperfusion periods in our experimental conditions . investigation of the no level both before and during the ischemic vasodilation in the presence and in the absence of the glucagon clearly demonstrated that : the no levels during the preischemic period were significantly elevated in the presence of glucagon compared to the control group ; the no level within the first 30 seconds of the reperfusion period was significantly higher , and after 90 seconds it was significantly lower in the test group , compared to the corresponding no levels in the control group . these findings were similar to the previous data reporting that the nitrite levels were higher under the glucagon treatment in the rat hepatocyte culture and that the no levels increased during the ischemia - reperfusion conditions , as well [ 7 , 9 , 34 , 35 ] . our experimental data also showed no apparent changes in the preischemic cpp values measured continuously during the glucagon administration . these results indicated that the elevated preischemic no level in the test group did not significantly affect the coronary vessel diameters in our experimental conditions . even though the elevated preischemic no level did not have the significant effects on the histamine release , its role in influencing the mast cells stabilization and consequently reducing the histamine release in the test group during ischemic vasodilation should not be neglected . our previous findings demonstrated the influence of l - arginine on the faster development of the perfusion pressure in the isolated blood vessels . also , our results showed that the histamine liberation during the ischemic vasodilation was significantly reduced in the presence of glucagon in the isolated rat heart . these data were in the accordance with the previous reports describing the influence of the glucagon on the inhibition of the histamine release during the anaphylactic response in a guinea - pig isolated heart and the protective effects of the glucagon pretreatment during cardiac anaphylaxis . changes in both no and histamine releases during both the ischemic vasodilation and the coronary autoregulation suggested a positive feedback between no and histamine . the initial increase of no within the first 30 seconds of the reperfusion period may lead to the increase of histamine release reaching the peak values between 3090 seconds . as mentioned earlier , the no level during the preischemic period was significantly elevated in the presence of glucagon . at the same time , there was neither a change in the cpp nor in the basal ( preischemic ) histamine concentrations . even though the preischemic no level was significantly elevated , the amount of no released seemed to be insufficient to dilate the coronary blood vessels , since there was no change in the cpp at that time . on the other hand , the increased amount of no might be sufficient to stabilize mast cells , therefore influencing the lack of the positive feedback relationship between no and the histamine in the presence of glucagon . this could be the cause of the significantly lower no release in the test group 90 seconds following the ischemic vasodilation . in the summary , the influence of the glucagon on the dynamic responses of the coronary vessels of the isolated rat heart was uncertain during the ischemic vasodilation . in this study , our results showed a faster restoration of the coronary perfusion pressure during the reperfusion period in the presence of glucagon compared to the control group . therefore , one of the possible postulated mechanism(s ) explaining this process could be as following : the glucagon increases no release both during the preischemic period and within the first 30 seconds of the ischemic vasodilation ; no stabilizes the mast cells and prevents the histamine release during the ischemic vasodilation ; further , the decreased histamine release then leads to the attenuated stimulation of the endothelial histamine receptor ( h1 ) and consequent decrease of the no release , thus switching off this vasodilatory mechanism ; simultaneously , the decreased histamine release leads to the attenuation of the h2/h3 mediated coronary vasodilation in the presence of glucagon ; as a result , the duration of the ischemic vasodilation is significantly shorter in the presence of glucagons ; moreover , the increased no release within the first 30 seconds of the ischemic vasodilation leads to the vasodilation in the presence of glucagon that in turn may also increase the rigidity of the coronary blood vessels , generating the faster development of cpp . finally , even though the mechanism(s ) of the glucagon influences on the coronary vessels during the ischemic vasodilation is still unclear , our results clearly indicate the key involvement of both histamine and no during this process .

every imaging modality depicts non - invasively a specific tissue property that results from an interaction between the tissue and the modality 's inherent feature . for example , x - ray and computed tomography depict the attenuation of x - rays , ultrasound depicts one of the mechanical properties ( reflectivity or acoustic impedance difference ) of the tissue , and positron emission tomography depicts the concentration of injected radio nucleotide in the body . similarly , photoacoustic ( pa ) imaging depicts optical absorption properties of a tissue at the wavelength of interrogating laser light similar to pure optical imaging but with noticeable differences . as the name suggests , the pa effect is a combination of both light and sound . when a laser pulse of short duration , approximately a few nanoseconds ( ns ) , interacts with the tissue , it results in localized heating of the tissue followed by rapid thermal expansion , generating thermo - elastic stress waves . this effect is known as a pa effect and the resulting stress ( ultrasound ) waves are referred to as pa waves . pa imaging is neither purely optical nor purely acoustical in nature , but a combination of the two . pure optical imaging uses light as the input and the backscattered radiation as the output for image formation , whereas pa imaging uses light as the input source for tissue excitation but detection and image formation are achieved using ultrasound waves generated by the tissue . for example , due to excessive scattering of light in turbid media , optical imaging suffers from poor penetration depths and low resolution . pa imaging has better penetration depths because the incident light has to propagate in only one direction into the tissue and the ultrasound waves do not attenuate as much in the tissue as backscattered light . a good resolution in pa imaging results from the fact that the ultrasound can be focused easily and speed of propagation does not deviate much ( for instance , it is ~ 1500 m / s in soft tissue ) so that the wave arrival time can provide information about the depth . the tissue and its major constituents ( water , oxy and de - oxy hemoglobin in blood , lipid , fat , melanin , collagen , etc ) have widely varying absorption spectra in the near infrared ( nir ) region that not only provide abundant contrast features , but also open the door for pa imaging based spectroscopy , non - invasive tissue characterization and functional imaging . photoacoustically generated ultrasound waves and typical echo - based ultrasound waves are similar from a detector standpoint as all that is needed to capture the signals is an ultrasound transducer . however , it is important to mention two vital characteristics of photoacoustically generated ultrasound waves that differentiate them from echo - based ultrasound waves . first , the frequency of echo - based ultrasound signal in a medical ultrasound imaging system lies within the bandwidth of the transmitted signal , whereas the frequency of the pa signal depends on the size of the biological absorber and the duration of the illuminating laser pulse . second , unlike conventional ultrasound where the signal strength or image intensity depends on the echogenicity ( ultrasound reflectivity ) of the echo - generating source in the medium , the pa signal strength depends on the optical absorption coefficient of the absorber in the medium and the difference in optical absorption of the incident light dictates the image contrast , as mentioned earlier . absorption coefficients of tissue constituents vary with wavelength of incident light . also , since angiogenesis is the fundamental step in the transition of a tumor from dormant to malignant state , a tumor that has blood vessels proliferated into it , interrogated at appropriate wavelengths will produce a stronger pa signal compared to the surrounding normal tissue.[35 ] the major part of a tissue is made up of water that has a lower absorption coefficient in the near infrared ( nir ) region , especially in the wavelength range of 650 - 950 nanometers ( nm ) , than blood and other tissue constituents . if the tissue is illuminated at these wavelengths , deeper penetration of light can be achieved and the tissue can be imaged with better contrast and the tissue characteristics such as oxy and deoxy - hemoglobin concentrations , oxygen saturation , and other parameters , can be extensively studied since pa imaging can yield images from depths of up to a few centimeters , it provides a map of the optical absorption by tissue constituents at the interrogating laser wavelength along with the depth or location information . therefore , the pa signal is more diagnostic in nature than the conventional ultrasound signal . a pulse - echo medical ultrasound system typically provides two - dimensional images of the underlying three - dimensional organ or tissue being scanned , which are technically called b - scan images . as the imaging depth increases , the lateral resolution in the b - scan images gets worse and to correct for it , electronic focusing techniques are implemented in the software . the advantages of the acoustic lens focusing over electronic focusing are : ( 1 ) it is faster , ( 2 ) it uses zero electrical power , ( 3 ) it is inexpensive to design and implement , and ( 4 ) there are no errors in the reconstructed image due to spatial and temporal sampling of the signal . however , our use of lens - based focusing is different from what has been done in the past in clinical ultrasound imaging . earlier , the focusing element was typically placed on the single element transducer , while the transducer was scanned in sectors to acquire the ultrasound image . this is time consuming . in our version , by separating the lens from the image plane , we can in principle acquire the focused image on the image plane ( coronal plane ) almost instantaneously with a two - dimensional sensor array . figure 1shows our acoustic lens - based pa imaging concept . when a near - infrared laser illuminates the tissue with very short high energy pulses of nanosecond duration , it generates pa waves within the tissue if any absorbers are present . an acoustic lens will collect the waves generated in the object plane and focus them onto an ultrasound detector which is placed in the image plane [ figure 1 ] . an appropriately designed lens will focus the wave front from a point source in the object plane to a point in the image plane . the object plane here corresponds to a coronal plane in the tissue unlike sagittal or transverse planes imaged by a conventional ultrasound imaging system . image plane is defined by time delay measured with respect to time t=0 when the laser illuminates the tissue . in our experience , the difference in travel time from different points in the object plane to their corresponding focus points in the image plane is usually less than the pulse duration of the pa signal . therefore , the entire image plane can be assumed to be in focus and is said to be the conjugate image plane corresponding to the object plane defined by the delay time . more details about the lens can be found under the pa image formation section . object plane lies within the tissue and an ultrasound detector placed in the image plane will collect focused pa signals . a detector array located in the image plane can capture this energy as a focused image that represents a thin coronal slice in the object . images of these coronal planes are referred to as c - scan images and represent laser absorption by different regions of the tissue that are intersected by that plane , much like ct scan images . the photoacoustically generated ultrasound waves travel with finite speed ( ~1500 m / s ) in soft tissue . therefore , a pragmatic way to capture focused signal from a predetermined coronal plane the assumption here is that all waves from the predetermined coronal plane arrive at the image plane at the same time . as a first step toward designing an in vivo pa imaging system , we have developed a prototype pa in vitro imaging system to detect any malignancies present in an excised tissue as illustrated in figure 2 . the system design was optimized to generate focused coronal plane ( c - scan ) images using acoustic lens technology . in principle , c - scan images are nothing but two - dimensional ( 2d ) images produced by spatially sampling the pa signal amplitude at a fixed time while the ultrasound sensor is raster - scanned over some surface . the in vitro prototype system comprise of the following components : a fiber optic cable for laser delivery , a sample holder to hold the excised tissue , a pa camera that is water - sealed with acoustic lens , and the ultrasound detector . a near infrared laser beam is delivered from the bottom of the tissue sample holder , which can hold a tissue up to 4 cm thick . the laser beam has a diameter of 5 mm with a repetition rate of 10 hz and pulse duration of 10 ns and is delivered onto the tissue via a fiber optic cable . at the sample surface , laser energy intensity is ~ 20 mj / cm , which is well below laser safety limits . we have designed the front - end and back - end electronics to acquire 16-channel signals simultaneously at each laser firing ; details of which are given in the subsequent sections . the entire tissue is scanned by the laser beam and the camera in tandem using linear actuators . typical time taken for imaging a 4 4 cm area of an excised specimen with this system is about 7 min , the delay being mainly due to the hardware limitation posed by the linear actuator speed and stabilization time . once the pa images are acquired , the pa camera head along with the laser delivery system is replaced by a portable ultrasound array head that captures ultrasound images that can be correlated and registered with the pa images . the ultrasound transducer array has 16 elements , each of size 0.5 mm 1.0 mm , pitch 0.7 mm , and the center frequency of 5 mhz with 60% bandwidth . the sensor element size was chosen such that the reception angular profile of the edge elements , subtended at the lens , was within 20% of what was for the center element . the pitch was chosen to minimize any aliasing due to spatial sampling of the image plane . the acoustic lens design was accomplished using oslo ( lambda research corporation , littleton , ma , usa ) ray tracing software simulation program and optimized for wavelength of 0.3 mm , corresponding to 5 mhz . the lens has a focal length ( f ) of 39.8 mm and a diameter of 32 mm corresponding to an active f - number of 1.2 . the lens design was optimized to achieve a magnification of 1 when both the lens - to - object plane and the lens - to - image plane distances are twice the focal length of the lens . hence , in our in vitro prototype , the transducer array was fixed at 79.6 ( 2f ) mm from the lens in the image plane and the ultrasound waves generated from any absorbers in the object plane that are 79.6 mm from the lens will be in sharp focus and are imaged with a magnification of 1 . the lens has a depth of field of approximately 5 mm on either side of the focal plane . the ultrasound sensor array located in the image plane acquires focused pa signals , much like optical image formation . to perform c - scan imaging , the pa camera is mounted on a holder that is controlled by two linear actuators ( zaber technologies inc . , vancouver , british columbia , canada ) and a raster scan is performed in the image plane to scan a 2d area by collecting pa signals at each pixel location . the pa signals acquired over all 16 channels at each linear actuator location are fed simultaneously to the custom - built back end electronics module that consists of a variable gain amplifier and a digitizer to generate pa b - scan images followed by an ultrasound scan . once the scanning is completed with both pa and ultrasound systems , the acquired pa images are fused with ultrasound images to illustrate anatomical as well as physiological information in a single image . a 16-channel simultaneous data acquisition ( daq ) system was custom - designed to be used with the pa imaging system whose layout is shown in figure 3 . the entire electronics can be categorized into four modules : ( 1 ) amplification of a raw pa signal ; ( 2 ) sampling / digitization of an amplified pa signal ; ( 3 ) dual axis stepper motor movement for c - scan imaging ; and ( 4 ) a host computer with labview ( national instruments , austin , tx , usa ) controlling the first three modules and to display real - time b - scan images . pa signals acquired by the linear array transducer are tapped using a 16-channel custom - fabricated distribution board and are amplified using two 8-channel vca8500evm ( texas instruments , dallas , tx , usa ) variable gain amplifiers . the variable gain amplifiers ( vgas ) have a maximum amplification gain of 50 db ( a pre - amplification gain of 20 db + selectable post gain amplification of 20 , 25 , 27 , or 30 db ) per channel and provide the user a selection of 10 mhz or 15 mhz low pass filtering . all the 16 channels of the two vgas are simultaneously controlled by the computer using a graphical user interface ( gui ) and hence the gain can be changed on - the - fly as well . the 16-channel amplified pa signal data is then simultaneously fed to two 8-channel pxi-5105 ( national instruments , austin , tx , usa ) digitizer boards , which are housed in an external chassis , at each laser firing . a digital trigger is provided by the laser to chassis , during each laser firing , for synchronization . the pxi-5105 digitizer board has 12-bit resolution and 60 mhz sampling rate with a 16 mb onboard memory buffer which allows storing the digitized pa signal samples onboard before transfer to the host computer using mxi interface . as mentioned earlier , the total acquisition time is largely limited by the hardware specifications of the actuators ; their maximum speed being only 4 mm / s , stabilization time of at least 250 ms , and a resolution of 0.1 m . the actuators are moved by 1 mm increments along the y - axis to cover 40 mm and by 11.2 mm increments ( 16 0.7 mm ) along the x - axis direction to cover 44.8 mm to scan an area of 4 cm 4 cm . the entire scanning mechanism including daq and linear actuator movement is controlled by the host computer using labview . the acquired pa b - scan images are transmitted to the host computer and processed further in matlab environment to reconstruct c - scan images and to achieve pa / ultrasound image fusion . in vitro experiments are conducted on excised tissue samples with this system and the results obtained are presented in the following section . a pulse - echo medical ultrasound system typically provides two - dimensional images of the underlying three - dimensional organ or tissue being scanned , which are technically called b - scan images . as the imaging depth increases , the lateral resolution in the b - scan images gets worse and to correct for it , electronic focusing techniques are implemented in the software . the advantages of the acoustic lens focusing over electronic focusing are : ( 1 ) it is faster , ( 2 ) it uses zero electrical power , ( 3 ) it is inexpensive to design and implement , and ( 4 ) there are no errors in the reconstructed image due to spatial and temporal sampling of the signal . however , our use of lens - based focusing is different from what has been done in the past in clinical ultrasound imaging . earlier , the focusing element was typically placed on the single element transducer , while the transducer was scanned in sectors to acquire the ultrasound image . this is time consuming . in our version , by separating the lens from the image plane , we can in principle acquire the focused image on the image plane ( coronal plane ) almost instantaneously with a two - dimensional sensor array . when a near - infrared laser illuminates the tissue with very short high energy pulses of nanosecond duration , it generates pa waves within the tissue if any absorbers are present . an acoustic lens will collect the waves generated in the object plane and focus them onto an ultrasound detector which is placed in the image plane [ figure 1 ] . an appropriately designed lens will focus the wave front from a point source in the object plane to a point in the image plane . the object plane here corresponds to a coronal plane in the tissue unlike sagittal or transverse planes imaged by a conventional ultrasound imaging system . image plane is defined by time delay measured with respect to time t=0 when the laser illuminates the tissue . in our experience , the difference in travel time from different points in the object plane to their corresponding focus points in the image plane is usually less than the pulse duration of the pa signal . therefore , the entire image plane can be assumed to be in focus and is said to be the conjugate image plane corresponding to the object plane defined by the delay time . more details about the lens can be found under the pa image formation section . object plane lies within the tissue and an ultrasound detector placed in the image plane will collect focused pa signals . a detector array located in the image plane can capture this energy as a focused image that represents a thin coronal slice in the object . images of these coronal planes are referred to as c - scan images and represent laser absorption by different regions of the tissue that are intersected by that plane , much like ct scan images . the photoacoustically generated ultrasound waves travel with finite speed ( ~1500 m / s ) in soft tissue . therefore , a pragmatic way to capture focused signal from a predetermined coronal plane is to time gate it when it is detected in the image plane . the assumption here is that all waves from the predetermined coronal plane arrive at the image plane at the same time . as a first step toward designing an in vivo pa imaging system , we have developed a prototype pa in vitro imaging system to detect any malignancies present in an excised tissue as illustrated in figure 2 . the system design was optimized to generate focused coronal plane ( c - scan ) images using acoustic lens technology . in principle , c - scan images are nothing but two - dimensional ( 2d ) images produced by spatially sampling the pa signal amplitude at a fixed time while the ultrasound sensor is raster - scanned over some surface . the in vitro prototype system comprise of the following components : a fiber optic cable for laser delivery , a sample holder to hold the excised tissue , a pa camera that is water - sealed with acoustic lens , and the ultrasound detector . a near infrared laser beam is delivered from the bottom of the tissue sample holder , which can hold a tissue up to 4 cm thick . the laser beam has a diameter of 5 mm with a repetition rate of 10 hz and pulse duration of 10 ns and is delivered onto the tissue via a fiber optic cable . at the sample surface , laser energy intensity is ~ 20 mj / cm , which is well below laser safety limits . we have designed the front - end and back - end electronics to acquire 16-channel signals simultaneously at each laser firing ; details of which are given in the subsequent sections . the entire tissue is scanned by the laser beam and the camera in tandem using linear actuators . typical time taken for imaging a 4 4 cm area of an excised specimen with this system is about 7 min , the delay being mainly due to the hardware limitation posed by the linear actuator speed and stabilization time . once the pa images are acquired , the pa camera head along with the laser delivery system is replaced by a portable ultrasound array head that captures ultrasound images that can be correlated and registered with the pa images . the ultrasound transducer array has 16 elements , each of size 0.5 mm 1.0 mm , pitch 0.7 mm , and the center frequency of 5 mhz with 60% bandwidth . the sensor element size was chosen such that the reception angular profile of the edge elements , subtended at the lens , was within 20% of what was for the center element . the pitch was chosen to minimize any aliasing due to spatial sampling of the image plane . the acoustic lens design was accomplished using oslo ( lambda research corporation , littleton , ma , usa ) ray tracing software simulation program and optimized for wavelength of 0.3 mm , corresponding to 5 mhz . the lens has a focal length ( f ) of 39.8 mm and a diameter of 32 mm corresponding to an active f - number of 1.2 . the lens design was optimized to achieve a magnification of 1 when both the lens - to - object plane and the lens - to - image plane distances are twice the focal length of the lens . hence , in our in vitro prototype , the transducer array was fixed at 79.6 ( 2f ) mm from the lens in the image plane and the ultrasound waves generated from any absorbers in the object plane that are 79.6 mm from the lens will be in sharp focus and are imaged with a magnification of 1 . the lens has a depth of field of approximately 5 mm on either side of the focal plane . the ultrasound sensor array located in the image plane acquires focused pa signals , much like optical image formation . to perform c - scan imaging , the pa camera is mounted on a holder that is controlled by two linear actuators ( zaber technologies inc . , vancouver , british columbia , canada ) and a raster scan is performed in the image plane to scan a 2d area by collecting pa signals at each pixel location . the pa signals acquired over all 16 channels at each linear actuator location are fed simultaneously to the custom - built back end electronics module that consists of a variable gain amplifier and a digitizer to generate pa b - scan images followed by an ultrasound scan . once the scanning is completed with both pa and ultrasound systems , the acquired pa images are fused with ultrasound images to illustrate anatomical as well as physiological information in a single image . a 16-channel simultaneous data acquisition ( daq ) system was custom - designed to be used with the pa imaging system whose layout is shown in figure 3 . the entire electronics can be categorized into four modules : ( 1 ) amplification of a raw pa signal ; ( 2 ) sampling / digitization of an amplified pa signal ; ( 3 ) dual axis stepper motor movement for c - scan imaging ; and ( 4 ) a host computer with labview ( national instruments , austin , tx , usa ) controlling the first three modules and to display real - time b - scan images . pa signals acquired by the linear array transducer are tapped using a 16-channel custom - fabricated distribution board and are amplified using two 8-channel vca8500evm ( texas instruments , dallas , tx , usa ) variable gain amplifiers . the variable gain amplifiers ( vgas ) have a maximum amplification gain of 50 db ( a pre - amplification gain of 20 db + selectable post gain amplification of 20 , 25 , 27 , or 30 db ) per channel and provide the user a selection of 10 mhz or 15 mhz low pass filtering . all the 16 channels of the two vgas are simultaneously controlled by the computer using a graphical user interface ( gui ) and hence the gain can be changed on - the - fly as well . the 16-channel amplified pa signal data is then simultaneously fed to two 8-channel pxi-5105 ( national instruments , austin , tx , usa ) digitizer boards , which are housed in an external chassis , at each laser firing . a digital trigger is provided by the laser to chassis , during each laser firing , for synchronization . the pxi-5105 digitizer board has 12-bit resolution and 60 mhz sampling rate with a 16 mb onboard memory buffer which allows storing the digitized pa signal samples onboard before transfer to the host computer using mxi interface . as mentioned earlier , the total acquisition time is largely limited by the hardware specifications of the actuators ; their maximum speed being only 4 mm / s , stabilization time of at least 250 ms , and a resolution of 0.1 m . the actuators are moved by 1 mm increments along the y - axis to cover 40 mm and by 11.2 mm increments ( 16 0.7 mm ) along the x - axis direction to cover 44.8 mm to scan an area of 4 cm 4 cm . the entire scanning mechanism including daq and linear actuator movement is controlled by the host computer using labview . the acquired pa b - scan images are transmitted to the host computer and processed further in matlab environment to reconstruct c - scan images and to achieve pa / ultrasound image fusion . in vitro experiments are conducted on excised tissue samples with this system and the results obtained are presented in the following section . with the in vitro prototype system , we have performed scanning of ( 1 ) lamb kidney with a 0.7 mm lead pencil embedded 1 cm deep inside and ( 2 ) fresh lamb kidney with no external objects . since the excised tissue is deprived of any blood within , a lead pencil was inserted into the excised kidney since the pa signal characteristics of lead are known to us by first - hand experience . once the pa images are acquired by the in vitro pa imaging system at a 1064 nm laser wavelength , a 40 mhz ultrasound scanner was used to acquire ultrasound images . figure 4a shows the photograph of a 2 cm thick lamb kidney with a lead pencil of diameter 0.7 mm embedded approximately 1 cm below the top surface of the kidney . figures 4b to 4h show the pa c - scan slices in depths varying from the anterior section to the posterior section of the kidney . each pa c - scan slice is 0.3 mm in thickness . the lead pencil is approximately 3 mm away from the lens - focal plane and yet it is clearly reproduced in these pa c - scan images with high resolution and a magnification of 1 . figure 4i shows the ultrasound image of the lamb kidney shown in figure 4a ; figure 4j shows the cumulative pa image obtained by adding images shown in figures 4b to 4h . from figure 4i , it can be noted that the area covered in the ultrasound image is only 1 cm 1 cm , whereas the area covered in the pa image shown in figure 4j is 4 cm 4 cm . so , in order to achieve image co - registration , a portion of the pa image shown in the square box that covers only 1 cm 1 cm area is cropped and then fused with the ultrasound image shown in figure 4i to obtain an image as shown in figure 4k . ( a ) a photograph of the lamb kidney with a lead pencil ( arrow ) embedded within it . ( b ) ( h ) the 0.3 mm thick c - scan slice traversing from the anterior to posterior plane of the lamb kidney . ( i ) sonogram of the lamb kidney with embedded lead pencil acquired with a 40 mhz probe . ( j ) pa image of lead pencil embedded within the lamb kidney by adding ( b)-(h ) . ( k ) fused ultrasound and pa image by combining ( i ) with a portion of pa image in ( j ) shown in a square box . the second experiment was performed to detect any intrinsic pa signal from the kidney without adding any external absorbing objects to evaluate the performance of our system in detecting in vivo abnormalities . figures 5b and 5c show two pa coronal images at a distance of 1.5 and 1.8 mm from the anterior surface of the kidney specimen . the bright spots in figures 5b and 5c are approximately coming from the red - colored square area indicated on the anterior surface of the kidney in figure 5a . ( b ) and ( c ) the pa images of the kidney obtained in the coronal plane from the anterior to posterior direction at an interval of 0.3 mm . the bright spots in the yellow - colored boxes of ( b ) , and ( c ) the pa signal obtained from the area within the red boxes as shown in figure ( a ) we have noticed that the pa signals from the tissue are typically 10 to 100 times smaller compared to the signal from a lead pencil . nevertheless , our system was capable of displaying pa images with low signal strength as well . this experiment illustrates the strength of pa imaging to perform in vivo imaging . to improve the strength of this intrinsic pa signal further so as to display the images with a higher contrast , an additional 20 db amplification of pa signal from the tissue might be needed . we believe that pa phenomenon has great promise for medical imaging in the area of early detection of cancer , guiding biopsy , imaging with targeted nano - particles , application of spectroscopy , and blood oxygen level determination , just to name a few . it is a safe and non - invasive or at worst , minimally invasive modality . we believe the trend is set for the development of more clinical pa imaging systems and more clinical trials are required that will be able to demonstrate the potential of pa phenomenon . we have presented in this paper for the first time an innovative acoustic lens - based focusing of a pa signal and a c - scan image generation concept . using this concept , we have described our experience in designing and fabricating a prototype pa imaging system for imaging an excised tissue . our current imaging system has a resolution of 0.7 mm in the coronal plane and can generate c - scan slices that are 0.1 mm apart . the advantage of our technology is that the need for software based image reconstruction is eliminated , it can image coronal planes along with sagittal or transverse planes and has better signal to noise ratio . implementation of this technology into an in vivo probe is currently in progress at our research facility .

in today 's world , technology is advancing at an exponential rate and medical imaging is no exception . during the last hundred years , the field of medical imaging has seen a tremendous technological growth with the invention of imaging modalities including but not limited to x - ray , ultrasound , computed tomography , magnetic resonance imaging , positron emission tomography , and single - photon emission computed tomography . these tools have led to better diagnosis and improved patient care . however , each of these modalities has its advantages as well as disadvantages and none of them can reveal all the information a physician would like to have . in the last decade , a new diagnostic technology called photoacoustic imaging has evolved which is moving rapidly from the research phase to the clinical trial phase . this article outlines the basics of photoacoustic imaging and describes our hands - on experience in developing a comprehensive photoacoustic imaging system to detect tissue abnormalities .

INTRODUCTION C-scan concept and acoustic lens-based photoacoustic imaging Photoacoustic imaging prototype system for Photoacoustic image formation Photoacoustic data acquisition and image display RESULTS AND DISCUSSION CONCLUSION

for example , x - ray and computed tomography depict the attenuation of x - rays , ultrasound depicts one of the mechanical properties ( reflectivity or acoustic impedance difference ) of the tissue , and positron emission tomography depicts the concentration of injected radio nucleotide in the body . as the name suggests , the pa effect is a combination of both light and sound . when a laser pulse of short duration , approximately a few nanoseconds ( ns ) , interacts with the tissue , it results in localized heating of the tissue followed by rapid thermal expansion , generating thermo - elastic stress waves . pa imaging is neither purely optical nor purely acoustical in nature , but a combination of the two . pa imaging has better penetration depths because the incident light has to propagate in only one direction into the tissue and the ultrasound waves do not attenuate as much in the tissue as backscattered light . a good resolution in pa imaging results from the fact that the ultrasound can be focused easily and speed of propagation does not deviate much ( for instance , it is ~ 1500 m / s in soft tissue ) so that the wave arrival time can provide information about the depth . the tissue and its major constituents ( water , oxy and de - oxy hemoglobin in blood , lipid , fat , melanin , collagen , etc ) have widely varying absorption spectra in the near infrared ( nir ) region that not only provide abundant contrast features , but also open the door for pa imaging based spectroscopy , non - invasive tissue characterization and functional imaging . however , it is important to mention two vital characteristics of photoacoustically generated ultrasound waves that differentiate them from echo - based ultrasound waves . first , the frequency of echo - based ultrasound signal in a medical ultrasound imaging system lies within the bandwidth of the transmitted signal , whereas the frequency of the pa signal depends on the size of the biological absorber and the duration of the illuminating laser pulse . second , unlike conventional ultrasound where the signal strength or image intensity depends on the echogenicity ( ultrasound reflectivity ) of the echo - generating source in the medium , the pa signal strength depends on the optical absorption coefficient of the absorber in the medium and the difference in optical absorption of the incident light dictates the image contrast , as mentioned earlier . also , since angiogenesis is the fundamental step in the transition of a tumor from dormant to malignant state , a tumor that has blood vessels proliferated into it , interrogated at appropriate wavelengths will produce a stronger pa signal compared to the surrounding normal tissue. [35 ] the major part of a tissue is made up of water that has a lower absorption coefficient in the near infrared ( nir ) region , especially in the wavelength range of 650 - 950 nanometers ( nm ) , than blood and other tissue constituents . if the tissue is illuminated at these wavelengths , deeper penetration of light can be achieved and the tissue can be imaged with better contrast and the tissue characteristics such as oxy and deoxy - hemoglobin concentrations , oxygen saturation , and other parameters , can be extensively studied since pa imaging can yield images from depths of up to a few centimeters , it provides a map of the optical absorption by tissue constituents at the interrogating laser wavelength along with the depth or location information . therefore , the pa signal is more diagnostic in nature than the conventional ultrasound signal . as the imaging depth increases , the lateral resolution in the b - scan images gets worse and to correct for it , electronic focusing techniques are implemented in the software . the advantages of the acoustic lens focusing over electronic focusing are : ( 1 ) it is faster , ( 2 ) it uses zero electrical power , ( 3 ) it is inexpensive to design and implement , and ( 4 ) there are no errors in the reconstructed image due to spatial and temporal sampling of the signal . however , our use of lens - based focusing is different from what has been done in the past in clinical ultrasound imaging . earlier , the focusing element was typically placed on the single element transducer , while the transducer was scanned in sectors to acquire the ultrasound image . in our version , by separating the lens from the image plane , we can in principle acquire the focused image on the image plane ( coronal plane ) almost instantaneously with a two - dimensional sensor array . an acoustic lens will collect the waves generated in the object plane and focus them onto an ultrasound detector which is placed in the image plane [ figure 1 ] . an appropriately designed lens will focus the wave front from a point source in the object plane to a point in the image plane . the object plane here corresponds to a coronal plane in the tissue unlike sagittal or transverse planes imaged by a conventional ultrasound imaging system . in our experience , the difference in travel time from different points in the object plane to their corresponding focus points in the image plane is usually less than the pulse duration of the pa signal . therefore , the entire image plane can be assumed to be in focus and is said to be the conjugate image plane corresponding to the object plane defined by the delay time . object plane lies within the tissue and an ultrasound detector placed in the image plane will collect focused pa signals . a detector array located in the image plane can capture this energy as a focused image that represents a thin coronal slice in the object . images of these coronal planes are referred to as c - scan images and represent laser absorption by different regions of the tissue that are intersected by that plane , much like ct scan images . therefore , a pragmatic way to capture focused signal from a predetermined coronal plane the assumption here is that all waves from the predetermined coronal plane arrive at the image plane at the same time . as a first step toward designing an in vivo pa imaging system , we have developed a prototype pa in vitro imaging system to detect any malignancies present in an excised tissue as illustrated in figure 2 . the in vitro prototype system comprise of the following components : a fiber optic cable for laser delivery , a sample holder to hold the excised tissue , a pa camera that is water - sealed with acoustic lens , and the ultrasound detector . a near infrared laser beam is delivered from the bottom of the tissue sample holder , which can hold a tissue up to 4 cm thick . at the sample surface , laser energy intensity is ~ 20 mj / cm , which is well below laser safety limits . we have designed the front - end and back - end electronics to acquire 16-channel signals simultaneously at each laser firing ; details of which are given in the subsequent sections . typical time taken for imaging a 4 4 cm area of an excised specimen with this system is about 7 min , the delay being mainly due to the hardware limitation posed by the linear actuator speed and stabilization time . once the pa images are acquired , the pa camera head along with the laser delivery system is replaced by a portable ultrasound array head that captures ultrasound images that can be correlated and registered with the pa images . the ultrasound transducer array has 16 elements , each of size 0.5 mm 1.0 mm , pitch 0.7 mm , and the center frequency of 5 mhz with 60% bandwidth . hence , in our in vitro prototype , the transducer array was fixed at 79.6 ( 2f ) mm from the lens in the image plane and the ultrasound waves generated from any absorbers in the object plane that are 79.6 mm from the lens will be in sharp focus and are imaged with a magnification of 1 . the lens has a depth of field of approximately 5 mm on either side of the focal plane . the ultrasound sensor array located in the image plane acquires focused pa signals , much like optical image formation . to perform c - scan imaging , the pa camera is mounted on a holder that is controlled by two linear actuators ( zaber technologies inc . , vancouver , british columbia , canada ) and a raster scan is performed in the image plane to scan a 2d area by collecting pa signals at each pixel location . the pa signals acquired over all 16 channels at each linear actuator location are fed simultaneously to the custom - built back end electronics module that consists of a variable gain amplifier and a digitizer to generate pa b - scan images followed by an ultrasound scan . once the scanning is completed with both pa and ultrasound systems , the acquired pa images are fused with ultrasound images to illustrate anatomical as well as physiological information in a single image . a 16-channel simultaneous data acquisition ( daq ) system was custom - designed to be used with the pa imaging system whose layout is shown in figure 3 . all the 16 channels of the two vgas are simultaneously controlled by the computer using a graphical user interface ( gui ) and hence the gain can be changed on - the - fly as well . the pxi-5105 digitizer board has 12-bit resolution and 60 mhz sampling rate with a 16 mb onboard memory buffer which allows storing the digitized pa signal samples onboard before transfer to the host computer using mxi interface . as mentioned earlier , the total acquisition time is largely limited by the hardware specifications of the actuators ; their maximum speed being only 4 mm / s , stabilization time of at least 250 ms , and a resolution of 0.1 m . the actuators are moved by 1 mm increments along the y - axis to cover 40 mm and by 11.2 mm increments ( 16 0.7 mm ) along the x - axis direction to cover 44.8 mm to scan an area of 4 cm 4 cm . the acquired pa b - scan images are transmitted to the host computer and processed further in matlab environment to reconstruct c - scan images and to achieve pa / ultrasound image fusion . in vitro experiments are conducted on excised tissue samples with this system and the results obtained are presented in the following section . as the imaging depth increases , the lateral resolution in the b - scan images gets worse and to correct for it , electronic focusing techniques are implemented in the software . the advantages of the acoustic lens focusing over electronic focusing are : ( 1 ) it is faster , ( 2 ) it uses zero electrical power , ( 3 ) it is inexpensive to design and implement , and ( 4 ) there are no errors in the reconstructed image due to spatial and temporal sampling of the signal . however , our use of lens - based focusing is different from what has been done in the past in clinical ultrasound imaging . earlier , the focusing element was typically placed on the single element transducer , while the transducer was scanned in sectors to acquire the ultrasound image . in our version , by separating the lens from the image plane , we can in principle acquire the focused image on the image plane ( coronal plane ) almost instantaneously with a two - dimensional sensor array . an acoustic lens will collect the waves generated in the object plane and focus them onto an ultrasound detector which is placed in the image plane [ figure 1 ] . an appropriately designed lens will focus the wave front from a point source in the object plane to a point in the image plane . the object plane here corresponds to a coronal plane in the tissue unlike sagittal or transverse planes imaged by a conventional ultrasound imaging system . in our experience , the difference in travel time from different points in the object plane to their corresponding focus points in the image plane is usually less than the pulse duration of the pa signal . therefore , the entire image plane can be assumed to be in focus and is said to be the conjugate image plane corresponding to the object plane defined by the delay time . object plane lies within the tissue and an ultrasound detector placed in the image plane will collect focused pa signals . a detector array located in the image plane can capture this energy as a focused image that represents a thin coronal slice in the object . images of these coronal planes are referred to as c - scan images and represent laser absorption by different regions of the tissue that are intersected by that plane , much like ct scan images . therefore , a pragmatic way to capture focused signal from a predetermined coronal plane is to time gate it when it is detected in the image plane . the assumption here is that all waves from the predetermined coronal plane arrive at the image plane at the same time . as a first step toward designing an in vivo pa imaging system , we have developed a prototype pa in vitro imaging system to detect any malignancies present in an excised tissue as illustrated in figure 2 . the in vitro prototype system comprise of the following components : a fiber optic cable for laser delivery , a sample holder to hold the excised tissue , a pa camera that is water - sealed with acoustic lens , and the ultrasound detector . a near infrared laser beam is delivered from the bottom of the tissue sample holder , which can hold a tissue up to 4 cm thick . at the sample surface , laser energy intensity is ~ 20 mj / cm , which is well below laser safety limits . we have designed the front - end and back - end electronics to acquire 16-channel signals simultaneously at each laser firing ; details of which are given in the subsequent sections . typical time taken for imaging a 4 4 cm area of an excised specimen with this system is about 7 min , the delay being mainly due to the hardware limitation posed by the linear actuator speed and stabilization time . once the pa images are acquired , the pa camera head along with the laser delivery system is replaced by a portable ultrasound array head that captures ultrasound images that can be correlated and registered with the pa images . the ultrasound transducer array has 16 elements , each of size 0.5 mm 1.0 mm , pitch 0.7 mm , and the center frequency of 5 mhz with 60% bandwidth . the acoustic lens design was accomplished using oslo ( lambda research corporation , littleton , ma , usa ) ray tracing software simulation program and optimized for wavelength of 0.3 mm , corresponding to 5 mhz . hence , in our in vitro prototype , the transducer array was fixed at 79.6 ( 2f ) mm from the lens in the image plane and the ultrasound waves generated from any absorbers in the object plane that are 79.6 mm from the lens will be in sharp focus and are imaged with a magnification of 1 . the lens has a depth of field of approximately 5 mm on either side of the focal plane . the ultrasound sensor array located in the image plane acquires focused pa signals , much like optical image formation . to perform c - scan imaging , the pa camera is mounted on a holder that is controlled by two linear actuators ( zaber technologies inc . , vancouver , british columbia , canada ) and a raster scan is performed in the image plane to scan a 2d area by collecting pa signals at each pixel location . the pa signals acquired over all 16 channels at each linear actuator location are fed simultaneously to the custom - built back end electronics module that consists of a variable gain amplifier and a digitizer to generate pa b - scan images followed by an ultrasound scan . once the scanning is completed with both pa and ultrasound systems , the acquired pa images are fused with ultrasound images to illustrate anatomical as well as physiological information in a single image . a 16-channel simultaneous data acquisition ( daq ) system was custom - designed to be used with the pa imaging system whose layout is shown in figure 3 . all the 16 channels of the two vgas are simultaneously controlled by the computer using a graphical user interface ( gui ) and hence the gain can be changed on - the - fly as well . the pxi-5105 digitizer board has 12-bit resolution and 60 mhz sampling rate with a 16 mb onboard memory buffer which allows storing the digitized pa signal samples onboard before transfer to the host computer using mxi interface . as mentioned earlier , the total acquisition time is largely limited by the hardware specifications of the actuators ; their maximum speed being only 4 mm / s , stabilization time of at least 250 ms , and a resolution of 0.1 m . the actuators are moved by 1 mm increments along the y - axis to cover 40 mm and by 11.2 mm increments ( 16 0.7 mm ) along the x - axis direction to cover 44.8 mm to scan an area of 4 cm 4 cm . the acquired pa b - scan images are transmitted to the host computer and processed further in matlab environment to reconstruct c - scan images and to achieve pa / ultrasound image fusion . in vitro experiments are conducted on excised tissue samples with this system and the results obtained are presented in the following section . with the in vitro prototype system , we have performed scanning of ( 1 ) lamb kidney with a 0.7 mm lead pencil embedded 1 cm deep inside and ( 2 ) fresh lamb kidney with no external objects . since the excised tissue is deprived of any blood within , a lead pencil was inserted into the excised kidney since the pa signal characteristics of lead are known to us by first - hand experience . once the pa images are acquired by the in vitro pa imaging system at a 1064 nm laser wavelength , a 40 mhz ultrasound scanner was used to acquire ultrasound images . figures 4b to 4h show the pa c - scan slices in depths varying from the anterior section to the posterior section of the kidney . the lead pencil is approximately 3 mm away from the lens - focal plane and yet it is clearly reproduced in these pa c - scan images with high resolution and a magnification of 1 . from figure 4i , it can be noted that the area covered in the ultrasound image is only 1 cm 1 cm , whereas the area covered in the pa image shown in figure 4j is 4 cm 4 cm . so , in order to achieve image co - registration , a portion of the pa image shown in the square box that covers only 1 cm 1 cm area is cropped and then fused with the ultrasound image shown in figure 4i to obtain an image as shown in figure 4k . ( b ) ( h ) the 0.3 mm thick c - scan slice traversing from the anterior to posterior plane of the lamb kidney . the second experiment was performed to detect any intrinsic pa signal from the kidney without adding any external absorbing objects to evaluate the performance of our system in detecting in vivo abnormalities . figures 5b and 5c show two pa coronal images at a distance of 1.5 and 1.8 mm from the anterior surface of the kidney specimen . the bright spots in figures 5b and 5c are approximately coming from the red - colored square area indicated on the anterior surface of the kidney in figure 5a . ( b ) and ( c ) the pa images of the kidney obtained in the coronal plane from the anterior to posterior direction at an interval of 0.3 mm . the bright spots in the yellow - colored boxes of ( b ) , and ( c ) the pa signal obtained from the area within the red boxes as shown in figure ( a ) we have noticed that the pa signals from the tissue are typically 10 to 100 times smaller compared to the signal from a lead pencil . nevertheless , our system was capable of displaying pa images with low signal strength as well . to improve the strength of this intrinsic pa signal further so as to display the images with a higher contrast , an additional 20 db amplification of pa signal from the tissue might be needed . we believe that pa phenomenon has great promise for medical imaging in the area of early detection of cancer , guiding biopsy , imaging with targeted nano - particles , application of spectroscopy , and blood oxygen level determination , just to name a few . using this concept , we have described our experience in designing and fabricating a prototype pa imaging system for imaging an excised tissue . our current imaging system has a resolution of 0.7 mm in the coronal plane and can generate c - scan slices that are 0.1 mm apart . the advantage of our technology is that the need for software based image reconstruction is eliminated , it can image coronal planes along with sagittal or transverse planes and has better signal to noise ratio .

glioblastoma ( gbm ) is the most frequent and aggressive malignant primary brain tumor with only about 12% of patients surviving beyond 36 months ( long - term survivors ) . according to the latest central brain tumor registry of the usa statistical report the incidence of gbm increases with age and peaks at 7584 years ( 14.93/100,000 ) , being more common in males ( 3.97/100,000 ) . the current treatment strategy for gbm patients combines maximal surgical resection , followed by radiotherapy ( rt ) with concomitant and adjuvant temozolomide ( tmz ) . complete surgical resection is virtually impossible due to the infiltrative nature of these tumors , yet gross total resection is still a positive prognostic marker . concurrent adjuvant rt in combination with tmz represents the standard of care for patients with newly diagnosed gbm , but still < 5% of patients survive for longer than 5 years after diagnosis . decades of molecular studies have identified key genetic abnormalities in human gbms , including the following : ( 1 ) dysregulation of growth factor signaling pathways via amplification and mutational activation of receptor tyrosine kinase ( rtk ) genes ; ( 2 ) activation of the phosphatidylinositol-3-oh kinase ( pi3k ) pathway ; and ( 3 ) inactivation of the p53 and retinoblastoma tumor suppressor pathways . during recent years , large - scale research efforts spearheaded by the cancer genome atlas ( tcga ) and chinese glioma genome atlas ( cgga ) have made rapid advances in understanding gbm tumor genetics . the discovery of new genetic alterations and their mapping against clinical outcome will trigger an avalanche of novel perceptions of the genomic and epigenomic landscape , biological subgroups and putative cells of origin of gbm , which has encouraged hopes for more effective treatment strategies in the near future . this review mainly discusses the recent advances in gbm molecular research and current trends in personalized therapy of this disease . malignant gliomas are histologically heterogeneous and invasive tumors that are derived from glia . the world health organization ( who ) classification system groups gliomas into 4 histological grades defined by increasing degrees of undifferentiation , anaplasia , and aggressiveness . malignant gliomas , the most common form of gliomas , consist of who grade iv tumors ( gbm ) and grade iii tumors ( anaplastic astrocytoma , oligodendroglioma , and oligoastrocytoma ) . gbms account for approximately 6070% of malignant gliomas and is characterized histologically by considerable cellularity and mitotic activity , microvascular proliferation , necrosis and they are also recalcitrant to radio / chemotherapy . primary ( de novo , approximately 95% of cases ) gbms manifest rapidly , without evidence of less malignant precursor lesions , after a short clinical history . secondary gbms ( approximately 5% of cases ) develop more slowly by progression from low - grade diffuse astrocytoma and anaplastic astrocytoma . gbm and other malignant gliomas are highly invasive , infiltrating surrounding brain parenchyma , yet they are typically confined to the central nervous system ( cns ) and do not metastasize . unfortunately , who morphological classification is based on subjective criteria , lacks reproducibility , and remains imperfect in its ability to predict individual outcomes . the first genome - wide exon sequencing effort for glioma identified heterozygous hotspot mutations at codon 132 ( most commonly r132h ) in isocitrate dehydrogenase 1 ( idh1 ) in 12% of gbm . these homologous enzymes decarboxylate isocitrate to -ketoglutarate ( kg ) , and this neomorphic mutation renders the idh enzyme to reduce kg into 2-hydroxyglutarate in the nicotinamide adenine dinucleotide phosphate - dependent manner . mutant idh1 or idh2 are correlated with increased histone methylation , causing epigenetic alterations in both dna and histones , altering gene expression and promoting oncogenic transformation . nowadays , mutations in idh1 are commonly established as a hallmark molecular feature of secondary gbm ( ~70% of secondary gbm , compared with 520% in primary gbm ) who have predominant localization of gbm in the frontal and temporal lobes . since primary gbm is a clinically defined entity and the presence of idh1/2 mutations has been shown to be inversely related to or even mutually exclusive of epidermal growth factor receptor ( egfr ) and phosphatase and tensin homolog ( pten ) abnormalities , which are hallmarks of primary gbm , idh - mutated gbm lesions may represent genetically secondary gbm tumors . moreover , the idh mutation status is stable during the progression of lower - grade gliomas to secondary gbms . mutations in the idh genes are thought to cause glioma - cpg island methylator phenotype ( g - cimp ) within the proneural gbm subgroup . idh mutations seem to require cooperating mutations in tp53 and atrx , and they are less frequently detected in primary gbms . the o(6)-methylguanine - dna methyltransferase ( mgmt ) is a dna repair enzyme , preventing errors during dna replication . abnormal methylation of the mgmt promoter caused its silencing , a reduction of the mgmt enzyme expression , and subsequently to less repair activity of dna damage , including that induced by tmz . mgmt promoter methylation in gbm is a prognostic and predictive biomarker indicating a response to chemoradiation . the trial of the effect of tmz on newly diagnosed gbm showed that mgmt promoter methylation was an independent favorable prognostic factor . patients with tumors with methylated mgmt promoter had a survival benefit when treated with tmz and rt , compared to those who received rt only factor . a recent report from the neuro - oncology working group ( noa ) of the german cancer society confirmed a predictive value of mgmt methylation for benefit from chemotherapy in patients with a wild - type idh , independent of tumor grade . recently , novel somatic mutations in the promoter region of telomerase reverse transcriptase ( tert ) have been identified in malignant melanomas , as well as being associated with increased telomerase expression and activity . the tumors derived from cell populations with low self - renewal capacity generally depend on alterations that keep telomerase activity , while epigenetic alteration maintains telomerase activity in tumor types arisen from self - renewing stem cells . the two most common mutations are located at c228 t and c250 t , with identical hotspots also found in gliomas . the highest incidence was identified among most tumors harboring 1p/19q co - deletion and idh mutations ( 98% ) , as well as idh wild - type ( idh wt ) tumors with egfr amplification ( 92% ) . the frequency of tert mutations is relatively low in diffuse and anaplastic astrocytomas ( 19% and 25% , respectively ) . in the study by killela et al . , patients with tert promoter mutations alone ( i.e. , no idh mutation ) had the poorest overall survival ( os ) ( median 11.3 months ) , patients with tumors without tert or idh1/2 mutations had a slightly better survival ( median 16.6 months ) , while patients with only idh mutant gbm had the best survival ( median 42.3 months ) . although another study with 358 patients found no significant difference in os between tert mutant and tert wild - type ( idh wt ) gbm , the role of tert promoter mutations may provide a tool to identify non - idh mutant gbms . the range of high - amplitude focal copy - number aberrations in adult gbm highlights a key role of egfr amplifications ( 43% of cases ) which co - occurred with egfr intragenic deletions and/or point mutations . egfr mutations were accompanied by regional dna amplification in the majority of cases , leading to a wide range of mutation allelic frequencies . the prominent intragenic deletions in gbm target parts of the gene encoding either the extracellular domain of egfr ( exons 27 , the deletion of which forms egfr variant iii ) or the carboxyl terminus , and these deletions are always correlated with amplification and co - expression of the wild - type egfr . egfr was recently shown to be activated by recurrent translocations in 7% of gbm samples : it was most frequently fused in - frame to septin 14 or phosphoserine phosphatase as the 3 gene segment . overall , 57% of gbm showed evidence of mutation , rearrangement , altered splicing , and/or focal amplification of egfr . loss of heterozygosity ( loh ) at chromosome 10q23 occurs at high frequency in a variety of human tumors . pten is a negative regulator of the phosphoinositide 3-kinase pathway , a major signaling pathway that stimulates cellular proliferation in response to growth factor stimulation . pten deletions were more common in gbm , except classical grade ii / iii gliomas . pten deletions were fairly common across all gene expressions subtypes , but absent in idh1 mutant tumors . several studies demonstrated that patients with loss of function mutations of pten generally had shorter survival than patients with pten retention . however , pten loss was not associated with worse os in newly diagnosed gbm patients of the tmz era . in a smaller fraction of primary gbms ( about 3% ) , a fusion of the tyrosine kinase coding region of fibroblast growth factor receptor 1 ( fgfr1 ) to the transforming acidic coiled - coil ( tacc ) coding domain of tacc1 ( or fusion of fgfr3 to tacc3 ) results in constitutive kinase activity . in transcriptome profiling of 272 gliomas from cgga , 67 in - frame fusion transcripts were identified , including three recurrent fusion transcripts : fgfr3-tacc3 , rnf213-slc26a11 , and ptprz1-met ( fusion transcript involving the protein tyrosine phosphatase , receptor - type , z polypeptide 1 gene and the met proto - oncogene , zm ) . exogenous expression of the zm fusion in the u87 mg gbm line enhanced cell migration and invasion . clinically , patients afflicted with zm fusion harboring gbms survived poorly relative to those afflicted with non - zm - harboring . therefore , recurrent fusion events that involve rtk - encoding genes might be a promising therapeutic target and provide a strong rationale for the inclusion of these patients in future stratified clinical trials using different rtk inhibitors . table 1 summarizes all of the above described and other genetic alterations and related altered signaling pathways in primary versus secondary gbm . genetic abnormalities and the major signaling pathways involved in the pathogenesis of gbm idh : isocitrate dehydrogenase ; cdkn2a : cyclin - dependent kinase inhibitor 2a ; pten : phosphatase and tensin homolog ; nf1 : neurofibromatosis 1 ; rb1 : retinoblastoma 1 ; tert : telomerase reverse transcriptase ; ampl . : amplification ; egfr : epidermal growth factor receptor ; pdgfra : platelet - derived growth factor receptor alpha ; fgfr3 : fibroblast growth factor receptor 3 ; tacc3 : transforming acidic coiled - coil 3 ; rtk : receptor tyrosine kinase ; gbm : glioblastoma ; mapk : mitogen - activated protein kinase . the first genome - wide exon sequencing effort for glioma identified heterozygous hotspot mutations at codon 132 ( most commonly r132h ) in isocitrate dehydrogenase 1 ( idh1 ) in 12% of gbm . these homologous enzymes decarboxylate isocitrate to -ketoglutarate ( kg ) , and this neomorphic mutation renders the idh enzyme to reduce kg into 2-hydroxyglutarate in the nicotinamide adenine dinucleotide phosphate - dependent manner . mutant idh1 or idh2 are correlated with increased histone methylation , causing epigenetic alterations in both dna and histones , altering gene expression and promoting oncogenic transformation . nowadays , mutations in idh1 are commonly established as a hallmark molecular feature of secondary gbm ( ~70% of secondary gbm , compared with 520% in primary gbm ) who have predominant localization of gbm in the frontal and temporal lobes . since primary gbm is a clinically defined entity and the presence of idh1/2 mutations has been shown to be inversely related to or even mutually exclusive of epidermal growth factor receptor ( egfr ) and phosphatase and tensin homolog ( pten ) abnormalities , which are hallmarks of primary gbm , idh - mutated gbm lesions may represent genetically secondary gbm tumors . moreover , the idh mutation status is stable during the progression of lower - grade gliomas to secondary gbms . mutations in the idh genes are thought to cause glioma - cpg island methylator phenotype ( g - cimp ) within the proneural gbm subgroup . idh mutations seem to require cooperating mutations in tp53 and atrx , and they are less frequently detected in primary gbms . the o(6)-methylguanine - dna methyltransferase ( mgmt ) is a dna repair enzyme , preventing errors during dna replication . abnormal methylation of the mgmt promoter caused its silencing , a reduction of the mgmt enzyme expression , and subsequently to less repair activity of dna damage , including that induced by tmz . mgmt promoter methylation in gbm is a prognostic and predictive biomarker indicating a response to chemoradiation . the trial of the effect of tmz on newly diagnosed gbm showed that mgmt promoter methylation was an independent favorable prognostic factor . patients with tumors with methylated mgmt promoter had a survival benefit when treated with tmz and rt , compared to those who received rt only factor . a recent report from the neuro - oncology working group ( noa ) of the german cancer society confirmed a predictive value of mgmt methylation for benefit from chemotherapy in patients with a wild - type idh , independent of tumor grade . recently , novel somatic mutations in the promoter region of telomerase reverse transcriptase ( tert ) have been identified in malignant melanomas , as well as being associated with increased telomerase expression and activity . the tumors derived from cell populations with low self - renewal capacity generally depend on alterations that keep telomerase activity , while epigenetic alteration maintains telomerase activity in tumor types arisen from self - renewing stem cells . the two most common mutations are located at c228 t and c250 t , with identical hotspots also found in gliomas . the highest incidence was identified among most tumors harboring 1p/19q co - deletion and idh mutations ( 98% ) , as well as idh wild - type ( idh wt ) tumors with egfr amplification ( 92% ) . the frequency of tert mutations is relatively low in diffuse and anaplastic astrocytomas ( 19% and 25% , respectively ) . in the study by killela et al . , patients with tert promoter mutations alone ( i.e. , no idh mutation ) had the poorest overall survival ( os ) ( median 11.3 months ) , patients with tumors without tert or idh1/2 mutations had a slightly better survival ( median 16.6 months ) , while patients with only idh mutant gbm had the best survival ( median 42.3 months ) . although another study with 358 patients found no significant difference in os between tert mutant and tert wild - type ( idh wt ) gbm , the role of tert promoter mutations may provide a tool to identify non - idh mutant gbms . the range of high - amplitude focal copy - number aberrations in adult gbm highlights a key role of egfr amplifications ( 43% of cases ) which co - occurred with egfr intragenic deletions and/or point mutations . egfr mutations were accompanied by regional dna amplification in the majority of cases , leading to a wide range of mutation allelic frequencies . the prominent intragenic deletions in gbm target parts of the gene encoding either the extracellular domain of egfr ( exons 27 , the deletion of which forms egfr variant iii ) or the carboxyl terminus , and these deletions are always correlated with amplification and co - expression of the wild - type egfr . egfr was recently shown to be activated by recurrent translocations in 7% of gbm samples : it was most frequently fused in - frame to septin 14 or phosphoserine phosphatase as the 3 gene segment . overall , 57% of gbm showed evidence of mutation , rearrangement , altered splicing , and/or focal amplification of egfr . loss of heterozygosity ( loh ) at chromosome 10q23 occurs at high frequency in a variety of human tumors . pten is a negative regulator of the phosphoinositide 3-kinase pathway , a major signaling pathway that stimulates cellular proliferation in response to growth factor stimulation . pten deletions were more common in gbm , except classical grade ii / iii gliomas . pten deletions were fairly common across all gene expressions subtypes , but absent in idh1 mutant tumors . several studies demonstrated that patients with loss of function mutations of pten generally had shorter survival than patients with pten retention . however , pten loss was not associated with worse os in newly diagnosed gbm patients of the tmz era . in a smaller fraction of primary gbms ( about 3% ) , a fusion of the tyrosine kinase coding region of fibroblast growth factor receptor 1 ( fgfr1 ) to the transforming acidic coiled - coil ( tacc ) coding domain of tacc1 ( or fusion of fgfr3 to tacc3 ) results in constitutive kinase activity . in transcriptome profiling of 272 gliomas from cgga , 67 in - frame fusion transcripts were identified , including three recurrent fusion transcripts : fgfr3-tacc3 , rnf213-slc26a11 , and ptprz1-met ( fusion transcript involving the protein tyrosine phosphatase , receptor - type , z polypeptide 1 gene and the met proto - oncogene , zm ) . exogenous expression of the zm fusion in the u87 mg gbm line enhanced cell migration and invasion . clinically , patients afflicted with zm fusion harboring gbms survived poorly relative to those afflicted with non - zm - harboring . therefore , recurrent fusion events that involve rtk - encoding genes might be a promising therapeutic target and provide a strong rationale for the inclusion of these patients in future stratified clinical trials using different rtk inhibitors . table 1 summarizes all of the above described and other genetic alterations and related altered signaling pathways in primary versus secondary gbm . genetic abnormalities and the major signaling pathways involved in the pathogenesis of gbm idh : isocitrate dehydrogenase ; cdkn2a : cyclin - dependent kinase inhibitor 2a ; pten : phosphatase and tensin homolog ; nf1 : neurofibromatosis 1 ; rb1 : retinoblastoma 1 ; tert : telomerase reverse transcriptase ; ampl . : amplification ; egfr : epidermal growth factor receptor ; pdgfra : platelet - derived growth factor receptor alpha ; fgfr3 : fibroblast growth factor receptor 3 ; tacc3 : transforming acidic coiled - coil 3 ; rtk : receptor tyrosine kinase ; gbm : glioblastoma ; mapk : mitogen - activated protein kinase . the phenotype of a tumor is the result of the genotype and the influence of the tumor 's environment on the tumor . one would expect that molecular diagnostics will contribute to a better classification of brain tumors [ tables 24 ] . phillips described three subclasses of high - grade gliomas ( termed proneural , mesenchymal , and proliferative ) associated with different outcomes ; specifically , prolonged survival of the proneural subclass . similar classification of gbms was also detected in a larger cohort of mixed gliomas . in 2010 , unsupervised clustering of gene expression data from adult gbm samples from the tcga identified four different molecular subtypes : proneural , neural , classical , and mesenchymal . proneural gbms were subdivided into g - cimp - positive and g - cimp - negative gbm subsets on the basis of characteristic dna methylation patterns that strongly correspond with idh1 mutation status . another later study , which compared dna methylation patterns across both pediatric and adult patients with gbm , found a similar clustering in tumors from adult patients and further identified three more distinct clusters that predominantly consisted of children and adolescents . recently , liu et al . profiled the genetic features of multifocal gbm and found that m - gbms had no idh1 , atrx , or pdgfra mutations , significantly associated with the mesenchymal subtype . they also identified the cyb5r2 gene to be hypomethylated and overexpressed in m - gbms . phillips classifications of gbm based on transcription profiling egfr : epidermal growth factor receptor ; pten : phosphatase and tensin homolog ; gbm : glioblastoma . tcga classifications of gbm based on transcription and methylation profiling tcga : the cancer genome atlas ; gbm : glioblastoma ; g - cimp : glioma - cpg island methylator phenotype ; ampl . : amplification ; idh : isocitrate dehydrogenase ; nf1 : neurofibromatosis 1 ; rb1 : retinoblastoma 1 . dkfz classifications of gbm based on methylation profiling dkfz : deutsches krebsforschungszentrum ( german cancer research center ) ; gbm : glioblastoma ; rtk : receptor tyrosine kinase ; pdgfra : platelet - derived growth factor receptor alpha ; egfr : epidermal growth factor receptor ; idh : isocitrate dehydrogenase ; ampl . : the recent reports published on the nature genetics and nejm were comprehensively analyzed by whole - exome sequencing and/or targeted deep sequencing as well as array comparative genomic hybridization . in the nature genetics article , grade ii and iii gliomas were divided into and exhausted by the genetically well - defined type i iii subtypes . type iii tumors represented the idh wild - type grade ii and iii tumors in the current cohort , showing an os rate more similar to that of gbm . similarly , the report from tcga research network independently identified similar groups , using unsupervised clustering analyses of dna mutation , rna expression , dna copy number , and dna methylation data . the integration of genome - wide data from multiple platforms delineated three molecular classes of lower - grade gliomas ( grade ii / iii gliomas ) that were more concordant with idh , 1p/19q , and tp53 status than with histologic class . this multi - platform approach yielded three groups similar to those initially described by jiao 's model . the large majority of lower - grade gliomas without an idh mutation had genomic aberrations and clinical behavior strikingly similar to those found in primary gbm . the report from mayo clinic and ucsf defined a priori groups that were based on the presence or absence of tert promoter mutations , idh mutations , and 1p/19q codeletion and found consistent associations between the molecular groups and age at diagnosis , survival , patterns of acquired alterations , and germline variants across the three data sets . the group with only tert mutations has a high prevalence of loss of chromosome 4 and acquired pik3ca or pik3r1 mutations . these tests ( for idh mutations , 1p/19q codeletion , and tert promoter alterations ) can be used to define five principal groups of gliomas with characteristic distributions of age at diagnosis , clinical behavior , acquired genetic alterations , and associated germline variants . over the past decade , insights into the molecular pathology of gliomas have significantly improved both our biological understanding of neoplasms as well as our abilities to diagnose tumors and estimate their prognosis and likelihood of response to specific therapies . to discuss the inclusion of molecular information into the next who classification of cns tumors , a meeting under the sponsorship of the international society of neuropathology ( isn ) has been held in haarlem , the netherlands . according to the isn - haarlem consensus , integrated diagnosis was established through the usage of atrx , idh1-r132h ihc , 1p/19q analyses , and idh sequencing in the diagnosis of diffuse gliomas . rt plus concomitant and adjuvant tmz chemotherapy is the current standard of care for patients with gbm . several clinical trials have displayed that mgmt promoter methylation correlated with prolonged progression - free and os in patients with gbm receiving alkylating drug chemotherapy . in 2012 , two independent randomized trials in elderly patients with gbm assessed rt alone versus tmz chemotherapy alone as an initial treatment . subgroup analyses of both trials showed better outcome for chemotherapy in patients with mgmt promoter methylated tumors but reduced survival in patients with unmethylated tumors . recently , the cgga project delineated that patients with idh wild - type gbm who underwent rt + tmz exhibited significantly longer survival times compared to the patients who were assigned to the rt alone treatment . however , among patients with idh mutation tumors , the survival pattern of patients undergoing rt + tmz or rt was comparable . these results strongly suggest that treatment strategies for elderly patients with gbm should be individualized dependent on idh and mgmt . in addition , due to the high heterogeneity of gbm , each of which may respond differently to one targeted therapy , there has been considerable interest in identifying molecular markers that predict response to a specific molecular targeted therapy . bevacizumab , a monoclonal antibody against vascular endothelial growth factor , being granted approval by the us food and drug administration for treating recurrent gbm in 2009 . however , it does not benefit os in either recurrent gbm or newly diagnosed gbm . the presence of egfr overexpression and egfr mutations in gbm could predict the activity of egfr - targeted drugs in patients with these aberrations . however , these potential treatment approaches still have not been clear with contradictory findings in previous clinical trials . it was demonstrated that a point mutation in idh1r132h , expressed in gliomas and other tumors , is presented on human major histocompatibility complex ( mhc ) class ii and induces a mutation - specific cd4 antitumor t - cell response in patients and a syngeneic tumor model in mhc - humanized mice . conceptually , patients with low - grade and anaplastic gliomas , secondary gbm with a high prevalence of the idh1 ( r132h ) mutation represent a patient population that may particularly benefit from an idh1r132h specific tumor vaccine . these recurrent genetic aberrations occur in a specific context of cellular origin , co - oncogenic hits and are present in distinct patient populations . primary and secondary gbms are distinct disease entities that affect different age groups of patients and develop through distinct genetic aberrations . these differences are important , especially because they may affect sensitivity to radio- and chemo - therapy and should thus be considered in the identification of targets for novel therapeutic approaches . the biological distinction of gbm subgroups should therefore guide the design of future clinical trials .

objective : to summary the recent advances in molecular research of glioblastoma ( gbm ) and current trends in personalized therapy of this disease.data sources : data cited in this review were obtained mainly from pubmed in english up to 2015 , with keywords molecular , genetics , gbm , isocitrate dehydrogenase , telomerase reverse transcriptase , epidermal growth factor receptor , ptprz1-met , and clinical treatment.study selection : articles regarding the morphological pathology of gbm , the epidemiology of gbm , genetic alteration of gbm , and the development of treatment for gbm patients were identified , retrieved , and reviewed.results:there is a large amount of data supporting the view that these recurrent genetic aberrations occur in a specific context of cellular origin , co - oncogenic hits and are present in distinct patient populations . primary and secondary gbms are distinct disease entities that affect different age groups of patients and develop through distinct genetic aberrations . these differences are important , especially because they may affect sensitivity to radio- and chemo - therapy and should thus be considered in the identification of targets for novel therapeutic approaches.conclusion:this review highlights the molecular and genetic alterations of gbm , indicating that they are of potential value in the diagnosis and treatment for patients with gbm .

I M G Isocitrate dehydrogenase mutations O(6)-Methylguanine-DNA methyltransferase promoter methylation Telomerase reverse transcriptase promoter mutations Epidermal growth factor receptor aberrations PTEN alterations Other novel genetic aberrations M A C Financial support and sponsorship Conflicts of interest

glioblastoma ( gbm ) is the most frequent and aggressive malignant primary brain tumor with only about 12% of patients surviving beyond 36 months ( long - term survivors ) . the current treatment strategy for gbm patients combines maximal surgical resection , followed by radiotherapy ( rt ) with concomitant and adjuvant temozolomide ( tmz ) . concurrent adjuvant rt in combination with tmz represents the standard of care for patients with newly diagnosed gbm , but still < 5% of patients survive for longer than 5 years after diagnosis . decades of molecular studies have identified key genetic abnormalities in human gbms , including the following : ( 1 ) dysregulation of growth factor signaling pathways via amplification and mutational activation of receptor tyrosine kinase ( rtk ) genes ; ( 2 ) activation of the phosphatidylinositol-3-oh kinase ( pi3k ) pathway ; and ( 3 ) inactivation of the p53 and retinoblastoma tumor suppressor pathways . during recent years , large - scale research efforts spearheaded by the cancer genome atlas ( tcga ) and chinese glioma genome atlas ( cgga ) have made rapid advances in understanding gbm tumor genetics . the discovery of new genetic alterations and their mapping against clinical outcome will trigger an avalanche of novel perceptions of the genomic and epigenomic landscape , biological subgroups and putative cells of origin of gbm , which has encouraged hopes for more effective treatment strategies in the near future . this review mainly discusses the recent advances in gbm molecular research and current trends in personalized therapy of this disease . the world health organization ( who ) classification system groups gliomas into 4 histological grades defined by increasing degrees of undifferentiation , anaplasia , and aggressiveness . malignant gliomas , the most common form of gliomas , consist of who grade iv tumors ( gbm ) and grade iii tumors ( anaplastic astrocytoma , oligodendroglioma , and oligoastrocytoma ) . secondary gbms ( approximately 5% of cases ) develop more slowly by progression from low - grade diffuse astrocytoma and anaplastic astrocytoma . gbm and other malignant gliomas are highly invasive , infiltrating surrounding brain parenchyma , yet they are typically confined to the central nervous system ( cns ) and do not metastasize . unfortunately , who morphological classification is based on subjective criteria , lacks reproducibility , and remains imperfect in its ability to predict individual outcomes . the first genome - wide exon sequencing effort for glioma identified heterozygous hotspot mutations at codon 132 ( most commonly r132h ) in isocitrate dehydrogenase 1 ( idh1 ) in 12% of gbm . these homologous enzymes decarboxylate isocitrate to -ketoglutarate ( kg ) , and this neomorphic mutation renders the idh enzyme to reduce kg into 2-hydroxyglutarate in the nicotinamide adenine dinucleotide phosphate - dependent manner . nowadays , mutations in idh1 are commonly established as a hallmark molecular feature of secondary gbm ( ~70% of secondary gbm , compared with 520% in primary gbm ) who have predominant localization of gbm in the frontal and temporal lobes . since primary gbm is a clinically defined entity and the presence of idh1/2 mutations has been shown to be inversely related to or even mutually exclusive of epidermal growth factor receptor ( egfr ) and phosphatase and tensin homolog ( pten ) abnormalities , which are hallmarks of primary gbm , idh - mutated gbm lesions may represent genetically secondary gbm tumors . moreover , the idh mutation status is stable during the progression of lower - grade gliomas to secondary gbms . idh mutations seem to require cooperating mutations in tp53 and atrx , and they are less frequently detected in primary gbms . abnormal methylation of the mgmt promoter caused its silencing , a reduction of the mgmt enzyme expression , and subsequently to less repair activity of dna damage , including that induced by tmz . mgmt promoter methylation in gbm is a prognostic and predictive biomarker indicating a response to chemoradiation . patients with tumors with methylated mgmt promoter had a survival benefit when treated with tmz and rt , compared to those who received rt only factor . recently , novel somatic mutations in the promoter region of telomerase reverse transcriptase ( tert ) have been identified in malignant melanomas , as well as being associated with increased telomerase expression and activity . the two most common mutations are located at c228 t and c250 t , with identical hotspots also found in gliomas . the highest incidence was identified among most tumors harboring 1p/19q co - deletion and idh mutations ( 98% ) , as well as idh wild - type ( idh wt ) tumors with egfr amplification ( 92% ) . , patients with tert promoter mutations alone ( i.e. , no idh mutation ) had the poorest overall survival ( os ) ( median 11.3 months ) , patients with tumors without tert or idh1/2 mutations had a slightly better survival ( median 16.6 months ) , while patients with only idh mutant gbm had the best survival ( median 42.3 months ) . although another study with 358 patients found no significant difference in os between tert mutant and tert wild - type ( idh wt ) gbm , the role of tert promoter mutations may provide a tool to identify non - idh mutant gbms . egfr mutations were accompanied by regional dna amplification in the majority of cases , leading to a wide range of mutation allelic frequencies . the prominent intragenic deletions in gbm target parts of the gene encoding either the extracellular domain of egfr ( exons 27 , the deletion of which forms egfr variant iii ) or the carboxyl terminus , and these deletions are always correlated with amplification and co - expression of the wild - type egfr . egfr was recently shown to be activated by recurrent translocations in 7% of gbm samples : it was most frequently fused in - frame to septin 14 or phosphoserine phosphatase as the 3 gene segment . overall , 57% of gbm showed evidence of mutation , rearrangement , altered splicing , and/or focal amplification of egfr . loss of heterozygosity ( loh ) at chromosome 10q23 occurs at high frequency in a variety of human tumors . pten is a negative regulator of the phosphoinositide 3-kinase pathway , a major signaling pathway that stimulates cellular proliferation in response to growth factor stimulation . however , pten loss was not associated with worse os in newly diagnosed gbm patients of the tmz era . in a smaller fraction of primary gbms ( about 3% ) , a fusion of the tyrosine kinase coding region of fibroblast growth factor receptor 1 ( fgfr1 ) to the transforming acidic coiled - coil ( tacc ) coding domain of tacc1 ( or fusion of fgfr3 to tacc3 ) results in constitutive kinase activity . in transcriptome profiling of 272 gliomas from cgga , 67 in - frame fusion transcripts were identified , including three recurrent fusion transcripts : fgfr3-tacc3 , rnf213-slc26a11 , and ptprz1-met ( fusion transcript involving the protein tyrosine phosphatase , receptor - type , z polypeptide 1 gene and the met proto - oncogene , zm ) . exogenous expression of the zm fusion in the u87 mg gbm line enhanced cell migration and invasion . genetic abnormalities and the major signaling pathways involved in the pathogenesis of gbm idh : isocitrate dehydrogenase ; cdkn2a : cyclin - dependent kinase inhibitor 2a ; pten : phosphatase and tensin homolog ; nf1 : neurofibromatosis 1 ; rb1 : retinoblastoma 1 ; tert : telomerase reverse transcriptase ; ampl . : amplification ; egfr : epidermal growth factor receptor ; pdgfra : platelet - derived growth factor receptor alpha ; fgfr3 : fibroblast growth factor receptor 3 ; tacc3 : transforming acidic coiled - coil 3 ; rtk : receptor tyrosine kinase ; gbm : glioblastoma ; mapk : mitogen - activated protein kinase . the first genome - wide exon sequencing effort for glioma identified heterozygous hotspot mutations at codon 132 ( most commonly r132h ) in isocitrate dehydrogenase 1 ( idh1 ) in 12% of gbm . these homologous enzymes decarboxylate isocitrate to -ketoglutarate ( kg ) , and this neomorphic mutation renders the idh enzyme to reduce kg into 2-hydroxyglutarate in the nicotinamide adenine dinucleotide phosphate - dependent manner . nowadays , mutations in idh1 are commonly established as a hallmark molecular feature of secondary gbm ( ~70% of secondary gbm , compared with 520% in primary gbm ) who have predominant localization of gbm in the frontal and temporal lobes . since primary gbm is a clinically defined entity and the presence of idh1/2 mutations has been shown to be inversely related to or even mutually exclusive of epidermal growth factor receptor ( egfr ) and phosphatase and tensin homolog ( pten ) abnormalities , which are hallmarks of primary gbm , idh - mutated gbm lesions may represent genetically secondary gbm tumors . moreover , the idh mutation status is stable during the progression of lower - grade gliomas to secondary gbms . mutations in the idh genes are thought to cause glioma - cpg island methylator phenotype ( g - cimp ) within the proneural gbm subgroup . idh mutations seem to require cooperating mutations in tp53 and atrx , and they are less frequently detected in primary gbms . abnormal methylation of the mgmt promoter caused its silencing , a reduction of the mgmt enzyme expression , and subsequently to less repair activity of dna damage , including that induced by tmz . patients with tumors with methylated mgmt promoter had a survival benefit when treated with tmz and rt , compared to those who received rt only factor . recently , novel somatic mutations in the promoter region of telomerase reverse transcriptase ( tert ) have been identified in malignant melanomas , as well as being associated with increased telomerase expression and activity . the highest incidence was identified among most tumors harboring 1p/19q co - deletion and idh mutations ( 98% ) , as well as idh wild - type ( idh wt ) tumors with egfr amplification ( 92% ) . in the study by killela et al . , no idh mutation ) had the poorest overall survival ( os ) ( median 11.3 months ) , patients with tumors without tert or idh1/2 mutations had a slightly better survival ( median 16.6 months ) , while patients with only idh mutant gbm had the best survival ( median 42.3 months ) . although another study with 358 patients found no significant difference in os between tert mutant and tert wild - type ( idh wt ) gbm , the role of tert promoter mutations may provide a tool to identify non - idh mutant gbms . the prominent intragenic deletions in gbm target parts of the gene encoding either the extracellular domain of egfr ( exons 27 , the deletion of which forms egfr variant iii ) or the carboxyl terminus , and these deletions are always correlated with amplification and co - expression of the wild - type egfr . loss of heterozygosity ( loh ) at chromosome 10q23 occurs at high frequency in a variety of human tumors . pten is a negative regulator of the phosphoinositide 3-kinase pathway , a major signaling pathway that stimulates cellular proliferation in response to growth factor stimulation . several studies demonstrated that patients with loss of function mutations of pten generally had shorter survival than patients with pten retention . in a smaller fraction of primary gbms ( about 3% ) , a fusion of the tyrosine kinase coding region of fibroblast growth factor receptor 1 ( fgfr1 ) to the transforming acidic coiled - coil ( tacc ) coding domain of tacc1 ( or fusion of fgfr3 to tacc3 ) results in constitutive kinase activity . in transcriptome profiling of 272 gliomas from cgga , 67 in - frame fusion transcripts were identified , including three recurrent fusion transcripts : fgfr3-tacc3 , rnf213-slc26a11 , and ptprz1-met ( fusion transcript involving the protein tyrosine phosphatase , receptor - type , z polypeptide 1 gene and the met proto - oncogene , zm ) . exogenous expression of the zm fusion in the u87 mg gbm line enhanced cell migration and invasion . table 1 summarizes all of the above described and other genetic alterations and related altered signaling pathways in primary versus secondary gbm . genetic abnormalities and the major signaling pathways involved in the pathogenesis of gbm idh : isocitrate dehydrogenase ; cdkn2a : cyclin - dependent kinase inhibitor 2a ; pten : phosphatase and tensin homolog ; nf1 : neurofibromatosis 1 ; rb1 : retinoblastoma 1 ; tert : telomerase reverse transcriptase ; ampl . : amplification ; egfr : epidermal growth factor receptor ; pdgfra : platelet - derived growth factor receptor alpha ; fgfr3 : fibroblast growth factor receptor 3 ; tacc3 : transforming acidic coiled - coil 3 ; rtk : receptor tyrosine kinase ; gbm : glioblastoma ; mapk : mitogen - activated protein kinase . phillips described three subclasses of high - grade gliomas ( termed proneural , mesenchymal , and proliferative ) associated with different outcomes ; specifically , prolonged survival of the proneural subclass . in 2010 , unsupervised clustering of gene expression data from adult gbm samples from the tcga identified four different molecular subtypes : proneural , neural , classical , and mesenchymal . another later study , which compared dna methylation patterns across both pediatric and adult patients with gbm , found a similar clustering in tumors from adult patients and further identified three more distinct clusters that predominantly consisted of children and adolescents . phillips classifications of gbm based on transcription profiling egfr : epidermal growth factor receptor ; pten : phosphatase and tensin homolog ; gbm : glioblastoma . tcga classifications of gbm based on transcription and methylation profiling tcga : the cancer genome atlas ; gbm : glioblastoma ; g - cimp : glioma - cpg island methylator phenotype ; ampl . dkfz classifications of gbm based on methylation profiling dkfz : deutsches krebsforschungszentrum ( german cancer research center ) ; gbm : glioblastoma ; rtk : receptor tyrosine kinase ; pdgfra : platelet - derived growth factor receptor alpha ; egfr : epidermal growth factor receptor ; idh : isocitrate dehydrogenase ; ampl . : the recent reports published on the nature genetics and nejm were comprehensively analyzed by whole - exome sequencing and/or targeted deep sequencing as well as array comparative genomic hybridization . type iii tumors represented the idh wild - type grade ii and iii tumors in the current cohort , showing an os rate more similar to that of gbm . similarly , the report from tcga research network independently identified similar groups , using unsupervised clustering analyses of dna mutation , rna expression , dna copy number , and dna methylation data . the report from mayo clinic and ucsf defined a priori groups that were based on the presence or absence of tert promoter mutations , idh mutations , and 1p/19q codeletion and found consistent associations between the molecular groups and age at diagnosis , survival , patterns of acquired alterations , and germline variants across the three data sets . these tests ( for idh mutations , 1p/19q codeletion , and tert promoter alterations ) can be used to define five principal groups of gliomas with characteristic distributions of age at diagnosis , clinical behavior , acquired genetic alterations , and associated germline variants . over the past decade , insights into the molecular pathology of gliomas have significantly improved both our biological understanding of neoplasms as well as our abilities to diagnose tumors and estimate their prognosis and likelihood of response to specific therapies . to discuss the inclusion of molecular information into the next who classification of cns tumors , a meeting under the sponsorship of the international society of neuropathology ( isn ) has been held in haarlem , the netherlands . according to the isn - haarlem consensus , integrated diagnosis was established through the usage of atrx , idh1-r132h ihc , 1p/19q analyses , and idh sequencing in the diagnosis of diffuse gliomas . rt plus concomitant and adjuvant tmz chemotherapy is the current standard of care for patients with gbm . several clinical trials have displayed that mgmt promoter methylation correlated with prolonged progression - free and os in patients with gbm receiving alkylating drug chemotherapy . in 2012 , two independent randomized trials in elderly patients with gbm assessed rt alone versus tmz chemotherapy alone as an initial treatment . subgroup analyses of both trials showed better outcome for chemotherapy in patients with mgmt promoter methylated tumors but reduced survival in patients with unmethylated tumors . recently , the cgga project delineated that patients with idh wild - type gbm who underwent rt + tmz exhibited significantly longer survival times compared to the patients who were assigned to the rt alone treatment . however , among patients with idh mutation tumors , the survival pattern of patients undergoing rt + tmz or rt was comparable . these results strongly suggest that treatment strategies for elderly patients with gbm should be individualized dependent on idh and mgmt . in addition , due to the high heterogeneity of gbm , each of which may respond differently to one targeted therapy , there has been considerable interest in identifying molecular markers that predict response to a specific molecular targeted therapy . the presence of egfr overexpression and egfr mutations in gbm could predict the activity of egfr - targeted drugs in patients with these aberrations . conceptually , patients with low - grade and anaplastic gliomas , secondary gbm with a high prevalence of the idh1 ( r132h ) mutation represent a patient population that may particularly benefit from an idh1r132h specific tumor vaccine . these recurrent genetic aberrations occur in a specific context of cellular origin , co - oncogenic hits and are present in distinct patient populations . primary and secondary gbms are distinct disease entities that affect different age groups of patients and develop through distinct genetic aberrations . these differences are important , especially because they may affect sensitivity to radio- and chemo - therapy and should thus be considered in the identification of targets for novel therapeutic approaches .

chronic pancreatitis ( cp ) is characterized by irreversible damage to the pancreas that may lead on to the development of abdominal pain as well as varying degrees of endocrine and exocrine insufficiency . however , pancreatic biopsy is usually not performed in routine clinical settings because obtaining a histological diagnosis of cp is risky and carries a high rate of potentially severe complications . the diagnosis of cp is thus based upon the demonstration of morphological and/or functional changes in the pancreas that occurs because of irreversible damage to the pancreatic parenchyma . the morphological changes in the pancreas can be appreciated by various imaging methods such as abdominal x - ray , transabdominal ultrasound ( ultrasonography ) , computed tomography ( ct ) , magnetic resonance imaging and endoscopic retrograde cholangio pancreatography . however , these imaging modalities can pick up advanced morphological changes only , and the diagnostic ability of these modalities to diagnose early and minimal cp is limited . the pancreatic function tests have been shown to improve the diagnostic sensitivity and specificity but because of associated technical challenges , patient discomfort and limited availability , these functional tests are infrequently performed . due to the ability to place the transducer in close proximity to the pancreas , endoscopic ultrasound ( eus ) is considered to be the most sensitive imaging modality for diagnosing pancreatic disorders . eus has been shown to demonstrate subtle alterations in the pancreatic parenchymal and ductal structure even before traditional imaging and functional testing demonstrate any abnormality . the eus diagnosis of cp is based upon the demonstration of ductal and parenchymal features , and a number of diagnostic criteria incorporating these eus features have been proposed to diagnose cp . improvement in eus equipment and availability of newer and sophisticated eus applications like color doppler , contrast eus and elastography , have further improved the diagnostic ability of eus . the eus features to diagnose cp [ figures 19 ] have evolved over a period from a pure qualitative approach to more advanced and complicated scoring systems incorporating multiple parenchymal and ductal eus features . increasing the number of these eus features required for diagnosis of cp increases the specificity and lowering the threshold number of criteria increases the sensitivity , but decreases the already poor specificity of eus . the rosemont criteria developed by leading experts has attempted to surmount the limitations of the current diagnostic criteria , define precisely each eus criterion and thus develop criteria that have good inter - observer agreement . the various eus features that have to be demonstrated for the diagnosis of cp are : examination of pancreatic body from stomach using radial echoendoscope . thus , this patient has one major a and 4 minor endoscopic ultrasound features and is consistent with chronic pancreatitis examination of pancreatic body from stomach using radial echoendoscope . thus , this patient has only 4 minor endoscopic ultrasound features and is indeterminate for chronic pancreatitis examination of pancreatic body from stomach using radial echoendoscope . thus , this patient has only 4 minor endoscopic ultrasound features and is indeterminate for chronic pancreatitis examination of pancreatic body from stomach using radial echoendoscope . thus , this patient has one major a and 2 minor endoscopic ultrasound features and is suggestive of chronic pancreatitis examination of pancreatic body from stomach using radial echoendoscope . the main pancreatic duct is dilated with an irregular contour and has echogenic structures suggestive of calculi . thus , this patient has two major a endoscopic ultrasound features and is consistent with chronic pancreatitis examination of pancreatic body from stomach using linear echoendoscope . thus , this patient has one major a and 2 minor endoscopic ultrasound features and is suggestive of chronic pancreatitis quantitative endoscopic ultrasound ( eus ) elastography and strain ratio measurement with radial echoendoscope at the body of pancreas : the eus shows echogenic foci with shadowing ( right side ) . a stable eus image for at least 5 s is obtained for eus quantitative analysis . the region of interest for the elastographic evaluation is manually selected to include the targeted area of the pancreas ( region a ) and soft ( red ) reference area corresponding to normal gastric wall ( region b ) . a strain ratio of 13.6 has been obtained quantitative endoscopic ultrasound ( eus ) elastography and strain ratio measurement with radial echoendoscope at the body of pancreas : the eus shows stranding with echogenic foci and lobularity ( right side ) . a stable eus image for at least 5 s is obtained for eus quantitative analysis . the region of interest for the elastographic evaluation is manually selected to include the targeted area of the pancreas ( region a ) and soft ( red ) reference area corresponding to normal gastric wall ( region b ) . a strain ratio of 147.7 has been obtained examination of pancreatic body from stomach using radial echoendoscope . thus , this patient has major a and b criteria along with 1 minor criteria and is consistent with chronic pancreatitis hyperechoic foci with shadowing ( major a ) : they have been defined as echogenic structures 2 mm in length and width that shadow . they histologically correlate with pancreatic parenchymal calcification.lobularity with honeycombing ( major b ) : well - circumscribed , 5 mm structures with rims that are hyperechoic relative to the echogenicity of its central areas are defined as lobules . at least three contiguous lobules that are present in the body or tail of the pancreas are labeled as honeycombing lobularity . the histological correlate of lobularity is not known.lobularity without honeycombing ( minor ) : lobules as defined above that are non - contiguous and present in body and tail.hyperechoic foci without shadowing ( minor ) : defined as above but without shadowing.cysts ( minor ) : anechoic , rounded / elliptic structures that should measure 2 mm in short axis.stranding ( minor ) : hyperechoic lines 3 mm in length seen in at least two different directions with respect to the imaged plane . hyperechoic foci with shadowing ( major a ) : they have been defined as echogenic structures 2 mm in length and width that shadow . lobularity with honeycombing ( major b ) : well - circumscribed , 5 mm structures with rims that are hyperechoic relative to the echogenicity of its central areas are defined as lobules . at least three contiguous lobules that are present in the body or tail of the pancreas are labeled as honeycombing lobularity . lobularity without honeycombing ( minor ) : lobules as defined above that are non - contiguous and present in body and tail . cysts ( minor ) : anechoic , rounded / elliptic structures that should measure 2 mm in short axis . stranding ( minor ) : hyperechoic lines 3 mm in length seen in at least two different directions with respect to the imaged plane . all other features should be looked in the body and tail of pancreas only main pancreatic duct ( mpd ) calculi ( major a ) : echogenic structures within mpd with acoustic shadowing located anywhere in the head , body and tail.irregular mpd contour ( minor ) : mpd that is uneven or irregular in outline and ectatic in its course . these features should be demonstrated in the body and tail of pancreas only.dilated side branches ( minor ) : presence of 3 tubular anechoic structures each measuring 1 mm in width and communicating with mpd . these should be demonstrated in the body and tail of pancreas only.main pancreatic duct dilatation ( minor ) : mpd diameter 3.5 mm in the pancreatic body or 1.5 mm in the pancreatic tail.hyper echoic duct margin ( minor ) : relatively hyperechoic duct wall found in > 50% of the entire mpd of the body and tail . main pancreatic duct ( mpd ) calculi ( major a ) : echogenic structures within mpd with acoustic shadowing located anywhere in the head , body and tail . irregular mpd contour ( minor ) : mpd that is uneven or irregular in outline and ectatic in its course . dilated side branches ( minor ) : presence of 3 tubular anechoic structures each measuring 1 mm in width and communicating with mpd . main pancreatic duct dilatation ( minor ) : mpd diameter 3.5 mm in the pancreatic body or 1.5 mm in the pancreatic tail . hyper echoic duct margin ( minor ) : relatively hyperechoic duct wall found in > 50% of the entire mpd of the body and tail . endoscopic ultrasound diagnosis of chronic pancreatitis based upon rosemont criteria consistent with cp : one major a feature with 3 minor features or 1 major a feature and major b feature or 2 major a features.suggestive of cp : one major a feature with <3 minor features or 1 major b feature with 3 minor features or 5 minor features.indeterminate for cp : 3 or 4 minor features with no major features or major b feature alone or with <3 minor features . consistent with cp : one major a feature with 3 minor features or 1 major a feature and major b feature or 2 major a features . suggestive of cp : one major a feature with <3 minor features or 1 major b feature with 3 minor features or 5 minor features . indeterminate for cp : 3 or 4 minor features with no major features or major b feature alone or with <3 minor features . a number of conditions such as aging , smoking , obesity , and chronic alcohol consumption may cause similar eus changes in the pancreas . although , rosemont criteria has refined the past criteria with more objectivity initial studies have failed to demonstrate any significant improvement in kappa scores . to decrease interobserver variability adjunctive imaging technologies like digital image analysis and elastography have been used for diagnosis of cp with encouraging results . however , further studies with larger sample size are needed to determine the exact diagnostic role of these ancillary eus techniques . hyperechoic foci with shadowing ( major a ) : they have been defined as echogenic structures 2 mm in length and width that shadow . they histologically correlate with pancreatic parenchymal calcification.lobularity with honeycombing ( major b ) : well - circumscribed , 5 mm structures with rims that are hyperechoic relative to the echogenicity of its central areas are defined as lobules . at least three contiguous lobules that are present in the body or tail of the pancreas are labeled as honeycombing lobularity . the histological correlate of lobularity is not known.lobularity without honeycombing ( minor ) : lobules as defined above that are non - contiguous and present in body and tail.hyperechoic foci without shadowing ( minor ) : defined as above but without shadowing.cysts ( minor ) : anechoic , rounded / elliptic structures that should measure 2 mm in short axis.stranding ( minor ) : hyperechoic lines 3 mm in length seen in at least two different directions with respect to the imaged plane . hyperechoic foci with shadowing ( major a ) : they have been defined as echogenic structures 2 mm in length and width that shadow . lobularity with honeycombing ( major b ) : well - circumscribed , 5 mm structures with rims that are hyperechoic relative to the echogenicity of its central areas are defined as lobules . at least three contiguous lobules that are present in the body or tail of the pancreas are labeled as honeycombing lobularity . lobularity without honeycombing ( minor ) : lobules as defined above that are non - contiguous and present in body and tail . cysts ( minor ) : anechoic , rounded / elliptic structures that should measure 2 mm in short axis . stranding ( minor ) : hyperechoic lines 3 mm in length seen in at least two different directions with respect to the imaged plane . all other features should be looked in the body and tail of pancreas only main pancreatic duct ( mpd ) calculi ( major a ) : echogenic structures within mpd with acoustic shadowing located anywhere in the head , body and tail.irregular mpd contour ( minor ) : mpd that is uneven or irregular in outline and ectatic in its course . these features should be demonstrated in the body and tail of pancreas only.dilated side branches ( minor ) : presence of 3 tubular anechoic structures each measuring 1 mm in width and communicating with mpd . these should be demonstrated in the body and tail of pancreas only.main pancreatic duct dilatation ( minor ) : mpd diameter 3.5 mm in the pancreatic body or 1.5 mm in the pancreatic tail.hyper echoic duct margin ( minor ) : relatively hyperechoic duct wall found in > 50% of the entire mpd of the body and tail . main pancreatic duct ( mpd ) calculi ( major a ) : echogenic structures within mpd with acoustic shadowing located anywhere in the head , body and tail . irregular mpd contour ( minor ) : mpd that is uneven or irregular in outline and ectatic in its course . dilated side branches ( minor ) : presence of 3 tubular anechoic structures each measuring 1 mm in width and communicating with mpd . main pancreatic duct dilatation ( minor ) : mpd diameter 3.5 mm in the pancreatic body or 1.5 mm in the pancreatic tail . hyper echoic duct margin ( minor ) : relatively hyperechoic duct wall found in > 50% of the entire mpd of the body and tail . endoscopic ultrasound diagnosis of chronic pancreatitis based upon rosemont criteria consistent with cp : one major a feature with 3 minor features or 1 major a feature and major b feature or 2 major a features.suggestive of cp : one major a feature with <3 minor features or 1 major b feature with 3 minor features or 5 minor features.indeterminate for cp : 3 or 4 minor features with no major features or major b feature alone or with <3 minor features . consistent with cp : one major a feature with 3 minor features or 1 major a feature and major b feature or 2 major a features . suggestive of cp : one major a feature with <3 minor features or 1 major b feature with 3 minor features or 5 minor features . indeterminate for cp : 3 or 4 minor features with no major features or major b feature alone or with <3 minor features . a number of conditions such as aging , smoking , obesity , and chronic alcohol consumption may cause similar eus changes in the pancreas . although , rosemont criteria has refined the past criteria with more objectivity initial studies have failed to demonstrate any significant improvement in kappa scores . to decrease interobserver variability adjunctive imaging technologies like digital image analysis and elastography have been used for diagnosis of cp with encouraging results . however , further studies with larger sample size are needed to determine the exact diagnostic role of these ancillary eus techniques . hyperechoic foci with shadowing ( major a ) : they have been defined as echogenic structures 2 mm in length and width that shadow . they histologically correlate with pancreatic parenchymal calcification.lobularity with honeycombing ( major b ) : well - circumscribed , 5 mm structures with rims that are hyperechoic relative to the echogenicity of its central areas are defined as lobules . at least three contiguous lobules that are present in the body or tail of the pancreas are labeled as honeycombing lobularity . the histological correlate of lobularity is not known.lobularity without honeycombing ( minor ) : lobules as defined above that are non - contiguous and present in body and tail.hyperechoic foci without shadowing ( minor ) : defined as above but without shadowing.cysts ( minor ) : anechoic , rounded / elliptic structures that should measure 2 mm in short axis.stranding ( minor ) : hyperechoic lines 3 mm in length seen in at least two different directions with respect to the imaged plane . hyperechoic foci with shadowing ( major a ) : they have been defined as echogenic structures 2 mm in length and width that shadow . lobularity with honeycombing ( major b ) : well - circumscribed , 5 mm structures with rims that are hyperechoic relative to the echogenicity of its central areas are defined as lobules . at least three contiguous lobules that are present in the body or tail of the pancreas are labeled as honeycombing lobularity . lobularity without honeycombing ( minor ) : lobules as defined above that are non - contiguous and present in body and tail . cysts ( minor ) : anechoic , rounded / elliptic structures that should measure 2 mm in short axis . stranding ( minor ) : hyperechoic lines 3 mm in length seen in at least two different directions with respect to the imaged plane . except for ductal calculi all other features should be looked in the body and tail of pancreas only main pancreatic duct ( mpd ) calculi ( major a ) : echogenic structures within mpd with acoustic shadowing located anywhere in the head , body and tail.irregular mpd contour ( minor ) : mpd that is uneven or irregular in outline and ectatic in its course . these features should be demonstrated in the body and tail of pancreas only.dilated side branches ( minor ) : presence of 3 tubular anechoic structures each measuring 1 mm in width and communicating with mpd . these should be demonstrated in the body and tail of pancreas only.main pancreatic duct dilatation ( minor ) : mpd diameter 3.5 mm in the pancreatic body or 1.5 mm in the pancreatic tail.hyper echoic duct margin ( minor ) : relatively hyperechoic duct wall found in > 50% of the entire mpd of the body and tail . main pancreatic duct ( mpd ) calculi ( major a ) : echogenic structures within mpd with acoustic shadowing located anywhere in the head , body and tail . irregular mpd contour ( minor ) : mpd that is uneven or irregular in outline and ectatic in its course . these features should be demonstrated in the body and tail of pancreas only . dilated side branches ( minor ) : presence of 3 tubular anechoic structures each measuring 1 mm in width and communicating with mpd . main pancreatic duct dilatation ( minor ) : mpd diameter 3.5 mm in the pancreatic body or 1.5 mm in the pancreatic tail . hyper echoic duct margin ( minor ) : relatively hyperechoic duct wall found in > 50% of the entire mpd of the body and tail . endoscopic ultrasound diagnosis of chronic pancreatitis based upon rosemont criteria consistent with cp : one major a feature with 3 minor features or 1 major a feature and major b feature or 2 major a features.suggestive of cp : one major a feature with <3 minor features or 1 major b feature with 3 minor features or 5 minor features.indeterminate for cp : 3 or 4 minor features with no major features or major b feature alone or with <3 minor features . consistent with cp : one major a feature with 3 minor features or 1 major a feature and major b feature or 2 major a features . suggestive of cp : one major a feature with <3 minor features or 1 major b feature with 3 minor features or 5 minor features . indeterminate for cp : 3 or 4 minor features with no major features or major a number of conditions such as aging , smoking , obesity , and chronic alcohol consumption may cause similar eus changes in the pancreas . although , rosemont criteria has refined the past criteria with more objectivity initial studies have failed to demonstrate any significant improvement in kappa scores . to decrease interobserver variability adjunctive imaging technologies like digital image analysis and elastography have been used for diagnosis of cp with encouraging results . however , further studies with larger sample size are needed to determine the exact diagnostic role of these ancillary eus techniques . the eus procedure for looking for changes of cp is similar to the standard eus examination of the pancreas . the complete eus examination of the pancreas is possible from gastric and duodenal stations with uncinate process being visualized from the second part of the duodenum . both radial and linear eus have been shown to be equally accurate for the diagnosis of cp . radial eus , however , provides cross sectional imaging similar to other cross sectional imaging modalities like ct and this makes the imaging of mpd easier as it is seen as a long linear structure in contrast to a dot like appearance on linear eus . in order to correctly identify the changes of cp , the endoscopist needs to know the normal appearances of the pancreas as well as the normal variations seen in a nonpathological pancreas . the important things to keep in mind are : the normal pancreas has a diffusely speckled salt and pepper pattern of the body and tail with a barely visualized single , smooth and anechoic mpd.with the current advanced eus imaging systems side branches of mpd can also be seen in normal individuals especially the elderly and only if side branches are 1 mm , they are considered to be abnormal.even in normal individuals the mpd can be mildly tortuous and therefore the mpd should be carefully evaluated for a gradual decrease in its diameter from the head to tail , which is an important feature of the normal duct.the duct of alternating sizes or beading is abnormal.the dorsal and ventral pancreas can have different echogenicity with the dorsal anlage of the pancreas being more echogenic than the ventral pancreas . the normal pancreas has a diffusely speckled salt and pepper pattern of the body and tail with a barely visualized single , smooth and anechoic mpd . with the current advanced eus imaging systems side branches of mpd can also be seen in normal individuals especially the elderly and only if side branches are 1 mm , they are considered to be abnormal . even in normal individuals the mpd can be mildly tortuous and therefore the mpd should be carefully evaluated for a gradual decrease in its diameter from the head to tail , which is an important feature of the normal duct . the dorsal and ventral pancreas can have different echogenicity with the dorsal anlage of the pancreas being more echogenic than the ventral pancreas . as mentioned above most of these eus features of cp are visualized in the body and tail and currently it is not clear how best to diagnose focal cp in the head as the isolated eus findings like lobularity in the head with normal body and tail of pancreas are generally reported as normal . endoscopic ultrasound elastography has also been shown to be an accurate tool for the diagnosis of cp and strain ratio obtained using quantitative eus elastography has been shown to vary significantly in various rosemont classification groups thus allowing quantification of pancreatic fibrosis . in quantitative elastography , the tissue stiffness is measured in the targeted area ( region of interest [ roi ] a ) and outside the targeted area in a region representing normal tissue ( roi b ) . thereafter , the strain ratio value is calculated as the quotient b / a , which seems to be better and more objective than visual color changes of qualitative elastography [ figures 7 and 8 ] . in a study on eus elastography in cp , region b selected was the normal surrounding gastroduodenal wall and strain ratio cut - off of 2.25 yielded an accuracy of 91.1% for diagnosis of cp . the results appear promising , but further studies with larger sample size are needed to confirm these interesting results . although eus can demonstrate subtle alterations in the pancreatic parenchymal and ductal structure , the exact role of eus in the diagnosis of cp is still not established.the eus features to diagnose cp have evolved over a period of time from a pure qualitative approach to more advanced and complicated scoring systems incorporating multiple parenchymal and ductal eus features.the rosemont criteria have attempted to define precisely each eus criterion and thus have good inter - observer agreement . but , initial studies have failed to demonstrate any significant improvement in the inter - observer variability.a number of conditions such as aging , smoking , obesity , and chronic alcohol consumption may cause eus changes similar to cp . therefore , eus findings should be interpreted in appropriate clinical context.most of the eus features of cp except ductal calculi and cysts should be located in the body and tail only.in order to correctly identify the changes of cp , the endoscopist needs to know the normal appearances of the pancreas as well as the normal variations seen in nonpathological pancreas.quantitative eus elastography appear promising technique for diagnosis of cp . although eus can demonstrate subtle alterations in the pancreatic parenchymal and ductal structure , the exact role of eus in the diagnosis of cp is still not established . the eus features to diagnose cp have evolved over a period of time from a pure qualitative approach to more advanced and complicated scoring systems incorporating multiple parenchymal and ductal eus features . the rosemont criteria have attempted to define precisely each eus criterion and thus have good inter - observer agreement . but , initial studies have failed to demonstrate any significant improvement in the inter - observer variability . a number of conditions such as aging , smoking , obesity , and chronic alcohol consumption may cause eus changes similar to cp . therefore most of the eus features of cp except ductal calculi and cysts should be located in the body and tail only . in order to correctly identify the changes of cp , the endoscopist needs to know the normal appearances of the pancreas as well as the normal variations seen in nonpathological pancreas .

as endoscopic ultrasound ( eus ) is the most sensitive imaging modality for diagnosing pancreatic disorders , it can demonstrate subtle alterations in the pancreatic parenchymal and ductal structure even before traditional imaging and functional testing demonstrate any abnormality . in spite of this fact and abundant literature , the exact role of eus in the diagnosis of chronic pancreatitis ( cp ) is still not established . the eus features to diagnose cp have evolved over a period from a pure qualitative approach to more advanced and complicated scoring systems incorporating multiple parenchymal and ductal eus features . the rosemont criteria have attempted to define precisely each eus criterion and thus have good inter - observer agreement . however , initial studies have failed to demonstrate any significant improvement in the inter - observer variability and further validation studies are needed to define the exact role of these criteria . the measurement of strain ratio using quantitative eus elastography and thus allowing quantification of pancreatic fibrosis seems to be a promising new technique .

INTRODUCTION ENDOSCOPIC ULTRASOUND FEATURES OF CHRONIC PANCREATITIS None Parenchymal features Ductal features TECHNICAL ISSUES IN THE ENDOSCOPIC PROCEDURE ROLE OF ENDOSCOPIC ULTRASOUND ELASTOGRAPHY IMPORTANT FACTS ABOUT ENDOSCOPIC ULTRASOUND IN CHRONIC PANCREATITIS

chronic pancreatitis ( cp ) is characterized by irreversible damage to the pancreas that may lead on to the development of abdominal pain as well as varying degrees of endocrine and exocrine insufficiency . however , pancreatic biopsy is usually not performed in routine clinical settings because obtaining a histological diagnosis of cp is risky and carries a high rate of potentially severe complications . the diagnosis of cp is thus based upon the demonstration of morphological and/or functional changes in the pancreas that occurs because of irreversible damage to the pancreatic parenchyma . the morphological changes in the pancreas can be appreciated by various imaging methods such as abdominal x - ray , transabdominal ultrasound ( ultrasonography ) , computed tomography ( ct ) , magnetic resonance imaging and endoscopic retrograde cholangio pancreatography . however , these imaging modalities can pick up advanced morphological changes only , and the diagnostic ability of these modalities to diagnose early and minimal cp is limited . the pancreatic function tests have been shown to improve the diagnostic sensitivity and specificity but because of associated technical challenges , patient discomfort and limited availability , these functional tests are infrequently performed . due to the ability to place the transducer in close proximity to the pancreas , endoscopic ultrasound ( eus ) is considered to be the most sensitive imaging modality for diagnosing pancreatic disorders . eus has been shown to demonstrate subtle alterations in the pancreatic parenchymal and ductal structure even before traditional imaging and functional testing demonstrate any abnormality . the eus diagnosis of cp is based upon the demonstration of ductal and parenchymal features , and a number of diagnostic criteria incorporating these eus features have been proposed to diagnose cp . improvement in eus equipment and availability of newer and sophisticated eus applications like color doppler , contrast eus and elastography , have further improved the diagnostic ability of eus . the eus features to diagnose cp [ figures 19 ] have evolved over a period from a pure qualitative approach to more advanced and complicated scoring systems incorporating multiple parenchymal and ductal eus features . increasing the number of these eus features required for diagnosis of cp increases the specificity and lowering the threshold number of criteria increases the sensitivity , but decreases the already poor specificity of eus . the rosemont criteria developed by leading experts has attempted to surmount the limitations of the current diagnostic criteria , define precisely each eus criterion and thus develop criteria that have good inter - observer agreement . the various eus features that have to be demonstrated for the diagnosis of cp are : examination of pancreatic body from stomach using radial echoendoscope . thus , this patient has one major a and 4 minor endoscopic ultrasound features and is consistent with chronic pancreatitis examination of pancreatic body from stomach using radial echoendoscope . thus , this patient has only 4 minor endoscopic ultrasound features and is indeterminate for chronic pancreatitis examination of pancreatic body from stomach using radial echoendoscope . thus , this patient has only 4 minor endoscopic ultrasound features and is indeterminate for chronic pancreatitis examination of pancreatic body from stomach using radial echoendoscope . thus , this patient has one major a and 2 minor endoscopic ultrasound features and is suggestive of chronic pancreatitis examination of pancreatic body from stomach using radial echoendoscope . thus , this patient has two major a endoscopic ultrasound features and is consistent with chronic pancreatitis examination of pancreatic body from stomach using linear echoendoscope . thus , this patient has one major a and 2 minor endoscopic ultrasound features and is suggestive of chronic pancreatitis quantitative endoscopic ultrasound ( eus ) elastography and strain ratio measurement with radial echoendoscope at the body of pancreas : the eus shows echogenic foci with shadowing ( right side ) . a stable eus image for at least 5 s is obtained for eus quantitative analysis . a strain ratio of 13.6 has been obtained quantitative endoscopic ultrasound ( eus ) elastography and strain ratio measurement with radial echoendoscope at the body of pancreas : the eus shows stranding with echogenic foci and lobularity ( right side ) . a strain ratio of 147.7 has been obtained examination of pancreatic body from stomach using radial echoendoscope . thus , this patient has major a and b criteria along with 1 minor criteria and is consistent with chronic pancreatitis hyperechoic foci with shadowing ( major a ) : they have been defined as echogenic structures 2 mm in length and width that shadow . they histologically correlate with pancreatic parenchymal calcification.lobularity with honeycombing ( major b ) : well - circumscribed , 5 mm structures with rims that are hyperechoic relative to the echogenicity of its central areas are defined as lobules . at least three contiguous lobules that are present in the body or tail of the pancreas are labeled as honeycombing lobularity . lobularity with honeycombing ( major b ) : well - circumscribed , 5 mm structures with rims that are hyperechoic relative to the echogenicity of its central areas are defined as lobules . at least three contiguous lobules that are present in the body or tail of the pancreas are labeled as honeycombing lobularity . stranding ( minor ) : hyperechoic lines 3 mm in length seen in at least two different directions with respect to the imaged plane . all other features should be looked in the body and tail of pancreas only main pancreatic duct ( mpd ) calculi ( major a ) : echogenic structures within mpd with acoustic shadowing located anywhere in the head , body and tail.irregular mpd contour ( minor ) : mpd that is uneven or irregular in outline and ectatic in its course . these features should be demonstrated in the body and tail of pancreas only.dilated side branches ( minor ) : presence of 3 tubular anechoic structures each measuring 1 mm in width and communicating with mpd . these should be demonstrated in the body and tail of pancreas only.main pancreatic duct dilatation ( minor ) : mpd diameter 3.5 mm in the pancreatic body or 1.5 mm in the pancreatic tail.hyper echoic duct margin ( minor ) : relatively hyperechoic duct wall found in > 50% of the entire mpd of the body and tail . main pancreatic duct ( mpd ) calculi ( major a ) : echogenic structures within mpd with acoustic shadowing located anywhere in the head , body and tail . main pancreatic duct dilatation ( minor ) : mpd diameter 3.5 mm in the pancreatic body or 1.5 mm in the pancreatic tail . endoscopic ultrasound diagnosis of chronic pancreatitis based upon rosemont criteria consistent with cp : one major a feature with 3 minor features or 1 major a feature and major b feature or 2 major a features.suggestive of cp : one major a feature with <3 minor features or 1 major b feature with 3 minor features or 5 minor features.indeterminate for cp : 3 or 4 minor features with no major features or major b feature alone or with <3 minor features . a number of conditions such as aging , smoking , obesity , and chronic alcohol consumption may cause similar eus changes in the pancreas . although , rosemont criteria has refined the past criteria with more objectivity initial studies have failed to demonstrate any significant improvement in kappa scores . to decrease interobserver variability adjunctive imaging technologies like digital image analysis and elastography have been used for diagnosis of cp with encouraging results . however , further studies with larger sample size are needed to determine the exact diagnostic role of these ancillary eus techniques . they histologically correlate with pancreatic parenchymal calcification.lobularity with honeycombing ( major b ) : well - circumscribed , 5 mm structures with rims that are hyperechoic relative to the echogenicity of its central areas are defined as lobules . at least three contiguous lobules that are present in the body or tail of the pancreas are labeled as honeycombing lobularity . at least three contiguous lobules that are present in the body or tail of the pancreas are labeled as honeycombing lobularity . all other features should be looked in the body and tail of pancreas only main pancreatic duct ( mpd ) calculi ( major a ) : echogenic structures within mpd with acoustic shadowing located anywhere in the head , body and tail.irregular mpd contour ( minor ) : mpd that is uneven or irregular in outline and ectatic in its course . these features should be demonstrated in the body and tail of pancreas only.dilated side branches ( minor ) : presence of 3 tubular anechoic structures each measuring 1 mm in width and communicating with mpd . these should be demonstrated in the body and tail of pancreas only.main pancreatic duct dilatation ( minor ) : mpd diameter 3.5 mm in the pancreatic body or 1.5 mm in the pancreatic tail.hyper echoic duct margin ( minor ) : relatively hyperechoic duct wall found in > 50% of the entire mpd of the body and tail . main pancreatic duct ( mpd ) calculi ( major a ) : echogenic structures within mpd with acoustic shadowing located anywhere in the head , body and tail . irregular mpd contour ( minor ) : mpd that is uneven or irregular in outline and ectatic in its course . dilated side branches ( minor ) : presence of 3 tubular anechoic structures each measuring 1 mm in width and communicating with mpd . main pancreatic duct dilatation ( minor ) : mpd diameter 3.5 mm in the pancreatic body or 1.5 mm in the pancreatic tail . endoscopic ultrasound diagnosis of chronic pancreatitis based upon rosemont criteria consistent with cp : one major a feature with 3 minor features or 1 major a feature and major b feature or 2 major a features.suggestive of cp : one major a feature with <3 minor features or 1 major b feature with 3 minor features or 5 minor features.indeterminate for cp : 3 or 4 minor features with no major features or major b feature alone or with <3 minor features . consistent with cp : one major a feature with 3 minor features or 1 major a feature and major b feature or 2 major a features . a number of conditions such as aging , smoking , obesity , and chronic alcohol consumption may cause similar eus changes in the pancreas . although , rosemont criteria has refined the past criteria with more objectivity initial studies have failed to demonstrate any significant improvement in kappa scores . to decrease interobserver variability adjunctive imaging technologies like digital image analysis and elastography have been used for diagnosis of cp with encouraging results . however , further studies with larger sample size are needed to determine the exact diagnostic role of these ancillary eus techniques . they histologically correlate with pancreatic parenchymal calcification.lobularity with honeycombing ( major b ) : well - circumscribed , 5 mm structures with rims that are hyperechoic relative to the echogenicity of its central areas are defined as lobules . at least three contiguous lobules that are present in the body or tail of the pancreas are labeled as honeycombing lobularity . at least three contiguous lobules that are present in the body or tail of the pancreas are labeled as honeycombing lobularity . cysts ( minor ) : anechoic , rounded / elliptic structures that should measure 2 mm in short axis . stranding ( minor ) : hyperechoic lines 3 mm in length seen in at least two different directions with respect to the imaged plane . except for ductal calculi all other features should be looked in the body and tail of pancreas only main pancreatic duct ( mpd ) calculi ( major a ) : echogenic structures within mpd with acoustic shadowing located anywhere in the head , body and tail.irregular mpd contour ( minor ) : mpd that is uneven or irregular in outline and ectatic in its course . these features should be demonstrated in the body and tail of pancreas only.dilated side branches ( minor ) : presence of 3 tubular anechoic structures each measuring 1 mm in width and communicating with mpd . these should be demonstrated in the body and tail of pancreas only.main pancreatic duct dilatation ( minor ) : mpd diameter 3.5 mm in the pancreatic body or 1.5 mm in the pancreatic tail.hyper echoic duct margin ( minor ) : relatively hyperechoic duct wall found in > 50% of the entire mpd of the body and tail . main pancreatic duct ( mpd ) calculi ( major a ) : echogenic structures within mpd with acoustic shadowing located anywhere in the head , body and tail . these features should be demonstrated in the body and tail of pancreas only . dilated side branches ( minor ) : presence of 3 tubular anechoic structures each measuring 1 mm in width and communicating with mpd . main pancreatic duct dilatation ( minor ) : mpd diameter 3.5 mm in the pancreatic body or 1.5 mm in the pancreatic tail . endoscopic ultrasound diagnosis of chronic pancreatitis based upon rosemont criteria consistent with cp : one major a feature with 3 minor features or 1 major a feature and major b feature or 2 major a features.suggestive of cp : one major a feature with <3 minor features or 1 major b feature with 3 minor features or 5 minor features.indeterminate for cp : 3 or 4 minor features with no major features or major b feature alone or with <3 minor features . indeterminate for cp : 3 or 4 minor features with no major features or major a number of conditions such as aging , smoking , obesity , and chronic alcohol consumption may cause similar eus changes in the pancreas . although , rosemont criteria has refined the past criteria with more objectivity initial studies have failed to demonstrate any significant improvement in kappa scores . to decrease interobserver variability adjunctive imaging technologies like digital image analysis and elastography have been used for diagnosis of cp with encouraging results . however , further studies with larger sample size are needed to determine the exact diagnostic role of these ancillary eus techniques . the eus procedure for looking for changes of cp is similar to the standard eus examination of the pancreas . both radial and linear eus have been shown to be equally accurate for the diagnosis of cp . radial eus , however , provides cross sectional imaging similar to other cross sectional imaging modalities like ct and this makes the imaging of mpd easier as it is seen as a long linear structure in contrast to a dot like appearance on linear eus . in order to correctly identify the changes of cp , the endoscopist needs to know the normal appearances of the pancreas as well as the normal variations seen in a nonpathological pancreas . the important things to keep in mind are : the normal pancreas has a diffusely speckled salt and pepper pattern of the body and tail with a barely visualized single , smooth and anechoic mpd.with the current advanced eus imaging systems side branches of mpd can also be seen in normal individuals especially the elderly and only if side branches are 1 mm , they are considered to be abnormal.even in normal individuals the mpd can be mildly tortuous and therefore the mpd should be carefully evaluated for a gradual decrease in its diameter from the head to tail , which is an important feature of the normal duct.the duct of alternating sizes or beading is abnormal.the dorsal and ventral pancreas can have different echogenicity with the dorsal anlage of the pancreas being more echogenic than the ventral pancreas . with the current advanced eus imaging systems side branches of mpd can also be seen in normal individuals especially the elderly and only if side branches are 1 mm , they are considered to be abnormal . as mentioned above most of these eus features of cp are visualized in the body and tail and currently it is not clear how best to diagnose focal cp in the head as the isolated eus findings like lobularity in the head with normal body and tail of pancreas are generally reported as normal . endoscopic ultrasound elastography has also been shown to be an accurate tool for the diagnosis of cp and strain ratio obtained using quantitative eus elastography has been shown to vary significantly in various rosemont classification groups thus allowing quantification of pancreatic fibrosis . in quantitative elastography , the tissue stiffness is measured in the targeted area ( region of interest [ roi ] a ) and outside the targeted area in a region representing normal tissue ( roi b ) . thereafter , the strain ratio value is calculated as the quotient b / a , which seems to be better and more objective than visual color changes of qualitative elastography [ figures 7 and 8 ] . in a study on eus elastography in cp , region b selected was the normal surrounding gastroduodenal wall and strain ratio cut - off of 2.25 yielded an accuracy of 91.1% for diagnosis of cp . the results appear promising , but further studies with larger sample size are needed to confirm these interesting results . although eus can demonstrate subtle alterations in the pancreatic parenchymal and ductal structure , the exact role of eus in the diagnosis of cp is still not established.the eus features to diagnose cp have evolved over a period of time from a pure qualitative approach to more advanced and complicated scoring systems incorporating multiple parenchymal and ductal eus features.the rosemont criteria have attempted to define precisely each eus criterion and thus have good inter - observer agreement . but , initial studies have failed to demonstrate any significant improvement in the inter - observer variability.a number of conditions such as aging , smoking , obesity , and chronic alcohol consumption may cause eus changes similar to cp . therefore , eus findings should be interpreted in appropriate clinical context.most of the eus features of cp except ductal calculi and cysts should be located in the body and tail only.in order to correctly identify the changes of cp , the endoscopist needs to know the normal appearances of the pancreas as well as the normal variations seen in nonpathological pancreas.quantitative eus elastography appear promising technique for diagnosis of cp . although eus can demonstrate subtle alterations in the pancreatic parenchymal and ductal structure , the exact role of eus in the diagnosis of cp is still not established . the eus features to diagnose cp have evolved over a period of time from a pure qualitative approach to more advanced and complicated scoring systems incorporating multiple parenchymal and ductal eus features . the rosemont criteria have attempted to define precisely each eus criterion and thus have good inter - observer agreement . but , initial studies have failed to demonstrate any significant improvement in the inter - observer variability . therefore most of the eus features of cp except ductal calculi and cysts should be located in the body and tail only . in order to correctly identify the changes of cp , the endoscopist needs to know the normal appearances of the pancreas as well as the normal variations seen in nonpathological pancreas .

although the hypothesis that gross neuroanatomical features may reflect the mental abilities of exceptionally talented individuals has held a long fascination ( amunts et al . , 2004 ; witelson et al . , 1999b ) , efforts to address it are frequently viewed with hesitation for several reasons : first , such studies have historically been associated with phrenology , which was rightfully dismissed at the end of the 19th century as a pseudoscience ( gould , 1981 ) . second , the extent to which one of the traditional foci of these studies , brain size , is correlated with intelligence is difficult to assess ( roth and dicke , 2005 ) . finally , although sulcal patterns have also been of interest , sulci usually do not correlate precisely with the borders of functionally defined cytoarchitectonic fields ( amunts et al . , 1999 ; zilles et al . , 1997 ) , some of which have been associated with exceptional competence , e.g. , the extraordinary cytoarchitectonic features in broca 's area of emil krebs ( 18671930 ) who was fluent in more than 60 languages ( amunts et al . , 2004 ) . despite these caveats , however , gross sulcal patterns have been associated with enlarged cortical representations that subserve functional specializations in mammals including carnivores ( welker and campos , 1963 ) and primates ( falk , 1982 ) , in a phenomenon called the principle of proper mass ( jerison , 1973 ) . raccoons , for example , have greatly enlarged primary somatosensory forepaw representations in which the various palm pad and digit areas are demarcated from one another by sulci , and this remarkable cortical morphology has been attributed to the fact that these animals use their forepaws extensively to explore their environments ( welker and campos , 1963 ) . it is also well known that dramatic changes may occur in sensory and motor cortices during a human 's lifetime as revealed by medical imaging studies of braille readers and upper limb amputees , which show that the cerebral cortex can exhibit long - term adaptations , including enlargement or relocation of specific representations such as those for hands ( amunts et al . , 1997 ) . further , gross cortical features entailing sulcal depths or patterns have been identified in people with exceptional abilities such as highly trained musicians ( amunts et al . , 1997 ; bangert and schlaug , 2006 ; schlaug , 2001 ) and the world - renowned physicist who is the subject of this report , albert einstein ( witelson et al . , 1999a , b ) . after his death in 1955 at 76 years of age , albert einstein 's brain was removed from his body by thomas harvey ( a pathologist ) , fixed , measured , photographed , and sectioned into 240 blocks that were embedded in celloidin ( lepore , 2001 ) . twelve sets of microscopic slides were prepared from the embedded blocks and distributed to various neuropathologists by harvey ( lepore , 2001 ) . it took decades , however , before papers began to appear on the histology or gross morphology of einstein 's brain . although the neuron : glial ratio was determined to be significantly smaller in einstein 's left than right brodmann 's area ( ba ) 39 using the kluver barrera stain ( diamond et al . , 1985 ) , this report has been criticized on methodological grounds ( hines , 1998 ) . another study determined that einstein 's prefrontal cortex had a greater neuronal density than those of normal controls because it packed approximately the same number of neurons into a thinner cortex ( anderson and harvey , 1996 ) , but one does not know the extent to which this was due to age , especially in people with superior intelligence ( shaw et al . , 2006 ) . because the blocks of einstein 's brain were embedded in celloidin , histological studies using golgi or other more revealing techniques would have been difficult if not impossible ( diamond et al . , 1985 ) . einstein 's cerebral cortex was thin ( anderson and harvey , 1996 ) and had widened sulci , which were normal for his age ( magnotta et al . , 1999 ) . his brain mass of 1230 g ( witelson et al . , 1999b ) was also unexceptional . gross anatomical studies of einstein 's brain included identifications for a number of sulci that were indicated on photographs ( witelson et al . , 1999a , b ) , and provided measurements that were obtained directly from the brain , as well as others that were measured from calibrated photographs ( witelson et al . the table in which the measurements appeared , however , did not indicate which ones were from each source , or when they were collected . after being removed and processed , what remained of einstein 's previously whole brain were histological slides , fragments of brain stored in a jar of formaldehyde , unspecified measurements that harvey obtained directly from the brain , and calibrated photographs ( lepore , 2001 ) . nevertheless , sulci may still be identified and interpreted from the extant photographs of einstein 's whole brain , in much the same way that cortical morphology is observed and studied on endocasts from fossils by paleoneurologists . it is hoped that the newly identified gyral and sulcal features reported below for einstein 's cerebral cortex will be of interest to future scholars . despite the fact that a large portion of einstein 's cerebral cortex was superficially unremarkable , regions in and near his primary somatosensory and motor cortices were highly unusual , and it is tentatively suggested that these may have contributed to the neuroanatomical substrates for some of his remarkable abilities . previously unrecognized gyral asymmetries and sulci are identified on published photographs of dorsal and lateral views of einstein 's whole brain ( witelson et al . , 1999b ) , using traditional landmarks ( brodmann , 1909 ; connolly , 1950 ; ono et al . , 1990 ; yousry et al . , , certain observations are quantified by comparing them to the range of measurements for normal humans published in ono et al . conflicting reports regarding einstein 's handedness have been resolved through photographic evidence that reveals einstein held pens , manipulated objects , and played the violin like a right - hander ( wolff and goodman , 2007 ) . results are tentatively interpreted in light of contemporary medical imaging studies ( bangert and schlaug , 2006 ; caulo et al . , 2007 ; falk et al . , 1991 ; steinmetz et al . , 1990 ; yousry et al . , 1997 ) and published details about einstein 's linguistic and musical abilities ( bucky , 1992 ; einstein , 1970 ; hadamard , 1945 ; wertheimer , 1959 ; wolff and goodman , 2007 ) . the controversial question of whether or not einstein had opercular cortices ( galaburda , 1999 ; witelson et al . my identifications for cortical sulci are indicated on photographs of einstein 's brain ( figure 1 ) . as far as i know , this is the first time that the following sulci have been identified on such photographs : angular ( a ) , anterior occipital ( a ) , diagonal ( d ) , descending terminal portion of the caudal sylvian ( dt ) , inferior frontal ( fi ) , middle frontal ( fm ) , superior frontal ( fs ) , precentral inferior and superior ( pci , pcs ) , marginal precentral ( pma ) , medial precentral ( pme ) , ascending ramus of sylvian fissure ( r ) , middle temporal ( tm ) , and superior temporal sulcus ( ts ) . the knob ( k ) representing hand motor cortex ( discussed below ) is also identified on einstein 's brain for the first time . photographs of einstein 's brain that were taken in 1955 , adapted from witelson et al . ( a ) dorsal view , ( b ) left lateral view , ( c ) right lateral view . sulci : angular ( a ) , anterior occipital ( a ) , ascending limb of the posterior sylvian fissure ( asyl ) , central fissure ( red lines ) , diagonal ( d ) , descending terminal portion of asyl ( dt ) , inferior frontal ( fi ) , middle frontal ( fm ) , superior frontal ( fs ) , horizontal limb of the posterior sylvian fissure ( hsyl ) , intraparietal ( ip ) , precentral inferior and superior ( pci , pcs ) , marginal precentral ( pma ) , medial precentral ( pme ) , postcentral inferior and superior ( pti , pts ) , ascending ramus of sylvian fissure ( r ) , subcentral posterior sulcus ( scp ) , middle temporal ( tm ) , superior temporal sulcus ( ts ) , unnamed sulcus in postcentral gyrus ( u ) . other features : branching point between hsyl and asyl ( white dots , b ) , hand motor cortex knob ( k , shaded in a , c ) , termination of asyl ( white dots , s ) . the central fissure ( c , red in figure 1 ) forms the boundary between the postcentral gyrus that represents primary somatosensory cortex and the precentral gyrus that contains motor cortex . on the lateral surface of the brain , einstein 's postcentral gyri were noticeably wider at their lateral compared to medial ends ( figures 1b , c ) , contrary to measurements from 25 human cadavers , which revealed that the widths of pre- and postcentral gyri are very similar along their entire lengths and manifest little asymmetry between hemispheres ( ono et al . einstein 's left postcentral gyrus contained a long unnamed sulcus ( u ) parallel to c ( figure 1b ) , which suggests expansion in depth as well as width in the cortical regions that normally represent face and tongue ( penfield and rasmussen , 1968 ) . [ although this unnamed sulcus has been identified elsewhere as the retrocentralis transversus ( rct ) ( witelson et al . , 1999a ) , rct is much shorter and triradiate , as detailed by connolly ( connolly , 1950:208209 ) . ] widths of pre- and postcentral gyri ( mm ) in left and right hemispheres from 25 human cadavers . as illustrated , the range for the medial measurement for the right m is 922 mm rather than 912 mm , which is a typo in ono et al . ( c. d. abernathey , personal communication ) . note that , contrary to einstein 's brain in which the postcentral gyri are noticeably wider in their lateral compared to medial ends , particularly in the left hemisphere ( figures 1b , c ) , the widths of pre- and postcentral gyri in normal individuals are very similar along their entire lengths and manifest little asymmetry between hemispheres . the medial parts of einstein 's sensory / motor strip manifested several unusual features ( figure 1a ) : on both sides , the precentral superior and inferior sulci ( pcs and pci ) were continuous , contrary to the normal condition in which the precentral sulcus is separated into two or more segments that characterized 98% of the 50 hemispheres scored by ono et al . the medial extent of the left postcentral gyrus was unusually narrow ( compare figures 1 and 2 ) , while on the right it was interrupted by a knob - shaped fold of precentral gyrus , or knob ( k ; shaded in figures 1a , c ) , that protruded into c , causing the latter 's middle knee ( genu ) to merge superficially with the postcentral superior sulcus ( pts ) . although this knob of hand motor cortex is usually better defined in deeper planes ( caulo et al . , 2007 ; yousry et al . , 1997 ) , it sometimes appears on the brain 's surface in the perirolandic region as an omega sign ( bangert and schlaug , 2006 ) , which was the case for einstein 's right hemisphere ( k ; figure 1a ) . functional imaging studies on normal humans reveal that the knob extends from the surface to the base of the precentral gyrus , and is typically larger in the hemisphere that is contralateral to the preferred hand ( caulo et al . , 2007 ; although einstein was right - handed ( wolff and goodman , 2007 ) , his superficial knob appeared larger in the right hemisphere , which may have been related to his musical training ( see below ) . my identifications of postcentral superior ( pts ) and inferior ( pti ) are based on their relationship to the intraparietal sulcus ( ip ) ( connolly , 1950 ) . as reported by witelson et al ( 1999b ) , einstein 's pti , which defines the caudal boundary of the postcentral gyrus , connected bilaterally with the termination ( s ) of the ascending limb of the posterior sylvian fissure ( asyl ) , instead of coursing separately and more rostral to asyl as is typical ( figures 1b , c ) . connolly discusses this variation and notes that , although rare , it is more likely to occur on the right hemisphere ( connolly , 1950:210 ) . additionally , he illustrates this pattern in the left hemisphere of one child ( p. 177 ) . my identification of the level at which asyl and pti meet in einstein 's right hemisphere [ at the white dot labeled s ( the caudal termination of the sylvian fissure ) in figure 1c ] is noticeably lower than witelson et al . it is not clear when or by whom the arrows were placed on witelson et al . 's photographs , or what methods were used to determine the terminal points of the sylvian fissures . ideally , one would determine the point ( s ) of confluence of asyl and pti by examining their submerged morphology ( connolly , 1950 ; steinmetz et al . einstein 's gross brain is no longer available , however , and there is no indication that the relevant blocks / sections were analyzed for submerged sulcal patterns . my location for right s is based on a number of observations : in lateral view , the sulcus at this level appears relatively wide compared to the medially located and slightly arched pti that merges with it , similar to einstein 's left hemisphere ( figures 1b , c ) . these locations result in lengths and positions of pts and pti that are relatively balanced in the two hemispheres ( figure 1 ) , and yield an asyl that is shorter in the right than left hemisphere , consistent with a statistically significant finding in a 3d mr study on eight normal volunteers ( falk et al . , 1991 ) . a descending terminal ( dt ) portion of asyl appears in einstein 's right hemisphere ( compare with ono et al . the distance between the lateral ends of c and pti ( which coincides with s in einstein 's brain ) appears shorter on the right than left side , which is consistent with the reported distance of 3.5 cm between these two points in einstein 's left hemisphere and 2.0 cm in the right hemisphere [ measurement 22 in witelson et al . 's ( 1999b ) table ] . on the other hand , witelson et al . , 1999b : figures 1b , c ) reveal an obviously longer distance between the lateral ends of c and pti ( = s indicated by arrow ) on the right , which contradicts measurement 22 in their table . the lateral end of einstein 's left c is also more rostral than its counterpart on the right ( compare figures 1b , c , this paper ) , which is another significant asymmetry that characterizes normal people ( falk et al . these data suggest that the correct location for s in einstein 's right hemisphere is the one presented here . of 58 brains from normal humans that could be scored bilaterally , a pattern of pti connecting with s similar to einstein 's appeared in 8 ( 13.7% ) of the right hemispheres and 1 ( 1.7% ) of the left hemispheres , but never appeared in both hemispheres of the same brain ( steinmetz et al . , if one generalizes from this sample and makes the assumption that the left and right occurrences are independent , then the odds of it occurring on both sides of one individual 's brain ( as it did on einstein 's ) are 0.137 0.017 = 0.002 ( or 0.2 of 1% ) . because of this unusual sulcal pattern , einstein 's asyls were not capped by continuous supramarginal gyri ( figure 3 ) that represent an important language area in the left hemisphere , brodmann 's area 40 ( ba 40 , brodmann , 1909 ) . as discussed below , this morphology may have been related to einstein 's delayed acquisition and use of language . ( a ) typical distribution of brodmann 's areas 39 , 40 , and 43 ( brodmann , 1909 ) . ba 40 constitutes the supramarginal gyrus , which caps the fissure between b and s. a minimal region that encloses ba 40 may be defined by the sylvian fissure , and lines that connect the end of scp with s and the latter with the end of an unnamed sulcus that extends caudally from b. ( b ) these landmarks are available in einstein 's left hemisphere and enclose an area that probably approximates a minimal surface representation of ba 40 . a supramarginal gyrus containing ba 40 did not cap asyl , however , because the latter was continuous with pti . whether or not einstein had parietal opercula has been debated ( galaburda , 1999 ; witelson et al . operculum derives from the latin operire , which means to close or shut and refers to a lid or cover . cortical opercula begin to develop around the fifth month in the human fetus as parietal , temporal and , eventually , frontal cortices expand and gradually cover the ( previously ) exposed insula ( connolly , 1950 ) . normally , the parietal operculum is located between the lateral end of c and the termination of asyl and includes lateral portions of the inferior postcentral gyrus ( ba 43 ) and rostral supramarginal gyrus ( ba 40 ) ( steinmetz et al . , 1990 ) ( figure 3a ) , although most of it is buried on the superior bank within the sylvian fissure ( eickhoff et al . , 2006 ) . it is reasonable to speculate that the supramarginal gyrus began to develop prenatally in einstein 's left hemisphere but was subsequently divided when pti merged with asyl , consistent with connolly 's discussion of the order in which prenatal opercula and postcentral sulci develop ( connolly , 1950 ) . numerous landmarks suggest that the cortex directly rostral to asyl in einstein 's brain was part of ba 40 and that a separate portion of this area also occurred caudal to asyl above an unnamed sulcus stemming from the branching point ( b ) in the left hemisphere at the junction of the horizontal limb of the posterior sylvian fissure ( hsyl ) and asyl ( brodmann , 1909 ; eickhoff et al . , 2006 ; ba 40 and 43 normally share a border near the subcentral posterior sulcus ( scp ) ( brodmann , 1909 ; eickhoff et al . , 2006 ) , which is present on einstein 's left hemisphere and permits tentative identification of ba 43 in addition to ba 40 ( figure 3b ) . these observations support galaburda 's identification of a left parietal operculum in einstein 's brain ( galaburda , 1999 ) . einstein 's right insula is also covered with an operculum that probably contained ba 43 which may , or may not , have extended onto the brain 's lateral surface ( eickhoff et al . , 2006 ) one would need to examine the cytoarchitecture within the superior bank of the sylvian fissure in this hemisphere to determine whether the operculum also contained part of ba 40 ( eickhoff et al . , 2006 ) . given how the brain was processed , however , it seems unlikely that such a study could be done . whether or not einstein had parietal opercula has been debated ( galaburda , 1999 ; witelson et al . operculum derives from the latin operire , which means to close or shut and refers to a lid or cover . cortical opercula begin to develop around the fifth month in the human fetus as parietal , temporal and , eventually , frontal cortices expand and gradually cover the ( previously ) exposed insula ( connolly , 1950 ) . normally , the parietal operculum is located between the lateral end of c and the termination of asyl and includes lateral portions of the inferior postcentral gyrus ( ba 43 ) and rostral supramarginal gyrus ( ba 40 ) ( steinmetz et al . , 1990 ) ( figure 3a ) , although most of it is buried on the superior bank within the sylvian fissure ( eickhoff et al . , 2006 ) . it is reasonable to speculate that the supramarginal gyrus began to develop prenatally in einstein 's left hemisphere but was subsequently divided when pti merged with asyl , consistent with connolly 's discussion of the order in which prenatal opercula and postcentral sulci develop ( connolly , 1950 ) . numerous landmarks suggest that the cortex directly rostral to asyl in einstein 's brain was part of ba 40 and that a separate portion of this area also occurred caudal to asyl above an unnamed sulcus stemming from the branching point ( b ) in the left hemisphere at the junction of the horizontal limb of the posterior sylvian fissure ( hsyl ) and asyl ( brodmann , 1909 ; eickhoff et al . , 2006 ; steinmetz et al . , 1990 ) ( figure 3b ) . ba 40 and 43 normally share a border near the subcentral posterior sulcus ( scp ) ( brodmann , 1909 ; eickhoff et al . , 2006 ) , which is present on einstein 's left hemisphere and permits tentative identification of ba 43 in addition to ba 40 ( figure 3b ) . these observations support galaburda 's identification of a left parietal operculum in einstein 's brain ( galaburda , 1999 ) . einstein 's right insula is also covered with an operculum that probably contained ba 43 which may , or may not , have extended onto the brain 's lateral surface ( eickhoff et al . , 2006 ) one would need to examine the cytoarchitecture within the superior bank of the sylvian fissure in this hemisphere to determine whether the operculum also contained part of ba 40 ( eickhoff et al . given how the brain was processed , however , it seems unlikely that such a study could be done . exceptional abilities are sometimes manifested in cortical symmetries or asymmetries that depart from those of normal controls ( amunts et al . , 2004 ) . for example , the better performances of musicians with perfect pitch are associated with relatively enlarged left planum temporale ( schlaug et al . , 1995 ) , and early commencement of musical training is sometimes associated with pronounced structural differences in hand representations ( schlaug , 2001 ) that are reflected in cortical sulcal patterns ( bangert and schlaug , 2006 ) . professional keyboard players have deeper and more symmetrical ( in terms of depth ) central sulci within their sensorimotor hand representations than normal controls , which appears to be related to increased skill of the nondominant hand as a result of early training ( amunts et al . , 1997 ; jancke et al . , the cytoarchitecture of ba 44 was more symmetric in the polyglot emil krebs than in controls , while that of his ba 45 was more asymmetric than theirs ( amunts et al . , 2004 ) . einstein 's brain was characterized by an unusual mixture of symmetrical and asymmetrical features . a rare convergence of the postcentral sulcus with the sylvian fissure ( steinmetz et al . , , 1999b ) , which nonetheless manifested a marked degree of asymmetry in the width of the lateral postcentral gyrus that favored the left hemisphere , and a pronounced knob in the right hemisphere . these asymmetries together with an atypical lack of uniformity in the medial and lateral widths of the pre - and post central sulci ( ono et al . , 1990 ) indicate that the gross anatomy of albert einstein 's brain in and around the primary somatosensory and motor cortices was , indeed , unusual . musicians have more pronounced cortical knobs than nonmusicians , and right - handed string - players ( as opposed to pianists ) tend to have differentially pronounced superficial knobs on their right rather than left hemispheres , especially if they began their musical training early in life ( bangert and schlaug , 2006 ) . correspondingly , somatosensory representations for the left digits of right - handed violinists are larger than those of controls ( especially if they began training before the age of 12 ) , presumably because their performances engage their left digits more than the right ones that manipulate the bow ( elbert et al . , einstein 's differentially enlarged knob ( k ) on the surface of his right hemisphere is consistent with the fact that he was a right - handed string - player who took violin lessons from age 6 to 14 years ( bangert and schlaug , 2006 ; bucky , 1992 ) . although there is no proof that musical training induces changes in hand representations of musicians , the correlation between early commencement of training and pronounced structural differences ( amunts et al . , 1997 ) , including superficially prominent knobs ( bangert and schlaug , 2006 ) , suggests that these features developed in response to individuals ' lifetime experiences ( schlaug , 2001 ) , which does not rule out a genetic component . an earlier report concluded that einstein 's visuospatial and mathematical cognition may have been influenced by relatively expanded parietal regions ( witelson et al . recent neuroanatomical and functional imaging studies suggest , further , that cortical features reported here may also have been related to einstein 's self - reported preference for thinking in sensory impressions including visual images rather than words ( einstein , 1970 ) . normally , ba 40 is one continuous region that is involved with short - term maintenance of phonemes and syllables during language tasks ( galaburda et al . , 2002 ) , and damage to the left supramarginal gyrus and its underlying white matter may result in a profound disability in which words are no longer a means of expression of thoughts , although the individual may still be capable of thinking ( crosby et al . , 1962 ) . it is therefore tempting to speculate that the unusual superficial cleaving of ba 40 and seamless melding of its rostral portion with the postcentral gyrus in einstein 's left hemisphere ( figures 1b and 3b ) may have been associated with his well - known delay at acquiring language and the fact that he repeated sentences to himself softly until the age of about seven ( wolff and goodman , 2007 ) . as an adult as they are written or spoken , do not seem to play any role in my mechanism of thought . the psychical entities which seem to serve as elements in thought are certain signs and more or less clear images which can be einstein laughed when informed that many people always think in words ( wertheimer , 1959 ) , and emphasized that concepts became meaningful for him only through their connection with sense - experiences ( einstein , 1970 ) . family members and friends have documented that , when stuck on a physics problem , einstein would play the violin until , suddenly , he would announce excitedly , it is interesting to contemplate that such synthesizing may have contributed to einstein 's insights , and that his extraordinary abilities may , to some degree , have been associated with the unusual gross anatomy of his cerebral cortex in and around the primary somatosensory and motor cortices . although these views are speculative , the identifications of previously unrecognized cortical morphology on einstein 's brain will , hopefully , be of use to future scholars who have access to new information and methodologies . the author declares that the research was conducted in the absence of commercial or financial relationships that could be construed as a potential conflict of interest .

in order to glean information about hominin ( or other ) brains that no longer exist , details of external neuroanatomy that are reproduced on endocranial casts ( endocasts ) from fossilized braincases may be described and interpreted . despite being , of necessity , speculative , such studies can be very informative when conducted in light of the literature on comparative neuroanatomy , paleontology , and functional imaging studies . albert einstein 's brain no longer exists in an intact state , but there are photographs of it in various views . applying techniques developed from paleoanthropology , previously unrecognized details of external neuroanatomy are identified on these photographs . this information should be of interest to paleoneurologists , comparative neuroanatomists , historians of science , and cognitive neuroscientists . the new identifications of cortical features should also be archived for future scholars who will have access to additional information from improved functional imaging technology . meanwhile , to the extent possible , einstein 's cerebral cortex is investigated in light of available data about variation in human sulcal patterns . although much of his cortical surface was unremarkable , regions in and near einstein 's primary somatosensory and motor cortices were unusual . it is possible that these atypical aspects of einstein 's cerebral cortex were related to the difficulty with which he acquired language , his preference for thinking in sensory impressions including visual images rather than words , and his early training on the violin .

Introduction Materials and Methods Results Did einstein have parietal opercula? Discussion Conflict of Interest Statement

, 1999b ) , efforts to address it are frequently viewed with hesitation for several reasons : first , such studies have historically been associated with phrenology , which was rightfully dismissed at the end of the 19th century as a pseudoscience ( gould , 1981 ) . second , the extent to which one of the traditional foci of these studies , brain size , is correlated with intelligence is difficult to assess ( roth and dicke , 2005 ) . finally , although sulcal patterns have also been of interest , sulci usually do not correlate precisely with the borders of functionally defined cytoarchitectonic fields ( amunts et al . despite these caveats , however , gross sulcal patterns have been associated with enlarged cortical representations that subserve functional specializations in mammals including carnivores ( welker and campos , 1963 ) and primates ( falk , 1982 ) , in a phenomenon called the principle of proper mass ( jerison , 1973 ) . raccoons , for example , have greatly enlarged primary somatosensory forepaw representations in which the various palm pad and digit areas are demarcated from one another by sulci , and this remarkable cortical morphology has been attributed to the fact that these animals use their forepaws extensively to explore their environments ( welker and campos , 1963 ) . it is also well known that dramatic changes may occur in sensory and motor cortices during a human 's lifetime as revealed by medical imaging studies of braille readers and upper limb amputees , which show that the cerebral cortex can exhibit long - term adaptations , including enlargement or relocation of specific representations such as those for hands ( amunts et al . , 1997 ; bangert and schlaug , 2006 ; schlaug , 2001 ) and the world - renowned physicist who is the subject of this report , albert einstein ( witelson et al . after his death in 1955 at 76 years of age , albert einstein 's brain was removed from his body by thomas harvey ( a pathologist ) , fixed , measured , photographed , and sectioned into 240 blocks that were embedded in celloidin ( lepore , 2001 ) . it took decades , however , before papers began to appear on the histology or gross morphology of einstein 's brain . although the neuron : glial ratio was determined to be significantly smaller in einstein 's left than right brodmann 's area ( ba ) 39 using the kluver barrera stain ( diamond et al . another study determined that einstein 's prefrontal cortex had a greater neuronal density than those of normal controls because it packed approximately the same number of neurons into a thinner cortex ( anderson and harvey , 1996 ) , but one does not know the extent to which this was due to age , especially in people with superior intelligence ( shaw et al . because the blocks of einstein 's brain were embedded in celloidin , histological studies using golgi or other more revealing techniques would have been difficult if not impossible ( diamond et al . einstein 's cerebral cortex was thin ( anderson and harvey , 1996 ) and had widened sulci , which were normal for his age ( magnotta et al . gross anatomical studies of einstein 's brain included identifications for a number of sulci that were indicated on photographs ( witelson et al . after being removed and processed , what remained of einstein 's previously whole brain were histological slides , fragments of brain stored in a jar of formaldehyde , unspecified measurements that harvey obtained directly from the brain , and calibrated photographs ( lepore , 2001 ) . nevertheless , sulci may still be identified and interpreted from the extant photographs of einstein 's whole brain , in much the same way that cortical morphology is observed and studied on endocasts from fossils by paleoneurologists . it is hoped that the newly identified gyral and sulcal features reported below for einstein 's cerebral cortex will be of interest to future scholars . despite the fact that a large portion of einstein 's cerebral cortex was superficially unremarkable , regions in and near his primary somatosensory and motor cortices were highly unusual , and it is tentatively suggested that these may have contributed to the neuroanatomical substrates for some of his remarkable abilities . previously unrecognized gyral asymmetries and sulci are identified on published photographs of dorsal and lateral views of einstein 's whole brain ( witelson et al . , , certain observations are quantified by comparing them to the range of measurements for normal humans published in ono et al . conflicting reports regarding einstein 's handedness have been resolved through photographic evidence that reveals einstein held pens , manipulated objects , and played the violin like a right - hander ( wolff and goodman , 2007 ) . results are tentatively interpreted in light of contemporary medical imaging studies ( bangert and schlaug , 2006 ; caulo et al . , 1997 ) and published details about einstein 's linguistic and musical abilities ( bucky , 1992 ; einstein , 1970 ; hadamard , 1945 ; wertheimer , 1959 ; wolff and goodman , 2007 ) . my identifications for cortical sulci are indicated on photographs of einstein 's brain ( figure 1 ) . as far as i know , this is the first time that the following sulci have been identified on such photographs : angular ( a ) , anterior occipital ( a ) , diagonal ( d ) , descending terminal portion of the caudal sylvian ( dt ) , inferior frontal ( fi ) , middle frontal ( fm ) , superior frontal ( fs ) , precentral inferior and superior ( pci , pcs ) , marginal precentral ( pma ) , medial precentral ( pme ) , ascending ramus of sylvian fissure ( r ) , middle temporal ( tm ) , and superior temporal sulcus ( ts ) . the knob ( k ) representing hand motor cortex ( discussed below ) is also identified on einstein 's brain for the first time . photographs of einstein 's brain that were taken in 1955 , adapted from witelson et al . on the lateral surface of the brain , einstein 's postcentral gyri were noticeably wider at their lateral compared to medial ends ( figures 1b , c ) , contrary to measurements from 25 human cadavers , which revealed that the widths of pre- and postcentral gyri are very similar along their entire lengths and manifest little asymmetry between hemispheres ( ono et al . as illustrated , the range for the medial measurement for the right m is 922 mm rather than 912 mm , which is a typo in ono et al . note that , contrary to einstein 's brain in which the postcentral gyri are noticeably wider in their lateral compared to medial ends , particularly in the left hemisphere ( figures 1b , c ) , the widths of pre- and postcentral gyri in normal individuals are very similar along their entire lengths and manifest little asymmetry between hemispheres . the medial parts of einstein 's sensory / motor strip manifested several unusual features ( figure 1a ) : on both sides , the precentral superior and inferior sulci ( pcs and pci ) were continuous , contrary to the normal condition in which the precentral sulcus is separated into two or more segments that characterized 98% of the 50 hemispheres scored by ono et al . the medial extent of the left postcentral gyrus was unusually narrow ( compare figures 1 and 2 ) , while on the right it was interrupted by a knob - shaped fold of precentral gyrus , or knob ( k ; shaded in figures 1a , c ) , that protruded into c , causing the latter 's middle knee ( genu ) to merge superficially with the postcentral superior sulcus ( pts ) . , 1997 ) , it sometimes appears on the brain 's surface in the perirolandic region as an omega sign ( bangert and schlaug , 2006 ) , which was the case for einstein 's right hemisphere ( k ; figure 1a ) . functional imaging studies on normal humans reveal that the knob extends from the surface to the base of the precentral gyrus , and is typically larger in the hemisphere that is contralateral to the preferred hand ( caulo et al . , 2007 ; although einstein was right - handed ( wolff and goodman , 2007 ) , his superficial knob appeared larger in the right hemisphere , which may have been related to his musical training ( see below ) . my identifications of postcentral superior ( pts ) and inferior ( pti ) are based on their relationship to the intraparietal sulcus ( ip ) ( connolly , 1950 ) . as reported by witelson et al ( 1999b ) , einstein 's pti , which defines the caudal boundary of the postcentral gyrus , connected bilaterally with the termination ( s ) of the ascending limb of the posterior sylvian fissure ( asyl ) , instead of coursing separately and more rostral to asyl as is typical ( figures 1b , c ) . connolly discusses this variation and notes that , although rare , it is more likely to occur on the right hemisphere ( connolly , 1950:210 ) . my identification of the level at which asyl and pti meet in einstein 's right hemisphere [ at the white dot labeled s ( the caudal termination of the sylvian fissure ) in figure 1c ] is noticeably lower than witelson et al . einstein 's gross brain is no longer available , however , and there is no indication that the relevant blocks / sections were analyzed for submerged sulcal patterns . my location for right s is based on a number of observations : in lateral view , the sulcus at this level appears relatively wide compared to the medially located and slightly arched pti that merges with it , similar to einstein 's left hemisphere ( figures 1b , c ) . these locations result in lengths and positions of pts and pti that are relatively balanced in the two hemispheres ( figure 1 ) , and yield an asyl that is shorter in the right than left hemisphere , consistent with a statistically significant finding in a 3d mr study on eight normal volunteers ( falk et al . the distance between the lateral ends of c and pti ( which coincides with s in einstein 's brain ) appears shorter on the right than left side , which is consistent with the reported distance of 3.5 cm between these two points in einstein 's left hemisphere and 2.0 cm in the right hemisphere [ measurement 22 in witelson et al . on the other hand , witelson et al . the lateral end of einstein 's left c is also more rostral than its counterpart on the right ( compare figures 1b , c , this paper ) , which is another significant asymmetry that characterizes normal people ( falk et al . these data suggest that the correct location for s in einstein 's right hemisphere is the one presented here . of 58 brains from normal humans that could be scored bilaterally , a pattern of pti connecting with s similar to einstein 's appeared in 8 ( 13.7% ) of the right hemispheres and 1 ( 1.7% ) of the left hemispheres , but never appeared in both hemispheres of the same brain ( steinmetz et al . , if one generalizes from this sample and makes the assumption that the left and right occurrences are independent , then the odds of it occurring on both sides of one individual 's brain ( as it did on einstein 's ) are 0.137 0.017 = 0.002 ( or 0.2 of 1% ) . because of this unusual sulcal pattern , einstein 's asyls were not capped by continuous supramarginal gyri ( figure 3 ) that represent an important language area in the left hemisphere , brodmann 's area 40 ( ba 40 , brodmann , 1909 ) . as discussed below , this morphology may have been related to einstein 's delayed acquisition and use of language . ba 40 constitutes the supramarginal gyrus , which caps the fissure between b and s. a minimal region that encloses ba 40 may be defined by the sylvian fissure , and lines that connect the end of scp with s and the latter with the end of an unnamed sulcus that extends caudally from b. ( b ) these landmarks are available in einstein 's left hemisphere and enclose an area that probably approximates a minimal surface representation of ba 40 . normally , the parietal operculum is located between the lateral end of c and the termination of asyl and includes lateral portions of the inferior postcentral gyrus ( ba 43 ) and rostral supramarginal gyrus ( ba 40 ) ( steinmetz et al . , 1990 ) ( figure 3a ) , although most of it is buried on the superior bank within the sylvian fissure ( eickhoff et al . it is reasonable to speculate that the supramarginal gyrus began to develop prenatally in einstein 's left hemisphere but was subsequently divided when pti merged with asyl , consistent with connolly 's discussion of the order in which prenatal opercula and postcentral sulci develop ( connolly , 1950 ) . numerous landmarks suggest that the cortex directly rostral to asyl in einstein 's brain was part of ba 40 and that a separate portion of this area also occurred caudal to asyl above an unnamed sulcus stemming from the branching point ( b ) in the left hemisphere at the junction of the horizontal limb of the posterior sylvian fissure ( hsyl ) and asyl ( brodmann , 1909 ; eickhoff et al . , 2006 ) , which is present on einstein 's left hemisphere and permits tentative identification of ba 43 in addition to ba 40 ( figure 3b ) . these observations support galaburda 's identification of a left parietal operculum in einstein 's brain ( galaburda , 1999 ) . , 2006 ) one would need to examine the cytoarchitecture within the superior bank of the sylvian fissure in this hemisphere to determine whether the operculum also contained part of ba 40 ( eickhoff et al . , 1990 ) ( figure 3a ) , although most of it is buried on the superior bank within the sylvian fissure ( eickhoff et al . it is reasonable to speculate that the supramarginal gyrus began to develop prenatally in einstein 's left hemisphere but was subsequently divided when pti merged with asyl , consistent with connolly 's discussion of the order in which prenatal opercula and postcentral sulci develop ( connolly , 1950 ) . numerous landmarks suggest that the cortex directly rostral to asyl in einstein 's brain was part of ba 40 and that a separate portion of this area also occurred caudal to asyl above an unnamed sulcus stemming from the branching point ( b ) in the left hemisphere at the junction of the horizontal limb of the posterior sylvian fissure ( hsyl ) and asyl ( brodmann , 1909 ; eickhoff et al . , 2006 ) , which is present on einstein 's left hemisphere and permits tentative identification of ba 43 in addition to ba 40 ( figure 3b ) . these observations support galaburda 's identification of a left parietal operculum in einstein 's brain ( galaburda , 1999 ) . einstein 's right insula is also covered with an operculum that probably contained ba 43 which may , or may not , have extended onto the brain 's lateral surface ( eickhoff et al . , 1995 ) , and early commencement of musical training is sometimes associated with pronounced structural differences in hand representations ( schlaug , 2001 ) that are reflected in cortical sulcal patterns ( bangert and schlaug , 2006 ) . professional keyboard players have deeper and more symmetrical ( in terms of depth ) central sulci within their sensorimotor hand representations than normal controls , which appears to be related to increased skill of the nondominant hand as a result of early training ( amunts et al . , the cytoarchitecture of ba 44 was more symmetric in the polyglot emil krebs than in controls , while that of his ba 45 was more asymmetric than theirs ( amunts et al . einstein 's brain was characterized by an unusual mixture of symmetrical and asymmetrical features . a rare convergence of the postcentral sulcus with the sylvian fissure ( steinmetz et al . , , 1999b ) , which nonetheless manifested a marked degree of asymmetry in the width of the lateral postcentral gyrus that favored the left hemisphere , and a pronounced knob in the right hemisphere . , 1990 ) indicate that the gross anatomy of albert einstein 's brain in and around the primary somatosensory and motor cortices was , indeed , unusual . musicians have more pronounced cortical knobs than nonmusicians , and right - handed string - players ( as opposed to pianists ) tend to have differentially pronounced superficial knobs on their right rather than left hemispheres , especially if they began their musical training early in life ( bangert and schlaug , 2006 ) . , einstein 's differentially enlarged knob ( k ) on the surface of his right hemisphere is consistent with the fact that he was a right - handed string - player who took violin lessons from age 6 to 14 years ( bangert and schlaug , 2006 ; bucky , 1992 ) . recent neuroanatomical and functional imaging studies suggest , further , that cortical features reported here may also have been related to einstein 's self - reported preference for thinking in sensory impressions including visual images rather than words ( einstein , 1970 ) . , 2002 ) , and damage to the left supramarginal gyrus and its underlying white matter may result in a profound disability in which words are no longer a means of expression of thoughts , although the individual may still be capable of thinking ( crosby et al . it is therefore tempting to speculate that the unusual superficial cleaving of ba 40 and seamless melding of its rostral portion with the postcentral gyrus in einstein 's left hemisphere ( figures 1b and 3b ) may have been associated with his well - known delay at acquiring language and the fact that he repeated sentences to himself softly until the age of about seven ( wolff and goodman , 2007 ) . the psychical entities which seem to serve as elements in thought are certain signs and more or less clear images which can be einstein laughed when informed that many people always think in words ( wertheimer , 1959 ) , and emphasized that concepts became meaningful for him only through their connection with sense - experiences ( einstein , 1970 ) . family members and friends have documented that , when stuck on a physics problem , einstein would play the violin until , suddenly , he would announce excitedly , it is interesting to contemplate that such synthesizing may have contributed to einstein 's insights , and that his extraordinary abilities may , to some degree , have been associated with the unusual gross anatomy of his cerebral cortex in and around the primary somatosensory and motor cortices . although these views are speculative , the identifications of previously unrecognized cortical morphology on einstein 's brain will , hopefully , be of use to future scholars who have access to new information and methodologies . the author declares that the research was conducted in the absence of commercial or financial relationships that could be construed as a potential conflict of interest .

participants were enrolled between september 2010 and january 2012 at 50 pah centers across the united states , all of which received institutional review board approval . patients were eligible if they had been diagnosed with pah ( group 1 pulmonary hypertension ) and were parenteral - naive or parenteral - transitioned from another formulation of epoprostenol or treprostinil therapy to rts - epo . patients were followed for a maximum of 1 year or until discontinuation of rts - epo , withdrawal of consent , loss to follow - up , death , or end of study . patient data were collected at enrollment and quarterly intervals ( months 3 , 6 , 9 , and 12 ) ( e - table 1 ) . clinical characteristics , medical history , and comorbid conditions , including pulmonary function tests , echocardiogram , new york heart association functional class ( nyha fc ) evaluation , and 6-min walk distance ( 6mwd ) were collected using the most recent clinical visits , hospitalization , and/or telephone contacts . the treating physician determined renal insufficiency by selecting a checkbox in the electronic case report form . among those patients who had a creatinine recorded , estimated dosing regimen and titration schedule for rts - epo and concomitant pah medications were also collected quarterly . clinical outcomes from prospect included demographics and disease characteristics of patients receiving rts - epo . safety outcomes included all - cause deaths and hospitalizations and blood stream infections ( bsis ) . hospitalizations were assigned by the treating physician as related to the use of rts - epo , due to pah , both , or neither . patients who discontinued the study for any reason were censored on the date of discontinuation . baseline demographic and clinical characteristics were summarized using percentages for categorical variables and means sd for continuous variables . the reveal risk score calculator was included , which incorporates 19 variables that predict 1-year survival in patients with pah . patient characteristics were summarized by hospitalization status ( hospitalized vs not hospitalized ) , survival status ( survived vs died ) , and sex . rates of on - study hospitalizations ( defined as those with a date of hospital admission after the date of enrollment ) per 1,000 patient - days were calculated for all patients and by prostacyclin history ( parenteral - naive or parenteral - transitioned ) . survival over the 1 year of follow - up was estimated based on kaplan - meier curves . for patients who died during follow - up , length of survival patients who did not die were censored at the earlier of their date of discontinuation from the study or the date of the last data collection . freedom from hospitalization , survival estimates , and ses were summarized for all patients , and by prostacyclin history , sex , and chronic renal insufficiency ( cri ) . participants were enrolled between september 2010 and january 2012 at 50 pah centers across the united states , all of which received institutional review board approval . patients were eligible if they had been diagnosed with pah ( group 1 pulmonary hypertension ) and were parenteral - naive or parenteral - transitioned from another formulation of epoprostenol or treprostinil therapy to rts - epo . patients were followed for a maximum of 1 year or until discontinuation of rts - epo , withdrawal of consent , loss to follow - up , death , or end of study . patient data were collected at enrollment and quarterly intervals ( months 3 , 6 , 9 , and 12 ) ( e - table 1 ) . no treatments or assessments clinical characteristics , medical history , and comorbid conditions , including pulmonary function tests , echocardiogram , new york heart association functional class ( nyha fc ) evaluation , and 6-min walk distance ( 6mwd ) were collected using the most recent clinical visits , hospitalization , and/or telephone contacts . the treating physician determined renal insufficiency by selecting a checkbox in the electronic case report form . among those patients who had a creatinine recorded , estimated dosing regimen and titration schedule for rts - epo and concomitant pah medications were also collected quarterly . clinical outcomes from prospect included demographics and disease characteristics of patients receiving rts - epo . safety outcomes included all - cause deaths and hospitalizations and blood stream infections ( bsis ) . hospitalizations were assigned by the treating physician as related to the use of rts - epo , due to pah , both , or neither . patients who discontinued the study for any reason were censored on the date of discontinuation . baseline demographic and clinical characteristics were summarized using percentages for categorical variables and means sd for continuous variables . the reveal risk score calculator was included , which incorporates 19 variables that predict 1-year survival in patients with pah . patient characteristics were summarized by hospitalization status ( hospitalized vs not hospitalized ) , survival status ( survived vs died ) , and sex . rates of on - study hospitalizations ( defined as those with a date of hospital admission after the date of enrollment ) per 1,000 patient - days were calculated for all patients and by prostacyclin history ( parenteral - naive or parenteral - transitioned ) . survival over the 1 year of follow - up was estimated based on kaplan - meier curves . for patients who died during follow - up , length of survival was calculated from the date of enrollment to the date of death reported . patients who did not die were censored at the earlier of their date of discontinuation from the study or the date of the last data collection . freedom from hospitalization , survival estimates , and ses were summarized for all patients , and by prostacyclin history , sex , and chronic renal insufficiency ( cri ) . a total of 354 patients enrolled in prospect ; 18 did not have pah . thus , the analytic cohort included the remaining 336 patients . the clinical characteristics of all patients , and by prostacyclin history at enrollment , are summarized in table 2 . patient characteristics at baseline by hospitalization status , survival status ( over 1 year of follow - up ) , and sex are summarized in table 3 . the most common reason for initiation to rts - epo was pah progression in the parenteral - naive group ( 57.1% ) and other in the parenteral - transitioned group ( 42.3% ) ( e - table 2 ) . among the 18 non - group 1 patients , chronic thromboembolic pulmonary hypertension and sarcoidosis each represented approximately one - third ( table 4 ) . concomitant pah medications at start of rts - epo in the non - group 1 patients are summarized in table 2 . a summary of concomitant medications at enrollment for the patients in the non patient demographics and comorbid conditions at enrollment ( n = 336 ) data are given as mean sd or no . clinical characteristics at enrollment for all patients and by prostacyclin history data are given as mean sd or no . 6mwd = 6-min walk distance ; era = endothelial receptor antagonist ; mpap = mean pulmonary arterial pressure ; nyha = new york heart association ; pde5i = type 5 phosphodiesterase inhibitor ; pah = pulmonary arterial hypertension ; pcwp = pulmonary capillary wedge pressure ; pvri = pulmonary vascular resistance index ( wood units m ) ; rap = right arterial pressure ; rts - epo = room temperature stable - epoprostenol ; svo2 = venous oxygen saturation . includes n = 119 patients who were prostacyclin - naive , n = 26 patients who transitioned from an inhaled prostacyclin , and n = 2 patients who transitioned from an oral prostacyclin at study entry . includes n = 180 patients who transitioned from epoprostenol and n = 9 patients who transitioned from treprostinil at study entry . the initial dose at time of initiation of prolonged rts - epo , which may not be at enrollment . baseline demographic and clinical characteristics categorized by hospitalization status , survival status ( > 1 y of follow - up ) , and sex data are given as mean sd or no . apah = associated pulmonary hypertension ; chd = congenital heart disease ; ctd = connective tissue disease ; hpah = heritable pulmonary arterial hypertension ; ipah = idiopathic pulmonary arterial hypertension ; poph = portopulmonary hypertension ; pphn = persistent pulmonary hypertension of the newborn ; reveal registry = registry to evaluate early and long - term pulmonary arterial hypertension disease management . concomitant medication history at enrollment for non - who group 1 patients data are given as no . chronic thromboembolic pulmonary hypertension ( 5 ) , sarcoidosis ( 6 ) , carcinoid syndrome ( 1 ) , pulmonary veno - occlusive disease / pulmonary capillary hemangiomatosis ( 1 ) , lung disease ( 1 ) , pulmonary hypoplasia , giant omphalocele ( 1 ) , left - sided heart disease / morbid obesity ( 1 ) , post - liver transplant pulmonary hypertension ( 1 ) , and pulmonary hypertension due to underlying infl ammatory myopathy ( 1 ) . patients requiring hospitalization in the first year were more likely to be men , have pah associated with connective tissue disease ( ctd ) , be nyha fc iii or iv , have a lower mean 6mwd , have a higher mean reveal registry risk score , and were more likely to be parenteral prostanoid - naive compared with patients who were not hospitalized . patients who died during the study were older , were more likely to have pah associated with ctd , be nyha fc iv , have a lower mean 6mwd , have a higher mean reveal registry risk score , and were more likely to be parenteral prostanoid - naive compared with patients who survived . men comprised about 20% of the surviving patients in prospect ; 43.1% of the deaths occurred in men . men were more likely than women to have familial pah , congenital pah , or pah associated with portopulmonary hypertension . a higher mean reveal registry risk score was observed in men , who were also more likely to be parenteral prostanoid - naive at enrollment compared with women . a total of 296 on - study all - cause hospitalizations occurred in 157 patients ( 150 were parenteral - naive patients and 146 were parenteral - transitioned patients ) . the median length of stay was longer for parenteral - naive patients than for parenteral - transitioned patients ( median 5.0 , interquartile range : 3.0 , 9.0 vs median 4.0 , interquartile range : 2.0 , 7.0 , respectively ) . of the hospitalized patients ( n = 157 ) , 77 ( 49.0% ) were not rehospitalized , 51 ( 32.5% ) were rehospitalized once , 12 ( 7.6% ) were rehospitalized twice , and 17 ( 10.8% ) were rehospitalized three or more times during the 1-year follow - up . there were no differences in frequencies of rehospitalization between parenteral - naive and parenteral - transitioned groups . the overall rate of all - cause hospitalizations in group 1 patients with pah was 2.8 per 1,000 patient - days ( 3.3 per 1,000 patient - days in parenteral - naive patients and 2.4 per 1,000 patient - days in parenteral - transitioned patients [ fig 1 ] ) . hospitalization rates for the non - group 1 patients was 2.0 per 1,000 patient - days . rates of on - study hospitalizations * ( per 1,000 patient - d ) in the prospect cohort by prostacyclin history . * on - study hospitalization defined as those with a date of hospital admission after the date of enrollment into prospect . the most common causes of hospitalizations were infections ( 51 ) and heart failures ( 48 ) . thirty - seven percent of hospitalizations were categorized as being related to pah ( 44% in the parenteral - naive group and 31% in the parenteral - transitioned group ) . all causes of hospitalizations ( with at least five occurrences ) in patients during 1 y of follow - up in prospect hlgt = high - level group term ; hlt = high - level term ; nec = not elsewhere classified . see table 3 legend for expansion of other abbreviations . hospitalizations were coded using the meddra coding dictionary based on hlgt , with additional detail provided from the hlt for the most common hlgts . other causes include : cardiac therapeutic procedures ( 4 ) ; device issues ( 4 ) ; respiratory tract therapeutic procedures ( 4 ) ; bone and joint injuries ( 3 ) ; bronchial disorders ( excluding neoplasms ) ( 3 ) ; diabetic complications ( 3 ) ; investigations , imaging , and histopathology procedures ( nec ) ( 3 ) ; pericardial disorders ( 3 ) ; viral infectious disorders ( 3 ) ; anal and rectal conditions ( nec ) ( 2 ) ; body temperature conditions ( 2 ) ; cardiac disorder signs and symptoms ( 2 ) ; endocrine gland therapeutic procedures ( 2 ) ; exocrine pancreas conditions ( 2 ) ; hepatic and hepatobiliary disorders ( 2 ) ; joint disorders ( 2 ) ; medication errors ( 2 ) ; therapeutic procedures and supportive care ( nec ) ( 2 ) ; cardiac and vascular investigations ( excluding enzyme tests ) ( 1 ) ; central nervous system vascular disorders ( 1 ) ; chemical injury and poisoning ( 1 ) ; ctds ( excluding congenital ) ( 1 ) ; coronary artery disorders ( 1 ) ; epidermal and dermal conditions ( 1 ) ; gi conditions ( nec ) ( 1 ) ; gi inflammatory conditions ( 1 ) ; gi motility and defecation conditions ( 1 ) ; gi neoplasms , malignant and unspecified ( 1 ) ; headaches ( 1 ) ; hepatobiliary investigations ( 1 ) ; hepatobiliary therapeutic procedures ( 1 ) ; lymphomas , non - hodgkin s unspecified histology ( 1 ) ; musculoskeletal and ctds ( nec ) ( 1 ) ; obstetric and gynecologic therapeutic procedures ( 1 ) ; parathyroid gland disorders ( 1 ) ; peritoneal and retroperitoneal conditions ( 1 ) ; platelet disorders ( 1 ) ; pleural disorders ( 1 ) ; rickettsial infectious disorders ( 1 ) ; skin and subcutaneous tissue disorders ( nec ) ( 1 ) ; therapeutic and nontherapeutic effects ( excluding toxicity ) ( 1 ) ; thyroid gland disorders ( 1 ) ; upper respiratory tract disorders ( excluding infections ) ( 1 ) . seven patients with a hospitalization admission date on the same date as enrollment were excluded from the outcomes analysis . a total of 21 bsis ( 0.20 per 1,000 patient - days on study ) developed in 16 patients , 19 of which resulted in hospitalizations ( 10 in parenteral - naive patients [ 0.24 per 1,000 patient - days ] and nine in parenteral - transitioned patients [ 0.16 per 1,000 patient - days ] ) . of the 21 bsis , 15 were gram - positive ( 71.4% ) , three were gram - negative ( 14.3% ) , and three were of unknown organism(s ) ( 14.3% ) . the 1-year freedom from hospitalization estimate ( se ) for the n = 336 group 1 patients was 51.0% 2.8% . the freedom from hospitalization estimate for patients who were parenteral - naive at enrollment was 42.8% 4.3% compared with 57.1% 3.7% for parenteral - transitioned patients the freedom from hospitalization estimate for men was 38.3% 5.9% ; for women , it was 54.6% 3.2% ( p < .015 ) ( fig 2b ) . patients with cri had a lower freedom from hospitalization compared with patients without cri ( 17.0% 8.4% vs 53.7% 2.9% ; p < .001 ) . a , b , freedom from hospitalization at 1 y by prostacyclin history ( a ) and sex ( b ) . the 1-year survival estimate ( se ) for all patients in prospect was 84.0% 2.1% . the 1-year survival estimate for patients who were classified as parenteral - naive at baseline was lower than the survival estimate for patients who were parenteral - transitioned ( 79.4% 3.4% vs 87.7% 2.5% ; p = .038 ) ( fig 3a ) . the 1-year survival estimate in men was lower than in women ( 71.0% 5.2% vs 88.0% 2.1% ; p < .001 ) ( fig 3b ) . a sensitivity analysis using an estimated glomerular filtration rate cutpoint rather than the qualitative definition of cri produced similarly significant results . a multivariate cox regression model of group 1 patients confirmed that sex and cri were independent risk factors , whereas there was no differences between naive and transition patients after adjusting for differences in risk profile . the 1-year survival estimate was lower than in patients without cri ( 63.4% 9.3% vs 86.0% 2.0% ; p < .001 ) . the 1-year survival estimate in the non - group 1 patients was 88.9% 7.4% . a , b , one - y survival by prostacyclin history ( a ) and sex ( b ) . multivariable predictors of mortality bnp = b - type natriuretic peptide ; nt = n - terminal pro b - type natriuretic pepetide . a total of 354 patients enrolled in prospect ; 18 did not have pah . thus , the analytic cohort included the remaining 336 patients . the clinical characteristics of all patients , and by prostacyclin history at enrollment , are summarized in table 2 . patient characteristics at baseline by hospitalization status , survival status ( over 1 year of follow - up ) , and sex are summarized in table 3 . the most common reason for initiation to rts - epo was pah progression in the parenteral - naive group ( 57.1% ) and other in the parenteral - transitioned group ( 42.3% ) ( e - table 2 ) . among the 18 non - group 1 patients , chronic thromboembolic pulmonary hypertension and sarcoidosis each represented approximately one - third ( table 4 ) . concomitant pah medications at start of rts - epo in the non - group 1 patients are summarized in table 2 . a summary of concomitant medications at enrollment for the patients in the non patient demographics and comorbid conditions at enrollment ( n = 336 ) data are given as mean sd or no . clinical characteristics at enrollment for all patients and by prostacyclin history data are given as mean sd or no . 6mwd = 6-min walk distance ; era = endothelial receptor antagonist ; mpap = mean pulmonary arterial pressure ; nyha = new york heart association ; pde5i = type 5 phosphodiesterase inhibitor ; pah = pulmonary arterial hypertension ; pcwp = pulmonary capillary wedge pressure ; pvri = pulmonary vascular resistance index ( wood units m ) ; rap = right arterial pressure ; rts - epo = room temperature stable - epoprostenol ; svo2 = venous oxygen saturation . includes n = 119 patients who were prostacyclin - naive , n = 26 patients who transitioned from an inhaled prostacyclin , and n = 2 patients who transitioned from an oral prostacyclin at study entry . includes n = 180 patients who transitioned from epoprostenol and n = 9 patients who transitioned from treprostinil at study entry . the initial dose at time of initiation of prolonged rts - epo , which may not be at enrollment . baseline demographic and clinical characteristics categorized by hospitalization status , survival status ( > 1 y of follow - up ) , and sex data are given as mean sd or no . apah = associated pulmonary hypertension ; chd = congenital heart disease ; ctd = connective tissue disease ; hpah = heritable pulmonary arterial hypertension ; ipah = idiopathic pulmonary arterial hypertension ; poph = portopulmonary hypertension ; pphn = persistent pulmonary hypertension of the newborn ; reveal registry = registry to evaluate early and long - term pulmonary arterial hypertension disease management . concomitant medication history at enrollment for non - who group 1 patients data are given as no . chronic thromboembolic pulmonary hypertension ( 5 ) , sarcoidosis ( 6 ) , carcinoid syndrome ( 1 ) , pulmonary veno - occlusive disease / pulmonary capillary hemangiomatosis ( 1 ) , lung disease ( 1 ) , pulmonary hypoplasia , giant omphalocele ( 1 ) , left - sided heart disease / morbid obesity ( 1 ) , post - liver transplant pulmonary hypertension ( 1 ) , and pulmonary hypertension due to underlying infl ammatory myopathy ( 1 ) . patients requiring hospitalization in the first year were more likely to be men , have pah associated with connective tissue disease ( ctd ) , be nyha fc iii or iv , have a lower mean 6mwd , have a higher mean reveal registry risk score , and were more likely to be parenteral prostanoid - naive compared with patients who were not hospitalized . patients who died during the study were older , were more likely to have pah associated with ctd , be nyha fc iv , have a lower mean 6mwd , have a higher mean reveal registry risk score , and were more likely to be parenteral prostanoid - naive compared with patients who survived . men comprised about 20% of the surviving patients in prospect ; 43.1% of the deaths occurred in men . men were more likely than women to have familial pah , congenital pah , or pah associated with portopulmonary hypertension . a higher mean reveal registry risk score was observed in men , who were also more likely to be parenteral prostanoid - naive at enrollment compared with women . a total of 296 on - study all - cause hospitalizations occurred in 157 patients ( 150 were parenteral - naive patients and 146 were parenteral - transitioned patients ) . the median length of stay was longer for parenteral - naive patients than for parenteral - transitioned patients ( median 5.0 , interquartile range : 3.0 , 9.0 vs median 4.0 , interquartile range : 2.0 , 7.0 , respectively ) . of the hospitalized patients ( n = 157 ) , 77 ( 49.0% ) were not rehospitalized , 51 ( 32.5% ) were rehospitalized once , 12 ( 7.6% ) were rehospitalized twice , and 17 ( 10.8% ) were rehospitalized three or more times during the 1-year follow - up . there were no differences in frequencies of rehospitalization between parenteral - naive and parenteral - transitioned groups . the overall rate of all - cause hospitalizations in group 1 patients with pah was 2.8 per 1,000 patient - days ( 3.3 per 1,000 patient - days in parenteral - naive patients and 2.4 per 1,000 patient - days in parenteral - transitioned patients [ fig 1 ] ) . hospitalization rates for the non - group 1 patients was 2.0 per 1,000 patient - days . rates of on - study hospitalizations * ( per 1,000 patient - d ) in the prospect cohort by prostacyclin history . * on - study hospitalization defined as those with a date of hospital admission after the date of enrollment into prospect . the most common causes of hospitalizations were infections ( 51 ) and heart failures ( 48 ) . thirty - seven percent of hospitalizations were categorized as being related to pah ( 44% in the parenteral - naive group and 31% in the parenteral - transitioned group ) . all causes of hospitalizations ( with at least five occurrences ) in patients during 1 y of follow - up in prospect hlgt = high - level group term ; hlt = high - level term ; nec = not elsewhere classified . see table 3 legend for expansion of other abbreviations . hospitalizations were coded using the meddra coding dictionary based on hlgt , with additional detail provided from the hlt for the most common hlgts . other causes include : cardiac therapeutic procedures ( 4 ) ; device issues ( 4 ) ; respiratory tract therapeutic procedures ( 4 ) ; bone and joint injuries ( 3 ) ; bronchial disorders ( excluding neoplasms ) ( 3 ) ; diabetic complications ( 3 ) ; investigations , imaging , and histopathology procedures ( nec ) ( 3 ) ; pericardial disorders ( 3 ) ; viral infectious disorders ( 3 ) ; anal and rectal conditions ( nec ) ( 2 ) ; body temperature conditions ( 2 ) ; cardiac disorder signs and symptoms ( 2 ) ; endocrine gland therapeutic procedures ( 2 ) ; exocrine pancreas conditions ( 2 ) ; hepatic and hepatobiliary disorders ( 2 ) ; joint disorders ( 2 ) ; medication errors ( 2 ) ; therapeutic procedures and supportive care ( nec ) ( 2 ) ; cardiac and vascular investigations ( excluding enzyme tests ) ( 1 ) ; central nervous system vascular disorders ( 1 ) ; chemical injury and poisoning ( 1 ) ; ctds ( excluding congenital ) ( 1 ) ; coronary artery disorders ( 1 ) ; epidermal and dermal conditions ( 1 ) ; gi conditions ( nec ) ( 1 ) ; gi inflammatory conditions ( 1 ) ; gi motility and defecation conditions ( 1 ) ; gi neoplasms , malignant and unspecified ( 1 ) ; headaches ( 1 ) ; hepatobiliary investigations ( 1 ) ; hepatobiliary therapeutic procedures ( 1 ) ; lymphomas , non - hodgkin s unspecified histology ( 1 ) ; musculoskeletal and ctds ( nec ) ( 1 ) ; obstetric and gynecologic therapeutic procedures ( 1 ) ; parathyroid gland disorders ( 1 ) ; peritoneal and retroperitoneal conditions ( 1 ) ; platelet disorders ( 1 ) ; pleural disorders ( 1 ) ; rickettsial infectious disorders ( 1 ) ; skin and subcutaneous tissue disorders ( nec ) ( 1 ) ; therapeutic and nontherapeutic effects ( excluding toxicity ) ( 1 ) ; thyroid gland disorders ( 1 ) ; upper respiratory tract disorders ( excluding infections ) ( 1 ) . seven patients with a hospitalization admission date on the same date as enrollment were excluded from the outcomes analysis . a total of 21 bsis ( 0.20 per 1,000 patient - days on study ) developed in 16 patients , 19 of which resulted in hospitalizations ( 10 in parenteral - naive patients [ 0.24 per 1,000 patient - days ] and nine in parenteral - transitioned patients [ 0.16 per 1,000 patient - days ] ) . of the 21 bsis , 15 were gram - positive ( 71.4% ) , three were gram - negative ( 14.3% ) , and three were of unknown organism(s ) ( 14.3% ) . the 1-year freedom from hospitalization estimate ( se ) for the n = 336 group 1 patients was 51.0% 2.8% . the freedom from hospitalization estimate for patients who were parenteral - naive at enrollment was 42.8% 4.3% compared with 57.1% 3.7% for parenteral - transitioned patients the freedom from hospitalization estimate for men was 38.3% 5.9% ; for women , it was 54.6% 3.2% ( p < .015 ) ( fig 2b ) . patients with cri had a lower freedom from hospitalization compared with patients without cri ( 17.0% 8.4% vs 53.7% 2.9% ; p < .001 ) . a , b , freedom from hospitalization at 1 y by prostacyclin history ( a ) and sex ( b ) . the 1-year survival estimate ( se ) for all patients in prospect was 84.0% 2.1% . the 1-year survival estimate for patients who were classified as parenteral - naive at baseline was lower than the survival estimate for patients who were parenteral - transitioned ( 79.4% 3.4% vs 87.7% 2.5% ; p = .038 ) ( fig 3a ) . the 1-year survival estimate in men was lower than in women ( 71.0% 5.2% vs 88.0% 2.1% ; p < .001 ) ( fig 3b ) . a sensitivity analysis using an estimated glomerular filtration rate cutpoint rather than the qualitative definition of cri produced similarly significant results . a multivariate cox regression model of group 1 patients confirmed that sex and cri were independent risk factors , whereas there was no differences between naive and transition patients after adjusting for differences in risk profile . the 1-year survival estimate was lower than in patients without cri ( 63.4% 9.3% vs 86.0% 2.0% ; p < .001 ) . the 1-year survival estimate in the non - group 1 patients was 88.9% 7.4% . a , b , one - y survival by prostacyclin history ( a ) and sex ( b ) . multivariable predictors of mortality bnp = b - type natriuretic peptide ; nt = n - terminal pro b - type natriuretic pepetide . see table 2 and 4 legends for expansion of other abbreviations . prospect is the first modern - day registry , to our knowledge , to assess outcomes in a large series of patients with pah taking epoprostenol and examine outcomes by prostacyclin history , risk of hospitalization , and mortality in patients in a real - world setting . the strengths of the study include the size of the cohort , broad inclusion criteria , and 1-year follow - up . this analysis shows a high burden of illness in these patients , with an overall 1-year probability of survival of 84% 2.1% and only about 50% remaining free of hospitalization at 1 year . specific subgroups within prospect were at higher risk of both hospitalization and mortality compared with their counterparts , in particular , patients who were parenteral - naive at enrollment , male patients , and patients with cri . mean reveal registry risk scores were higher in these subgroups , reconfirming the applicability of the score to this patient population . an interesting finding is the large proportion of hospitalizations due to various comorbid conditions which could be explained by patients with pah living longer and developing unrelated or unrecognized complications of pah . survival in patients with pah in the modern treatment era has improved compared with previously observed survival . however , as outcomes among pah subpopulations vary substantially , continuing rigorous disease management of pah is important . patient survival appears to be related to the ability of the right ventricle to adapt to the chronically elevated pulmonary artery pressure . hospitalizations have been recognized as not only an important outcome measure when assessing clinical efficacy in pah trials , but also an important prognostic factor in patients with pah . while prospect confirms that use of rts - epo is associated with greater survival rates than that historically expected overall survival of patients taking rts - epo was comparable to other studies of epoprostenol . however , these older studies were limited single - site studies of patients with pph and did not include higher - risk patients from associated pah subgroups . survival in patients with pph treated with epoprostenol depends on disease severity . in prospect , patients who were parenteral - naive at baseline had worse outcomes compared with patients who were parenteral - transitioned . these results support findings that earlier initiation of prostacyclin therapy or more aggressive therapy should be considered . a study found that treatment with oral therapy or parenteral prostacyclin as initial strategy was associated with a high survival rate in patients who were who functional class iii and whose pah was idiopathic , familial , or anorexigen associated . the authors suggest that the more potent prostacyclins be reserved for high - risk patients , such as those with evidence of disease progression or those with treatment failure . it should be noted that findings in this analysis may reflect survivor bias in those already on parenteral therapy or the more critical nature of the disease in patients who are parenteral - naive . sex differences observed in this analysis are consistent with other studies . although the incidence of pah is higher in women , survival is lower in men . the 5-year survival rate from diagnosis in the reveal registry was 52% in men compared with 62% in women . it is unclear why women have a better survival rate than men : some have suggested that women respond better to treatment options or that female sex hormones are mediating protective effects . a recent study suggests that differences in the right ventricular ejection fraction response to initiation of therapy in ipah explain a significant portion of the poorer survival observed in men . bsis are a critical concern and major contributor to hospitalizations in patients with pah receiving parenteral therapy . the rate of bsis observed in prospect was 0.20 per 1,000 patient - days , which is consistent with prior studies of bsis in epoprostenol - treated patients . in a retrospective cohort study from the centers for disease control , the rate of bsis was 0.42 bsis per 1,000 treatment - days for patients on iv epoprostenol . in the reveal registry , prospect is subject to the inherent limitations of uncontrolled , observational studies for data acquisition and interpretation . other limitations include the collection of hospitalization data , which was based on primary discharge diagnosis , and the broader categorization of causes of hospitalization , which removed some granularity in the data . although treatment initiation was proximate to enrollment for many patients , some survival bias likely exists . therefore , these results can not be generalized directly to newly treated or newly diagnosed patients . patients who are parenteral - naive at initiation of rts - epo therapy , male patients , and patients with cri require close monitoring and aggressive clinical management .

background : few studies have prospectively reported outcomes in patients with pulmonary arterial hypertension ( pah ) treated with epoprostenol in the modern - day era of oral therapy and combination treatments . the registry to prospectively describe use of epoprostenol for injection ( veletri , prolonged room temperature stable - epoprostenol [ rts - epo ] ) in patients with pulmonary arterial hypertension ( prospect ) was established to prospectively describe the course of pah in patients prescribed rts-epo.methods:prospect is a multicenter , us - based drug registry of primarily group 1 patients with pah treated with rts - epo who were parenteral - naive or parenteral - transitioned at enrollment . patients were followed until discontinuation of rts - epo , withdrawal , loss to follow - up , death , or end of study ( maximum 1 year ) . one - year freedom from hospitalization ( fh ) and survival estimates were summarized by prostacyclin history ( parenteral - naive or parenteral - transitioned ) , sex , and chronic renal insufficiency ( cri).results : a total of 336 patients were included . the overall 1-year fh estimate was 51.0% 2.8% and was lower in parenteral - naive patients than parenteral - transitioned patients ( 42.8% 4.3% vs 57.1% 3.7% , respectively ; p = .002 ) . fh estimates were lower in male patients than female patients ( 38.3% 5.9% vs 54.6% 3.2% , respectively ; p < .015 ) and in patients with cri than patients without cri ( 17.0% 8.4% vs 53.7% 2.9% , respectively ; p < .001 ) . the overall 1-year survival estimate was 84.0% 2.1% . survival was poorer in parenteral - naive patients , male patients , and patients with cri.conclusions:risk of hospitalization and mortality remain high in patients with pah . in particular , patients who are parenteral - naive at initiation of rts - epo therapy , male patients , and patients with cri require close monitoring and aggressive clinical management .

Materials and Methods Registry Design and Participants Data Collection Outcomes Statistical Analysis Results Patient Demographic and Clinical Characteristics Hospitalizations Rates and All Causes of Hospitalizations Freedom From Hospitalization Survival Estimates Discussion Conclusions Supplementary Material

patients were eligible if they had been diagnosed with pah ( group 1 pulmonary hypertension ) and were parenteral - naive or parenteral - transitioned from another formulation of epoprostenol or treprostinil therapy to rts - epo . patients were followed for a maximum of 1 year or until discontinuation of rts - epo , withdrawal of consent , loss to follow - up , death , or end of study . among those patients who had a creatinine recorded , estimated dosing regimen and titration schedule for rts - epo and concomitant pah medications were also collected quarterly . hospitalizations were assigned by the treating physician as related to the use of rts - epo , due to pah , both , or neither . the reveal risk score calculator was included , which incorporates 19 variables that predict 1-year survival in patients with pah . patient characteristics were summarized by hospitalization status ( hospitalized vs not hospitalized ) , survival status ( survived vs died ) , and sex . rates of on - study hospitalizations ( defined as those with a date of hospital admission after the date of enrollment ) per 1,000 patient - days were calculated for all patients and by prostacyclin history ( parenteral - naive or parenteral - transitioned ) . for patients who died during follow - up , length of survival patients who did not die were censored at the earlier of their date of discontinuation from the study or the date of the last data collection . freedom from hospitalization , survival estimates , and ses were summarized for all patients , and by prostacyclin history , sex , and chronic renal insufficiency ( cri ) . patients were eligible if they had been diagnosed with pah ( group 1 pulmonary hypertension ) and were parenteral - naive or parenteral - transitioned from another formulation of epoprostenol or treprostinil therapy to rts - epo . patients were followed for a maximum of 1 year or until discontinuation of rts - epo , withdrawal of consent , loss to follow - up , death , or end of study . among those patients who had a creatinine recorded , estimated dosing regimen and titration schedule for rts - epo and concomitant pah medications were also collected quarterly . hospitalizations were assigned by the treating physician as related to the use of rts - epo , due to pah , both , or neither . the reveal risk score calculator was included , which incorporates 19 variables that predict 1-year survival in patients with pah . patient characteristics were summarized by hospitalization status ( hospitalized vs not hospitalized ) , survival status ( survived vs died ) , and sex . rates of on - study hospitalizations ( defined as those with a date of hospital admission after the date of enrollment ) per 1,000 patient - days were calculated for all patients and by prostacyclin history ( parenteral - naive or parenteral - transitioned ) . for patients who died during follow - up , length of survival was calculated from the date of enrollment to the date of death reported . freedom from hospitalization , survival estimates , and ses were summarized for all patients , and by prostacyclin history , sex , and chronic renal insufficiency ( cri ) . the clinical characteristics of all patients , and by prostacyclin history at enrollment , are summarized in table 2 . patient characteristics at baseline by hospitalization status , survival status ( over 1 year of follow - up ) , and sex are summarized in table 3 . the most common reason for initiation to rts - epo was pah progression in the parenteral - naive group ( 57.1% ) and other in the parenteral - transitioned group ( 42.3% ) ( e - table 2 ) . among the 18 non - group 1 patients , chronic thromboembolic pulmonary hypertension and sarcoidosis each represented approximately one - third ( table 4 ) . concomitant pah medications at start of rts - epo in the non - group 1 patients are summarized in table 2 . 6mwd = 6-min walk distance ; era = endothelial receptor antagonist ; mpap = mean pulmonary arterial pressure ; nyha = new york heart association ; pde5i = type 5 phosphodiesterase inhibitor ; pah = pulmonary arterial hypertension ; pcwp = pulmonary capillary wedge pressure ; pvri = pulmonary vascular resistance index ( wood units m ) ; rap = right arterial pressure ; rts - epo = room temperature stable - epoprostenol ; svo2 = venous oxygen saturation . includes n = 119 patients who were prostacyclin - naive , n = 26 patients who transitioned from an inhaled prostacyclin , and n = 2 patients who transitioned from an oral prostacyclin at study entry . the initial dose at time of initiation of prolonged rts - epo , which may not be at enrollment . baseline demographic and clinical characteristics categorized by hospitalization status , survival status ( > 1 y of follow - up ) , and sex data are given as mean sd or no . chronic thromboembolic pulmonary hypertension ( 5 ) , sarcoidosis ( 6 ) , carcinoid syndrome ( 1 ) , pulmonary veno - occlusive disease / pulmonary capillary hemangiomatosis ( 1 ) , lung disease ( 1 ) , pulmonary hypoplasia , giant omphalocele ( 1 ) , left - sided heart disease / morbid obesity ( 1 ) , post - liver transplant pulmonary hypertension ( 1 ) , and pulmonary hypertension due to underlying infl ammatory myopathy ( 1 ) . patients requiring hospitalization in the first year were more likely to be men , have pah associated with connective tissue disease ( ctd ) , be nyha fc iii or iv , have a lower mean 6mwd , have a higher mean reveal registry risk score , and were more likely to be parenteral prostanoid - naive compared with patients who were not hospitalized . patients who died during the study were older , were more likely to have pah associated with ctd , be nyha fc iv , have a lower mean 6mwd , have a higher mean reveal registry risk score , and were more likely to be parenteral prostanoid - naive compared with patients who survived . a higher mean reveal registry risk score was observed in men , who were also more likely to be parenteral prostanoid - naive at enrollment compared with women . a total of 296 on - study all - cause hospitalizations occurred in 157 patients ( 150 were parenteral - naive patients and 146 were parenteral - transitioned patients ) . the median length of stay was longer for parenteral - naive patients than for parenteral - transitioned patients ( median 5.0 , interquartile range : 3.0 , 9.0 vs median 4.0 , interquartile range : 2.0 , 7.0 , respectively ) . of the hospitalized patients ( n = 157 ) , 77 ( 49.0% ) were not rehospitalized , 51 ( 32.5% ) were rehospitalized once , 12 ( 7.6% ) were rehospitalized twice , and 17 ( 10.8% ) were rehospitalized three or more times during the 1-year follow - up . the overall rate of all - cause hospitalizations in group 1 patients with pah was 2.8 per 1,000 patient - days ( 3.3 per 1,000 patient - days in parenteral - naive patients and 2.4 per 1,000 patient - days in parenteral - transitioned patients [ fig 1 ] ) . rates of on - study hospitalizations * ( per 1,000 patient - d ) in the prospect cohort by prostacyclin history . thirty - seven percent of hospitalizations were categorized as being related to pah ( 44% in the parenteral - naive group and 31% in the parenteral - transitioned group ) . all causes of hospitalizations ( with at least five occurrences ) in patients during 1 y of follow - up in prospect hlgt = high - level group term ; hlt = high - level term ; nec = not elsewhere classified . a total of 21 bsis ( 0.20 per 1,000 patient - days on study ) developed in 16 patients , 19 of which resulted in hospitalizations ( 10 in parenteral - naive patients [ 0.24 per 1,000 patient - days ] and nine in parenteral - transitioned patients [ 0.16 per 1,000 patient - days ] ) . the 1-year freedom from hospitalization estimate ( se ) for the n = 336 group 1 patients was 51.0% 2.8% . the freedom from hospitalization estimate for patients who were parenteral - naive at enrollment was 42.8% 4.3% compared with 57.1% 3.7% for parenteral - transitioned patients the freedom from hospitalization estimate for men was 38.3% 5.9% ; for women , it was 54.6% 3.2% ( p < .015 ) ( fig 2b ) . patients with cri had a lower freedom from hospitalization compared with patients without cri ( 17.0% 8.4% vs 53.7% 2.9% ; p < .001 ) . a , b , freedom from hospitalization at 1 y by prostacyclin history ( a ) and sex ( b ) . the 1-year survival estimate ( se ) for all patients in prospect was 84.0% 2.1% . the 1-year survival estimate for patients who were classified as parenteral - naive at baseline was lower than the survival estimate for patients who were parenteral - transitioned ( 79.4% 3.4% vs 87.7% 2.5% ; p = .038 ) ( fig 3a ) . the 1-year survival estimate in men was lower than in women ( 71.0% 5.2% vs 88.0% 2.1% ; p < .001 ) ( fig 3b ) . the 1-year survival estimate was lower than in patients without cri ( 63.4% 9.3% vs 86.0% 2.0% ; p < .001 ) . the 1-year survival estimate in the non - group 1 patients was 88.9% 7.4% . a , b , one - y survival by prostacyclin history ( a ) and sex ( b ) . the clinical characteristics of all patients , and by prostacyclin history at enrollment , are summarized in table 2 . patient characteristics at baseline by hospitalization status , survival status ( over 1 year of follow - up ) , and sex are summarized in table 3 . the most common reason for initiation to rts - epo was pah progression in the parenteral - naive group ( 57.1% ) and other in the parenteral - transitioned group ( 42.3% ) ( e - table 2 ) . among the 18 non - group 1 patients , chronic thromboembolic pulmonary hypertension and sarcoidosis each represented approximately one - third ( table 4 ) . concomitant pah medications at start of rts - epo in the non - group 1 patients are summarized in table 2 . clinical characteristics at enrollment for all patients and by prostacyclin history data are given as mean sd or no . 6mwd = 6-min walk distance ; era = endothelial receptor antagonist ; mpap = mean pulmonary arterial pressure ; nyha = new york heart association ; pde5i = type 5 phosphodiesterase inhibitor ; pah = pulmonary arterial hypertension ; pcwp = pulmonary capillary wedge pressure ; pvri = pulmonary vascular resistance index ( wood units m ) ; rap = right arterial pressure ; rts - epo = room temperature stable - epoprostenol ; svo2 = venous oxygen saturation . includes n = 119 patients who were prostacyclin - naive , n = 26 patients who transitioned from an inhaled prostacyclin , and n = 2 patients who transitioned from an oral prostacyclin at study entry . the initial dose at time of initiation of prolonged rts - epo , which may not be at enrollment . baseline demographic and clinical characteristics categorized by hospitalization status , survival status ( > 1 y of follow - up ) , and sex data are given as mean sd or no . patients requiring hospitalization in the first year were more likely to be men , have pah associated with connective tissue disease ( ctd ) , be nyha fc iii or iv , have a lower mean 6mwd , have a higher mean reveal registry risk score , and were more likely to be parenteral prostanoid - naive compared with patients who were not hospitalized . patients who died during the study were older , were more likely to have pah associated with ctd , be nyha fc iv , have a lower mean 6mwd , have a higher mean reveal registry risk score , and were more likely to be parenteral prostanoid - naive compared with patients who survived . a higher mean reveal registry risk score was observed in men , who were also more likely to be parenteral prostanoid - naive at enrollment compared with women . a total of 296 on - study all - cause hospitalizations occurred in 157 patients ( 150 were parenteral - naive patients and 146 were parenteral - transitioned patients ) . the median length of stay was longer for parenteral - naive patients than for parenteral - transitioned patients ( median 5.0 , interquartile range : 3.0 , 9.0 vs median 4.0 , interquartile range : 2.0 , 7.0 , respectively ) . of the hospitalized patients ( n = 157 ) , 77 ( 49.0% ) were not rehospitalized , 51 ( 32.5% ) were rehospitalized once , 12 ( 7.6% ) were rehospitalized twice , and 17 ( 10.8% ) were rehospitalized three or more times during the 1-year follow - up . there were no differences in frequencies of rehospitalization between parenteral - naive and parenteral - transitioned groups . the overall rate of all - cause hospitalizations in group 1 patients with pah was 2.8 per 1,000 patient - days ( 3.3 per 1,000 patient - days in parenteral - naive patients and 2.4 per 1,000 patient - days in parenteral - transitioned patients [ fig 1 ] ) . rates of on - study hospitalizations * ( per 1,000 patient - d ) in the prospect cohort by prostacyclin history . thirty - seven percent of hospitalizations were categorized as being related to pah ( 44% in the parenteral - naive group and 31% in the parenteral - transitioned group ) . all causes of hospitalizations ( with at least five occurrences ) in patients during 1 y of follow - up in prospect hlgt = high - level group term ; hlt = high - level term ; nec = not elsewhere classified . a total of 21 bsis ( 0.20 per 1,000 patient - days on study ) developed in 16 patients , 19 of which resulted in hospitalizations ( 10 in parenteral - naive patients [ 0.24 per 1,000 patient - days ] and nine in parenteral - transitioned patients [ 0.16 per 1,000 patient - days ] ) . the 1-year freedom from hospitalization estimate ( se ) for the n = 336 group 1 patients was 51.0% 2.8% . the freedom from hospitalization estimate for patients who were parenteral - naive at enrollment was 42.8% 4.3% compared with 57.1% 3.7% for parenteral - transitioned patients the freedom from hospitalization estimate for men was 38.3% 5.9% ; for women , it was 54.6% 3.2% ( p < .015 ) ( fig 2b ) . patients with cri had a lower freedom from hospitalization compared with patients without cri ( 17.0% 8.4% vs 53.7% 2.9% ; p < .001 ) . a , b , freedom from hospitalization at 1 y by prostacyclin history ( a ) and sex ( b ) . the 1-year survival estimate ( se ) for all patients in prospect was 84.0% 2.1% . the 1-year survival estimate for patients who were classified as parenteral - naive at baseline was lower than the survival estimate for patients who were parenteral - transitioned ( 79.4% 3.4% vs 87.7% 2.5% ; p = .038 ) ( fig 3a ) . the 1-year survival estimate in men was lower than in women ( 71.0% 5.2% vs 88.0% 2.1% ; p < .001 ) ( fig 3b ) . the 1-year survival estimate was lower than in patients without cri ( 63.4% 9.3% vs 86.0% 2.0% ; p < .001 ) . the 1-year survival estimate in the non - group 1 patients was 88.9% 7.4% . a , b , one - y survival by prostacyclin history ( a ) and sex ( b ) . prospect is the first modern - day registry , to our knowledge , to assess outcomes in a large series of patients with pah taking epoprostenol and examine outcomes by prostacyclin history , risk of hospitalization , and mortality in patients in a real - world setting . the strengths of the study include the size of the cohort , broad inclusion criteria , and 1-year follow - up . this analysis shows a high burden of illness in these patients , with an overall 1-year probability of survival of 84% 2.1% and only about 50% remaining free of hospitalization at 1 year . specific subgroups within prospect were at higher risk of both hospitalization and mortality compared with their counterparts , in particular , patients who were parenteral - naive at enrollment , male patients , and patients with cri . survival in patients with pah in the modern treatment era has improved compared with previously observed survival . hospitalizations have been recognized as not only an important outcome measure when assessing clinical efficacy in pah trials , but also an important prognostic factor in patients with pah . while prospect confirms that use of rts - epo is associated with greater survival rates than that historically expected overall survival of patients taking rts - epo was comparable to other studies of epoprostenol . survival in patients with pph treated with epoprostenol depends on disease severity . in prospect , patients who were parenteral - naive at baseline had worse outcomes compared with patients who were parenteral - transitioned . a study found that treatment with oral therapy or parenteral prostacyclin as initial strategy was associated with a high survival rate in patients who were who functional class iii and whose pah was idiopathic , familial , or anorexigen associated . it should be noted that findings in this analysis may reflect survivor bias in those already on parenteral therapy or the more critical nature of the disease in patients who are parenteral - naive . patients who are parenteral - naive at initiation of rts - epo therapy , male patients , and patients with cri require close monitoring and aggressive clinical management .

gender inequity impacts negatively on the health of women and children , including during pregnancy and the perinatal period ( caro 2009 ; gill , pande , and malhotra 2007 ; unfpa and promundo 2010 ) . a review of gender influences on child survival , for example , has documented the negative impacts of women 's limited capacity to influence household decision making , women 's lack of access to health - promoting resources , women 's heavy work load , restrictive gender norms and gender discrimination ( unicef and liverpool school of tropical medicine 2011 ) . addressing gender inequity is thus an essential part of strategies to improve maternal and newborn health ( greene et al . 2004 ) . historically , strategies to address the health impacts of gender inequity were focused on empowering women . this emphasis on increasing women 's autonomy has resulted in many documented gains for women . yet adopting an exclusive focus on women to address gender inequity is increasingly recognised as limited ( eves 2005 ; mumtaz and salway 2009 ; sternberg and hubley 2004 ) . while a separate focus on women and girls is important , an exclusive focus on women , rather than on gender as a social construct that affects both men and women , can not fully address gender inequity ( barker , ricardo , and nascimento 2007 ; barker et al . therefore , working with men as well as women has been recognised as key to successfully challenging and transforming gender roles and norms ( barker 2014 ; barker et al . 2010 ; eves 2005 ) . involving men in the health of women and newborns around the time of childbirth including but not limited to support for women during and after pregnancy , seeking skilled care for birth and complications , newborn care , nutrition and breastfeeding , family planning after childbirth and maternal mental health has the potential to directly address gender influences on maternal and newborn health outcomes.1 drawing on programme experience and theoretical work completed by barker and colleagues ( barker 2014 ; barker , ricardo , and nascimento 2007 ; barker et al . 2010 ) , this potential can be understood as threefold . first , working with men as well as women makes it possible for a programme to engage with how men and women interact within relationships and thereby directly target gender relations , which are continually reconstructed through the ways that women and men relate to each other ( barker , ricardo , and nascimento 2007 ; barker et al . 2010 ) . second , involving men in programming that is intended to address gender inequity acknowledges men 's capacity to act as agents of change and can support men to challenge pre - existing roles and norms surrounding masculinity , intimate partner relationships and parenting ( barker 2014 ) . third , given the dominance of men within most social structures , such as political and religious institutions , involving men is a means for a programme to engage with male - dominated social structures and potentially leverage that engagement to support men to ally with women in order to challenge patriarchal structures that reproduce gender inequities ( barker , ricardo , and nascimento 2007 ) . gender - transformative interventions actively examine and promote the transformation of harmful gender norms and seek to reduce inequalities between men and women to achieve desired outcomes ( kraft et al . 2014 , 125 ) . because male involvement provides opportunities to support improved maternal and newborn health outcomes by changing gender relations , gender roles and norms , and the structures that reproduce them , it can be defined as potentially gender - transformative . in this paper , we have used these three opportunities as a framework to guide our assessment of the emerging evidence base for male involvement against the potential of male involvement interventions to address gender influences on maternal and newborn health outcomes . the principle of involving men in maternal and newborn health ( as well as sexual and reproductive health ) as part of a wider strategy to address gender influences on health outcomes was endorsed two decades ago at the 1994 international conference for population and development ( sternberg and hubley 2004 ) . men were recognised to be not only clients with a right to healthcare and partners with a responsibility to support women 's and children 's health , but also agents of positive change with the ability to transform underlying gendered constraints on health ( greene et al . the recent upswell of interest in male involvement in maternal and newborn health can be traced to the 1994 conference , with its explicit emphasis on gender . despite this , the potential for male involvement to address gender inequity seems to rarely be made explicit in the maternal and newborn health sector . many health policymakers , researchers and programme planners have sought to encourage the positive involvement of men around the time of childbirth as a strategy to improve maternal and newborn health , without articulating whether or how men 's involvement is expected to change gender influences on health outcomes . indeed , a recurring critique has emerged that male involvement interventions commonly adopt a reductionist and instrumentalist approach that is focused on altering men 's behaviours , without addressing the underlying gender influences that drive these behaviours . such an approach can be the most feasible choice under certain programmatic conditions , including short - term interventions implemented in settings where gender - transformative approaches are unlikely to be readily accepted ( adeleye , aldoory , and parakoyi 2011 ; unfpa and promundo 2010 ) , but it can also undermine male involvement as a strategy to effect gender - transformative change ( barker and das 2004 ) . this paper has been developed following a recent systematic review of the evidence for male involvement in maternal and newborn health . the review sought to consolidate the evidence base for male involvement in maternal and newborn health . in an era of evidence - based policy and practice , this emerging evidence base will likely be used to inform global policy guidelines , to influence national and subnational policymaking and programming and to guide future research . approaches that are not supported by the emerging evidence base are less likely to be promoted or adopted . consequently , the studies that constitute the evidence base will influence how male involvement is understood and implemented in the maternal and newborn health sector . this paper assesses the emerging evidence base against the potential of male involvement strategies to address gender influences on maternal and newborn health outcomes . we conducted a secondary qualitative analysis of the evidence base for world health organization recommendations relating to male involvement interventions for maternal and newborn health ( world health organization 2015 ) . the impact of male involvement interventions around the time of childbirth on maternal and newborn health outcomes was examined as one part of a systematic review of maternal health intervention studies commissioned by the world health organization . the first stage identified , screened and mapped all maternal health intervention studies conducted in low- and middle - income countries between 2000 and 2012 . a broad and inclusive search strategy , described further in the online protocol ( mascot study group 2014 ) , encompassed both published and unpublished literature , drawn from academic and other databases and expert recommendation . all of these articles were screened on title and abstract , of which 4172 were screened on full text and 2340 were included in the mapping ( mascot / wotro 2013 ) . the second stage of the review sought to answer a series of specific review questions , one of which related to male involvement interventions for improved maternal and newborn health : what interventions employed with women , men , communities and community leaders to increase male involvement have been effective in increasing care - seeking behaviour during pregnancy , for child birth and after birth for the woman and newborn and in improving key maternal and newborn health outcomes?in this question , male involvement was defined broadly as strategies to increase the involvement of men . a total of 92 articles from the first stage of the review were eligible for screening . an additional 68 articles were sourced from existing systematic reviews and the reference lists of included articles . after exclusion of the duplicates , 119 articles were screened on full text , and pre - defined inclusion and exclusion criteria were applied . studies were only included where they reported on an intervention testing the impact of male involvement around the time of childbirth on pre - specified maternal and newborn health outcomes . studies were required to report on the impact of the intervention on one or more of the following outcomes : birth with a skilled attendant or in a facility , use of antenatal or postnatal care for the mother and newborn , uptake of essential maternal and child health interventions , maternal nutrition , newborn nutrition , birth and complication preparedness , maternal mortality , maternal morbidity , neonatal mortality and perinatal mortality . additionally , male involvement intervention studies were excluded where men 's involvement was sought only for the promotion of family planning or the prevention or treatment of sexually transmitted infections , including hiv . what interventions employed with women , men , communities and community leaders to increase male involvement have been effective in increasing care - seeking behaviour during pregnancy , for child birth and after birth for the woman and newborn and in improving key maternal and newborn health outcomes ? thirteen studies were identified as eligible following a rigorous systematic process intended to collate the available evidence for the impact of male involvement on maternal and newborn health , and have informed world health organization recommendations on this topic ( world health organization 2015 ) . the 13 included studies therefore constitute an important evidence base for male involvement interventions in maternal and newborn health programmes . we conducted a content analysis of the material extracted as part of the systematic review . this material included a description of the intervention , details about the people targeted by the intervention and people included in the study , and outcome measures relevant to maternal and newborn health or male involvement . the authors applied a critical gender lens to assess how the studies position men , and men 's involvement in maternal and newborn health , against the framework describing the potential of male involvement strategies detailed above : to engage with relations between men and women ; to support men to transform gender norms and roles ; and to challenge social structures dominated by men that reproduce gender inequities . three of the included studies describe facility - based interventions in south africa , india and nepal that delivered education sessions to men , usually by reaching men together with their pregnant female partners through existing antenatal care services ( kunene et al . education sessions covered topics including care and nutrition during pregnancy , birth preparedness and complications readiness , and family planning . one study describes a workplace - based intervention in turkey designed to deliver education sessions to groups of men but not women , with workplace physicians delivering information on topics including communication techniques , infant healthcare and fatherhood ( sahip and turan 2007 ) . two studies describe interventions in nepal and indonesia that used social marketing or mass media campaigns to reach men and other key family and community members with safe motherhood and birth preparedness and complications readiness messages ( sood et al . the remaining seven studies from india , bangladesh , nepal , pakistan , tanzania and eritrea describe community - based education and community outreach strategies to increase male involvement in pregnancy care , seeking skilled care for birth and complications , postnatal care and reproductive health , as well as to increase men 's awareness of maternal health issues more broadly ( fullerton , killian , and gass 2005 ; hossain and ross 2006 ; midhet and becker 2010 ; mushi , mpembeni , and jahn 2010 ; purdin , khan , and saucier 2009 ; sinha 2008 ; turan , tesfagiorghis , and polan 2011 ) . most of these interventions were focused primarily on male partners of pregnant women , and some additionally described a focus on male community members or community leaders . the majority of studies were designed to increase the involvement of male partners of pregnant women . however , studies also sought to reach men who were expectant fathers ( reached separately from their female partners ) , community health workers , religious or community leaders and general community members . interventions variously aimed to reach men as individuals , within family or household structures or through social networks or leadership groups . the involvement that interventions aimed to elicit from men was not always clearly defined ; across the studies , involvement included providing care and support to female partners during pregnancy , supporting uptake of health interventions and being present during antenatal care , postnatal care and childbirth , among other measures . there is clearly a range of different strategies to increase the involvement of men in maternal and newborn health . additionally , the studies were premised on a range of different explicit or implicit understandings of why male involvement in maternal and newborn health is desirable or appropriate . most studies viewed men as the gatekeepers to women 's health , as male partners or fathers who control the resources or make the decisions that allow women and newborns to access health care . for example : husbands are often the decision - makers when it comes to seeking medical care ( midhet and becker 2010 , n.p.)2 [ w]omen depend heavily on men for access to healthcare ( varkey et al . 2004 , 1 ) [ i]f women do not have support within the family , they often can not use the knowledge and skills that they gain ( sahip and turan 2007 , 845 ) [ w]omen 's ability to seek health care or implement lessons learned from health education interventions is often determined by the household head , usually the husband ( mullany , becker , and hindin 2007 , 166)engaging with men was thus seen in most studies as a way of facilitating decision - making at the household level to support health - promoting behaviours and care seeking by men 's female partners . beyond a focus on men as decision - makers , however , studies provided a range of different justifications for male involvement . husbands are often the decision - makers when it comes to seeking medical care ( midhet and becker 2010 , n.p.)2 [ w]omen depend heavily on men for access to healthcare ( varkey et al . 2004 , 1 ) [ i]f women do not have support within the family , they often can not use the knowledge and skills that they gain ( sahip and turan 2007 , 845 ) [ w]omen 's ability to seek health care or implement lessons learned from health education interventions is often determined by the household head , usually the husband ( mullany , becker , and hindin 2007 , 166 ) two studies rationalised the inclusion of men by describing their shared responsibility for the health and wellbeing of their female partners ( sood et al . two more studies conceptualised men as part of a larger community , to which they could make a positive contribution , rather than as an individual contributing to their immediate family ( hossain and ross 2006 ; sinha 2008 ) . two studies described benefits to men 's own health as a justification for increased male involvement in their family 's health and engagement with the health system:[m]en have relatively low use of reproductive health services and few contacts with reproductive health service providers ( sahip and turan 2007 , 844 ) in addressing men 's involvement it is important to consider how to frame their contact with the health system so that it will encourage their future and continued involvement ( kunene et al . 2004 , 2)only one study noted that male involvement interventions are warranted because men have a preference for greater involvement in maternal and newborn health:[m]en themselves would prefer that they play a more active role during pregnancy , delivery and infant care . 2004 , 2)the absence of a clear consensus among the 13 studies on what a male involvement strategy is , and why a strategy to increase male involvement is being adopted , is likely to , in part , reflect differences in the social and cultural contexts in which the studies were implemented . yet it is also illustrative of an ambiguity of intention in male involvement interventions that has been noted elsewhere ( montgomery , van der straten , and torjesen 2011 ) . when assessed against the three - point framework detailed above , the 13 studies do not describe interventions in a way that clearly documents the gender - transformative potential of male involvement . [ m]en have relatively low use of reproductive health services and few contacts with reproductive health service providers ( sahip and turan 2007 , 844 ) in addressing men 's involvement it is important to consider how to frame their contact with the health system so that it will encourage their future and continued involvement ( kunene et al . 2004 , 2 ) [ m]en themselves would prefer that they play a more active role during pregnancy , delivery and infant care . 2004 , 2 ) first , as described above , interventions focused on male involvement have the potential to work with men and women to directly address gender relations . the included studies were focused on men in relationships ; most interventions invited male participants to be involved as part of a couple , household or family unit , and authors in all studies tended to describe men in terms of their relationships with women . yet for example , despite the fact that most studies justified male involvement as a strategy to address household decision - making , only one study engaged directly with the dynamic of shared decision - making by measuring joint decision - making within couples ( varkey et al . several interventions included components such as couple counselling that could plausibly influence how men and women relate to each other , yet , with the exception of varkey and colleagues ( 2004 ) , findings relating to men 's relationships were not reported in the studies . anticipated outcomes for the interventions were generally defined as specific instances of support provided by men to women , such as saving money for emergency transportation in case of birth complications , rather than more substantive changes in how men and women relate to each other , such as changed patterns of communication and decision - making about what support a woman may want or need from her male partner during pregnancy and how he can best provide this . the fact that changes in how men and women relate to each other were generally beyond the scope of the 13 studies is an important gap in the emerging evidence base . where men 's relationships with women are not reported or considered an outcome of interest , this leaves little opportunity for studies to document any changes in gender relations following male involvement interventions . the second area of potential for male involvement interventions identified above is the opportunity provided by these interventions to support men 's ability to challenge gender roles and norms alongside women , premised on the recognition of men 's capacity to act as agents of change . yet men 's capacity to internalise and act on a desire for gender - transformative change was not well recognised in the 13 studies . the studies describe men 's external behaviours rather than their internalised identities , attitudes and subjective experiences . many of the studies did not survey or interview men during data collection , although the interventions clearly targeted men as participants . this was the case despite the fact that several studies acknowledged the importance of men 's attitudes on key study outcomes . additionally , numerous studies collected and reported sociodemographic information about female participants as a means to unpack the experiences of different groups of women , but this level of detail on male participants was not presented in any study . there was no qualitative reporting of men 's experiences with the male involvement programmes described , whereas in several studies limited qualitative information was reported for women . few studies explored men 's attitudes related to becoming involved in maternal and newborn health within their families . as noted above , only two studies made reference to men 's own preferences and identities as partners or fathers . overall , men were generally defined in terms of their utility for women and children , with men 's own subjective wishes and needs usually going unrecognised . the 13 studies did not capture changes in men 's internal identities , attitudes or motivations that may be associated with male involvement interventions . this means that men 's capacity to actively pursue gender - transformative change is not well documented within the emerging evidence base . the third area in which male involvement interventions are considered to have gender - transformative potential , as detailed above , is through supporting men 's ability to ally with women and challenge patriarchal social structures that reproduce gender inequity . several studies demonstrated an awareness of the role of existing social structures , such as religious groups and leadership committees , in reproducing gender roles and norms . while in some studies this awareness was confined to the background description of the study setting , others attempted to engage with these structures as part of the intervention ( hossain and ross 2006 ; purdin , khan , and saucier 2009 ; sood et al . 2004a , 2004b ) . by working with religious leaders , male elders or other influential figures , these interventions aimed to shift gender roles and norms to become more supportive of anticipated changes in behaviour among male partners targeted by the intervention . it was notable , however , that the studies did not aim to support men to challenge these structures , for example by advocating an increased role for women . rather , with two exceptions , interventions that engaged with social structures worked within or through existing structures , and no study reported that the intervention had included women as well as men in work done within these existing structures . the two exceptions to this were studies reporting that new social structures had been developed through the interventions maternal health volunteer discussion groups in eritrea ( turan , tesfagiorghis , and polan 2011 ) and community support systems for obstetric emergencies in bangladesh ( hossain and ross 2006 ) but it was unclear whether these new structures were intended to provide space for gender equitable discussion and decision - making . based on what was documented , studies that engaged with social structures did not aim to encourage men to ally with women to challenge the patriarchal nature of these structures . this indicates that the emerging evidence base is comprised of studies that did not capture the potential of male involvement strategies to engage with and transform social structures . in summary , the current evidence base does not describe interventions that directly address gender relations between men and women ; that support men to change their values , attitudes and identities , rather than simply their behaviours ; or that support men to ally with women to challenge patriarchal social structures . generally , although there was some variation between studies , the focus of the studies that constitute the emerging evidence base was to employ male involvement as a strategy to prevent men from taking actions that can harm women and newborns and support men to take actions that can improve maternal and newborn health . this strategic approach , which focuses almost exclusively on men 's actions and decisions , rather than their relationships or subjective experiences , has been critiqued in the literature on male involvement as reductionist and instrumentalist ( barker and das 2004 ) . first , an approach that focuses primarily on men 's actions rather than their subjective experiences is unlikely to create opportunities to engage with men 's agency and their capacity to reconstruct gender relations ( barker , ricardo , and nascimento 2007 ) . second , problematising individual men 's actions without recognising and challenging their broader context does not offer a way to address the patriarchal social structures through which individual men 's specific actions come to occur . an instrumentalist approach to male involvement neither constructs men as potential agents of positive change , nor supports transformative social change to challenge gender inequity ( barker and das 2004 ; greene et al . it is imperative to recognise , however , that an instrumentalist approach to male involvement can be the most feasible strategy to increase men 's engagement in maternal and newborn health in certain programmatic contexts . an exclusive focus on changing men 's behaviours may be particularly suited to short - term interventions implemented in settings where gender norms and roles are strongly enforced and transformative approaches are unlikely to be readily accepted by men , women and the broader community ( adeleye , aldoory , and parakoyi 2011 ; unfpa and promundo 2010 ) . additionally , in some cases pregnancy may not be a suitable time to encourage gender - transformative change , as both men and women can be vulnerable during this time and may not be open to change . in such scenarios , approaches that incorporate an understanding of the gender order in their particular setting , without seeking to transform it , may be the only viable short - term option for redressing some of the harms resulting from men 's gendered behaviours ( caro 2009 ) . 2014 , 125 ) . given that gender - accommodating approaches can be appropriate in certain contexts , the absence of a gender - transformative approach to male involvement should not be considered a gap in any individual study . in aggregate , however , the emerging evidence base is limited by the lack of evidence to support male involvement as a strategy for gender - transformative change . this shows a need for a body of work that approaches and documents male involvement differently , in order to broaden the current evidence base with studies illustrating the potential of male involvement to effect gender - transformative change . first , it is important to be able to describe models of male involvement in a way that clearly differentiates between instrumentalist and gender - transformative approaches . recognising that male involvement interventions will necessarily address gender relations , it is imperative that they be well theorised with respect to gender and gender - transformative change at interpersonal and social levels . maternal and newborn health could benefit from ample research and conceptual work in other areas of health that describes the impacts of male involvement in lower- and middle - income countries . male involvement has been explored more extensively in the gender - based violence , sexual and reproductive health and hiv / aids literature . for example , lundgren and colleagues ( 2005 ) and shattuck and colleagues ( 2011 ) provide good examples of gender - transformative family planning interventions in malawi and el salvador , respectively . both use clear theoretical models for proposing how and why the interventions would change attitudes as well as behaviours . there is also considerable research on male involvement in high - income countries , particularly in relation to fatherhood , and some of the findings and lessons from these experiences are likely to be applicable to other contexts . for example , alio and colleagues ( 2013 ) proposed a framework for male involvement consisting of four components ( accessibility , engagement , responsibility and couple 's relationship ) , which could be tested in other contexts . similarly , burgess ' ( 2004 , 2007 ) work on active fatherhood in the uk offers an example of a robust conceptualisation of male involvement in maternal and newborn health that could potentially be translated to other settings . makusha and richter 's study on maternal gatekeeping in kwazulu - natal in this special issue offers valuable insights into local conceptualisations of fatherhood in a low - income context ( makusha and richter , 2015 ) . in addition to the need for a clear theoretical approach , it is important to be able to measure male involvement interventions in a nuanced way that captures the difference between transformative and instrumentalist approaches . defining and measuring male involvement is a methodological challenge , as demonstrated by the diverse range of indicators adopted by the studies in this review and elsewhere in the literature . there is a tendency to use specific indicators of involvement , such as male attendance at the birth or assistance with transportation during pregnancy , without clear justification for how male involvement is defined or whether chosen indicators are representative of this definition . yet there are many ways in which men can be involved and many different motivations drive their involvement . a single , specific action is unlikely to be a meaningful measure for a man 's level of involvement . for example , the commonly used indicator of a man accompanying his female partner to antenatal care may be indicative of a man who is actively engaging in his partner 's pregnancy because he believes that he is a co - parent with his female partner ; the same indicator , however , could equally reflect a man 's view that independent mobility of his female partner is inappropriate , which does not necessarily correspond to his level of engagement with his partner 's pregnancy . some studies have attempted to measure the degree of engagement using cumulative measures of behaviours , such as a man accompanying his female partner to antenatal care , waiting in the waiting room , joining the appointment , talking to the health worker and discussing the appointment with his female partner afterwards ( byamugisha et al . 2010 ; iliyasu et al . however , this does not disentangle the motivations for these behaviours ( montgomery , van der straten , and torjesen 2011 ) . as has been discussed extensively elsewhere , some indicators of male involvement are more effective than others in capturing changes in gender relations , and more research needs to be done to identify and verify these indicators ( barker , ricardo , and nascimento 2007 ) . developing a strong conceptual base for male involvement interventions and integrating effective measures for documenting male involvement can be expected to support further development of male involvement as a promising area for addressing gender influences on maternal and newborn health . the 13 studies included in our secondary analysis were mostly identified as part of a rigorous systematic mapping designed to consolidate the evidence base for male involvement in maternal and newborn health , among other topics . this evidence base has informed global recommendations and will likely influence national and subnational policymaking and programming . the assessment of the evidence presented here must , however , be understood as limited by the particular framework through which the studies were identified . evaluation of conceptual approaches to male involvement was not the primary purpose of the original systematic review and did not specifically guide the inclusion or exclusion criteria of the review . the systematic review included studies that were intervention studies , rather than observational studies , commentaries or discussions of male involvement . additionally , studies were only included where they reported on pre - specified maternal and newborn health outcomes , which did not include certain key male involvement outcomes such as men 's support during pregnancy or joint decision - making about childbirth . for the above reasons , there may be other literature on the topic that is not included in the review , particularly qualitative studies or descriptive pieces that examine implementation processes and the theoretical basis of male involvement interventions in more detail . irrespective of the process used to identify these studies , however , they constitute an emerging evidence base , and the review demonstrates important gaps in this evidence base . an additional limitation is that articles did not necessarily document the complete male involvement strategy adopted . limited information was available on interventions , and it is not possible to know what aspects were unreported . in the absence of detailed information , there is a risk of reading the standard critique of instrumentalism into these studies , and we acknowledge that this may not accurately reflect how interventions were actually designed or implemented . nevertheless , given that these 13 studies comprise a body of evidence for male involvement in maternal and newborn health , the way that male involvement is documented and represented in this important sub - set of the literature matters separately from how the interventions were done : a critique of these studies ' reported approaches is a critique of how male involvement is currently understood in the emerging evidence base . the 13 studies included in our secondary analysis were mostly identified as part of a rigorous systematic mapping designed to consolidate the evidence base for male involvement in maternal and newborn health , among other topics . this evidence base has informed global recommendations and will likely influence national and subnational policymaking and programming . the assessment of the evidence presented here must , however , be understood as limited by the particular framework through which the studies were identified . evaluation of conceptual approaches to male involvement was not the primary purpose of the original systematic review and did not specifically guide the inclusion or exclusion criteria of the review . the systematic review included studies that were intervention studies , rather than observational studies , commentaries or discussions of male involvement . additionally , studies were only included where they reported on pre - specified maternal and newborn health outcomes , which did not include certain key male involvement outcomes such as men 's support during pregnancy or joint decision - making about childbirth . for the above reasons , there may be other literature on the topic that is not included in the review , particularly qualitative studies or descriptive pieces that examine implementation processes and the theoretical basis of male involvement interventions in more detail . irrespective of the process used to identify these studies , however , they constitute an emerging evidence base , and the review demonstrates important gaps in this evidence base . an additional limitation is that articles did not necessarily document the complete male involvement strategy adopted . limited information was available on interventions , and it is not possible to know what aspects were unreported . in the absence of detailed information , there is a risk of reading the standard critique of instrumentalism into these studies , and we acknowledge that this may not accurately reflect how interventions were actually designed or implemented . nevertheless , given that these 13 studies comprise a body of evidence for male involvement in maternal and newborn health , the way that male involvement is documented and represented in this important sub - set of the literature matters separately from how the interventions were done : a critique of these studies ' reported approaches is a critique of how male involvement is currently understood in the emerging evidence base . an assessment of the male involvement intervention studies identified through a comprehensive systematic review of maternal health interventions reveals important gaps in how male involvement is conceptualised in the emerging evidence base for male involvement in maternal and newborn health . emerging research , comprising the studies included in a systematic review commissioned to inform global recommendations relating to male involvement for maternal and newborn health , does not examine the gender - transformative potential of male involvement interventions . this points to the need for an approach to male involvement that is conceptualised and documented with closer reference to gender , in order to understand and document the potential of male involvement as a way of supporting health and gender equity . specifically , the included studies demonstrate the need for greater incorporation of gender - transformative conceptual approaches into future interventions , with effective measures built in to such interventions in order to develop the evidence base for their impact on a broad range of health and gender equity outcomes . we expect that the innovative approaches to male involvement included in this special issue will respond to some of these critical needs .

men 's involvement in the health of women and children is considered an important avenue for addressing gender influences on maternal and newborn health . the impact of male involvement around the time of childbirth on maternal and newborn health outcomes was examined as one part of a systematic review of maternal health intervention studies published between 2000 and 2012 . of 33,888 articles screened , 13 eligible studies relating to male involvement were identified . the interventions documented in these studies comprise an emerging evidence base for male involvement in maternal and newborn health . we conducted a secondary qualitative analysis of the 13 studies , reviewing content that had been systematically extracted . a critical assessment of this extracted content finds important gaps in the evidence base , which are likely to limit how male involvement is understood and implemented in maternal and newborn health policy , programmes and research . collectively , the studies point to the need for an evidence base that includes studies that clearly articulate and document the gender - transformative potential of involving men . this broader evidence base could support the use of male involvement as a strategy to improve both health and gender equity outcomes .

Background Methods Findings Discussion Limitations Conclusion Disclosure statement

gender inequity impacts negatively on the health of women and children , including during pregnancy and the perinatal period ( caro 2009 ; gill , pande , and malhotra 2007 ; unfpa and promundo 2010 ) . a review of gender influences on child survival , for example , has documented the negative impacts of women 's limited capacity to influence household decision making , women 's lack of access to health - promoting resources , women 's heavy work load , restrictive gender norms and gender discrimination ( unicef and liverpool school of tropical medicine 2011 ) . addressing gender inequity is thus an essential part of strategies to improve maternal and newborn health ( greene et al . involving men in the health of women and newborns around the time of childbirth including but not limited to support for women during and after pregnancy , seeking skilled care for birth and complications , newborn care , nutrition and breastfeeding , family planning after childbirth and maternal mental health has the potential to directly address gender influences on maternal and newborn health outcomes.1 drawing on programme experience and theoretical work completed by barker and colleagues ( barker 2014 ; barker , ricardo , and nascimento 2007 ; barker et al . first , working with men as well as women makes it possible for a programme to engage with how men and women interact within relationships and thereby directly target gender relations , which are continually reconstructed through the ways that women and men relate to each other ( barker , ricardo , and nascimento 2007 ; barker et al . because male involvement provides opportunities to support improved maternal and newborn health outcomes by changing gender relations , gender roles and norms , and the structures that reproduce them , it can be defined as potentially gender - transformative . in this paper , we have used these three opportunities as a framework to guide our assessment of the emerging evidence base for male involvement against the potential of male involvement interventions to address gender influences on maternal and newborn health outcomes . the principle of involving men in maternal and newborn health ( as well as sexual and reproductive health ) as part of a wider strategy to address gender influences on health outcomes was endorsed two decades ago at the 1994 international conference for population and development ( sternberg and hubley 2004 ) . the recent upswell of interest in male involvement in maternal and newborn health can be traced to the 1994 conference , with its explicit emphasis on gender . despite this , the potential for male involvement to address gender inequity seems to rarely be made explicit in the maternal and newborn health sector . many health policymakers , researchers and programme planners have sought to encourage the positive involvement of men around the time of childbirth as a strategy to improve maternal and newborn health , without articulating whether or how men 's involvement is expected to change gender influences on health outcomes . indeed , a recurring critique has emerged that male involvement interventions commonly adopt a reductionist and instrumentalist approach that is focused on altering men 's behaviours , without addressing the underlying gender influences that drive these behaviours . such an approach can be the most feasible choice under certain programmatic conditions , including short - term interventions implemented in settings where gender - transformative approaches are unlikely to be readily accepted ( adeleye , aldoory , and parakoyi 2011 ; unfpa and promundo 2010 ) , but it can also undermine male involvement as a strategy to effect gender - transformative change ( barker and das 2004 ) . this paper has been developed following a recent systematic review of the evidence for male involvement in maternal and newborn health . the review sought to consolidate the evidence base for male involvement in maternal and newborn health . approaches that are not supported by the emerging evidence base are less likely to be promoted or adopted . consequently , the studies that constitute the evidence base will influence how male involvement is understood and implemented in the maternal and newborn health sector . this paper assesses the emerging evidence base against the potential of male involvement strategies to address gender influences on maternal and newborn health outcomes . we conducted a secondary qualitative analysis of the evidence base for world health organization recommendations relating to male involvement interventions for maternal and newborn health ( world health organization 2015 ) . the impact of male involvement interventions around the time of childbirth on maternal and newborn health outcomes was examined as one part of a systematic review of maternal health intervention studies commissioned by the world health organization . the first stage identified , screened and mapped all maternal health intervention studies conducted in low- and middle - income countries between 2000 and 2012 . the second stage of the review sought to answer a series of specific review questions , one of which related to male involvement interventions for improved maternal and newborn health : what interventions employed with women , men , communities and community leaders to increase male involvement have been effective in increasing care - seeking behaviour during pregnancy , for child birth and after birth for the woman and newborn and in improving key maternal and newborn health outcomes?in this question , male involvement was defined broadly as strategies to increase the involvement of men . studies were only included where they reported on an intervention testing the impact of male involvement around the time of childbirth on pre - specified maternal and newborn health outcomes . studies were required to report on the impact of the intervention on one or more of the following outcomes : birth with a skilled attendant or in a facility , use of antenatal or postnatal care for the mother and newborn , uptake of essential maternal and child health interventions , maternal nutrition , newborn nutrition , birth and complication preparedness , maternal mortality , maternal morbidity , neonatal mortality and perinatal mortality . additionally , male involvement intervention studies were excluded where men 's involvement was sought only for the promotion of family planning or the prevention or treatment of sexually transmitted infections , including hiv . what interventions employed with women , men , communities and community leaders to increase male involvement have been effective in increasing care - seeking behaviour during pregnancy , for child birth and after birth for the woman and newborn and in improving key maternal and newborn health outcomes ? thirteen studies were identified as eligible following a rigorous systematic process intended to collate the available evidence for the impact of male involvement on maternal and newborn health , and have informed world health organization recommendations on this topic ( world health organization 2015 ) . the 13 included studies therefore constitute an important evidence base for male involvement interventions in maternal and newborn health programmes . we conducted a content analysis of the material extracted as part of the systematic review . this material included a description of the intervention , details about the people targeted by the intervention and people included in the study , and outcome measures relevant to maternal and newborn health or male involvement . the authors applied a critical gender lens to assess how the studies position men , and men 's involvement in maternal and newborn health , against the framework describing the potential of male involvement strategies detailed above : to engage with relations between men and women ; to support men to transform gender norms and roles ; and to challenge social structures dominated by men that reproduce gender inequities . the remaining seven studies from india , bangladesh , nepal , pakistan , tanzania and eritrea describe community - based education and community outreach strategies to increase male involvement in pregnancy care , seeking skilled care for birth and complications , postnatal care and reproductive health , as well as to increase men 's awareness of maternal health issues more broadly ( fullerton , killian , and gass 2005 ; hossain and ross 2006 ; midhet and becker 2010 ; mushi , mpembeni , and jahn 2010 ; purdin , khan , and saucier 2009 ; sinha 2008 ; turan , tesfagiorghis , and polan 2011 ) . there is clearly a range of different strategies to increase the involvement of men in maternal and newborn health . additionally , the studies were premised on a range of different explicit or implicit understandings of why male involvement in maternal and newborn health is desirable or appropriate . two studies described benefits to men 's own health as a justification for increased male involvement in their family 's health and engagement with the health system:[m]en have relatively low use of reproductive health services and few contacts with reproductive health service providers ( sahip and turan 2007 , 844 ) in addressing men 's involvement it is important to consider how to frame their contact with the health system so that it will encourage their future and continued involvement ( kunene et al . 2004 , 2)only one study noted that male involvement interventions are warranted because men have a preference for greater involvement in maternal and newborn health:[m]en themselves would prefer that they play a more active role during pregnancy , delivery and infant care . 2004 , 2)the absence of a clear consensus among the 13 studies on what a male involvement strategy is , and why a strategy to increase male involvement is being adopted , is likely to , in part , reflect differences in the social and cultural contexts in which the studies were implemented . when assessed against the three - point framework detailed above , the 13 studies do not describe interventions in a way that clearly documents the gender - transformative potential of male involvement . [ m]en have relatively low use of reproductive health services and few contacts with reproductive health service providers ( sahip and turan 2007 , 844 ) in addressing men 's involvement it is important to consider how to frame their contact with the health system so that it will encourage their future and continued involvement ( kunene et al . yet for example , despite the fact that most studies justified male involvement as a strategy to address household decision - making , only one study engaged directly with the dynamic of shared decision - making by measuring joint decision - making within couples ( varkey et al . several interventions included components such as couple counselling that could plausibly influence how men and women relate to each other , yet , with the exception of varkey and colleagues ( 2004 ) , findings relating to men 's relationships were not reported in the studies . the fact that changes in how men and women relate to each other were generally beyond the scope of the 13 studies is an important gap in the emerging evidence base . the second area of potential for male involvement interventions identified above is the opportunity provided by these interventions to support men 's ability to challenge gender roles and norms alongside women , premised on the recognition of men 's capacity to act as agents of change . yet men 's capacity to internalise and act on a desire for gender - transformative change was not well recognised in the 13 studies . many of the studies did not survey or interview men during data collection , although the interventions clearly targeted men as participants . few studies explored men 's attitudes related to becoming involved in maternal and newborn health within their families . the 13 studies did not capture changes in men 's internal identities , attitudes or motivations that may be associated with male involvement interventions . this means that men 's capacity to actively pursue gender - transformative change is not well documented within the emerging evidence base . the third area in which male involvement interventions are considered to have gender - transformative potential , as detailed above , is through supporting men 's ability to ally with women and challenge patriarchal social structures that reproduce gender inequity . while in some studies this awareness was confined to the background description of the study setting , others attempted to engage with these structures as part of the intervention ( hossain and ross 2006 ; purdin , khan , and saucier 2009 ; sood et al . the two exceptions to this were studies reporting that new social structures had been developed through the interventions maternal health volunteer discussion groups in eritrea ( turan , tesfagiorghis , and polan 2011 ) and community support systems for obstetric emergencies in bangladesh ( hossain and ross 2006 ) but it was unclear whether these new structures were intended to provide space for gender equitable discussion and decision - making . this indicates that the emerging evidence base is comprised of studies that did not capture the potential of male involvement strategies to engage with and transform social structures . generally , although there was some variation between studies , the focus of the studies that constitute the emerging evidence base was to employ male involvement as a strategy to prevent men from taking actions that can harm women and newborns and support men to take actions that can improve maternal and newborn health . this strategic approach , which focuses almost exclusively on men 's actions and decisions , rather than their relationships or subjective experiences , has been critiqued in the literature on male involvement as reductionist and instrumentalist ( barker and das 2004 ) . it is imperative to recognise , however , that an instrumentalist approach to male involvement can be the most feasible strategy to increase men 's engagement in maternal and newborn health in certain programmatic contexts . an exclusive focus on changing men 's behaviours may be particularly suited to short - term interventions implemented in settings where gender norms and roles are strongly enforced and transformative approaches are unlikely to be readily accepted by men , women and the broader community ( adeleye , aldoory , and parakoyi 2011 ; unfpa and promundo 2010 ) . in such scenarios , approaches that incorporate an understanding of the gender order in their particular setting , without seeking to transform it , may be the only viable short - term option for redressing some of the harms resulting from men 's gendered behaviours ( caro 2009 ) . given that gender - accommodating approaches can be appropriate in certain contexts , the absence of a gender - transformative approach to male involvement should not be considered a gap in any individual study . in aggregate , however , the emerging evidence base is limited by the lack of evidence to support male involvement as a strategy for gender - transformative change . this shows a need for a body of work that approaches and documents male involvement differently , in order to broaden the current evidence base with studies illustrating the potential of male involvement to effect gender - transformative change . first , it is important to be able to describe models of male involvement in a way that clearly differentiates between instrumentalist and gender - transformative approaches . recognising that male involvement interventions will necessarily address gender relations , it is imperative that they be well theorised with respect to gender and gender - transformative change at interpersonal and social levels . maternal and newborn health could benefit from ample research and conceptual work in other areas of health that describes the impacts of male involvement in lower- and middle - income countries . male involvement has been explored more extensively in the gender - based violence , sexual and reproductive health and hiv / aids literature . there is also considerable research on male involvement in high - income countries , particularly in relation to fatherhood , and some of the findings and lessons from these experiences are likely to be applicable to other contexts . for example , alio and colleagues ( 2013 ) proposed a framework for male involvement consisting of four components ( accessibility , engagement , responsibility and couple 's relationship ) , which could be tested in other contexts . similarly , burgess ' ( 2004 , 2007 ) work on active fatherhood in the uk offers an example of a robust conceptualisation of male involvement in maternal and newborn health that could potentially be translated to other settings . in addition to the need for a clear theoretical approach , it is important to be able to measure male involvement interventions in a nuanced way that captures the difference between transformative and instrumentalist approaches . defining and measuring male involvement is a methodological challenge , as demonstrated by the diverse range of indicators adopted by the studies in this review and elsewhere in the literature . there is a tendency to use specific indicators of involvement , such as male attendance at the birth or assistance with transportation during pregnancy , without clear justification for how male involvement is defined or whether chosen indicators are representative of this definition . some studies have attempted to measure the degree of engagement using cumulative measures of behaviours , such as a man accompanying his female partner to antenatal care , waiting in the waiting room , joining the appointment , talking to the health worker and discussing the appointment with his female partner afterwards ( byamugisha et al . developing a strong conceptual base for male involvement interventions and integrating effective measures for documenting male involvement can be expected to support further development of male involvement as a promising area for addressing gender influences on maternal and newborn health . the 13 studies included in our secondary analysis were mostly identified as part of a rigorous systematic mapping designed to consolidate the evidence base for male involvement in maternal and newborn health , among other topics . the assessment of the evidence presented here must , however , be understood as limited by the particular framework through which the studies were identified . evaluation of conceptual approaches to male involvement was not the primary purpose of the original systematic review and did not specifically guide the inclusion or exclusion criteria of the review . the systematic review included studies that were intervention studies , rather than observational studies , commentaries or discussions of male involvement . additionally , studies were only included where they reported on pre - specified maternal and newborn health outcomes , which did not include certain key male involvement outcomes such as men 's support during pregnancy or joint decision - making about childbirth . for the above reasons , there may be other literature on the topic that is not included in the review , particularly qualitative studies or descriptive pieces that examine implementation processes and the theoretical basis of male involvement interventions in more detail . irrespective of the process used to identify these studies , however , they constitute an emerging evidence base , and the review demonstrates important gaps in this evidence base . in the absence of detailed information , there is a risk of reading the standard critique of instrumentalism into these studies , and we acknowledge that this may not accurately reflect how interventions were actually designed or implemented . nevertheless , given that these 13 studies comprise a body of evidence for male involvement in maternal and newborn health , the way that male involvement is documented and represented in this important sub - set of the literature matters separately from how the interventions were done : a critique of these studies ' reported approaches is a critique of how male involvement is currently understood in the emerging evidence base . the 13 studies included in our secondary analysis were mostly identified as part of a rigorous systematic mapping designed to consolidate the evidence base for male involvement in maternal and newborn health , among other topics . the assessment of the evidence presented here must , however , be understood as limited by the particular framework through which the studies were identified . evaluation of conceptual approaches to male involvement was not the primary purpose of the original systematic review and did not specifically guide the inclusion or exclusion criteria of the review . the systematic review included studies that were intervention studies , rather than observational studies , commentaries or discussions of male involvement . additionally , studies were only included where they reported on pre - specified maternal and newborn health outcomes , which did not include certain key male involvement outcomes such as men 's support during pregnancy or joint decision - making about childbirth . irrespective of the process used to identify these studies , however , they constitute an emerging evidence base , and the review demonstrates important gaps in this evidence base . in the absence of detailed information , there is a risk of reading the standard critique of instrumentalism into these studies , and we acknowledge that this may not accurately reflect how interventions were actually designed or implemented . nevertheless , given that these 13 studies comprise a body of evidence for male involvement in maternal and newborn health , the way that male involvement is documented and represented in this important sub - set of the literature matters separately from how the interventions were done : a critique of these studies ' reported approaches is a critique of how male involvement is currently understood in the emerging evidence base . an assessment of the male involvement intervention studies identified through a comprehensive systematic review of maternal health interventions reveals important gaps in how male involvement is conceptualised in the emerging evidence base for male involvement in maternal and newborn health . emerging research , comprising the studies included in a systematic review commissioned to inform global recommendations relating to male involvement for maternal and newborn health , does not examine the gender - transformative potential of male involvement interventions . this points to the need for an approach to male involvement that is conceptualised and documented with closer reference to gender , in order to understand and document the potential of male involvement as a way of supporting health and gender equity . specifically , the included studies demonstrate the need for greater incorporation of gender - transformative conceptual approaches into future interventions , with effective measures built in to such interventions in order to develop the evidence base for their impact on a broad range of health and gender equity outcomes .

the term neurodenegerative disorder is used to describe diseases characterized by the progressive breakdown of neuronal function and structure . this term encompasses disorders such as alzheimer s , parkinson s , and huntington s diseases , as well as amyotrophic lateral sclerosis ( als ) , among others , although neuronal damage is also associated with stroke and ischemic events , cerebral palsy , and head trauma . although the human and economic cost of neurodegeneration continues to be astronomical , treatment is largely limited to palliative care and prevention of symptom progression . therefore , there is a constant demand for novel and effective approaches to slow or prevent the progression of these diseases . nitric oxide ( no ) is an important second messenger in the human body , and dysregulation of its production is implicated in many pathologies . no is produced by the nitric oxide synthase enzymes , of which there are three isoforms : endothelial nitric oxide synthase ( enos ) , which regulates blood pressure and flow , inducible nitric oxide synthase ( inos ) , involved in immune system activation , and nnos , which is required for normal neuronal signaling . nonetheless , overexpression of nnos in neural tissue and increased levels of no can result in protein nitration and oxidative damage to neurons , especially if peroxynitrite is formed from excess no . indeed , overexpression of nnos or excess no has been implicated in or associated with many neurodegenerative disorders . the inhibition of nnos is , therefore , a viable therapeutic strategy for preventing or treating neuronal damage . each monomer consists of both a reductase domain with fad , fmn , and nadph binding sites , and a heme - containing oxygenase domain , where the substrate ( l - arginine ) and cofactor ( 6r)-5,6,7,8-tetrahydrobiopterin ( h4b ) bind . activated and regulated by calmodulin binding , electron flow proceeds from one monomer s reductase domain to the other s oxygenase domain , catalyzing the oxidation of arginine to citrulline with concomitant production of no . not unexpectedly , most investigated nnos inhibitors are mimetics of arginine and act as competitive inhibitors . one major challenge in designing nnos inhibitors is that enos and inos share high sequence similarity and an identical overall architecture with nnos , especially in their substrate - binding sites . lack of isoform selectivity could have deleterious effects ; inhibition of enos can cause severe hypertension , and inos inhibition could impair immune system activation . previously , in our laboratories , fragment hopping and subsequent structure - based optimization afforded compounds 1 and 2 ( representative nnos inhibitors are shown in figure 1 ) . these compounds are highly potent and selective nnos inhibitors , and compound 1 reverses a hypoxic - ischemic brain damage phenotype in newborn rabbit kits when administered intravenously to the dam . representative nnos inhibitors discussed in this study . although effective , compounds 1 and 2 suffer from several drawbacks . like most arginine mimics , they are very polar and hydrophilic and contain numerous basic moieties and hydrogen - bond donors , as well as many rotatable bonds and a high total polar surface area ( tpsa ) , all properties that hamper both gi absorption and blood brain barrier permeation . many attempts to improve the bioavailability of these compounds have been made , including alkylation , fluorination , introduction of lipophilic tails , and replacement of the amines ; most of these strategies either diminished potency or selectivity or were synthetically challenging . the chiral scaffolds of 1 and 2 are also difficult ( > 12 steps ) to prepare , making them less desirable than simpler scaffolds , such as 3(26 ) and the astrazeneca candidate 4 ( ar - r17477 ; potencies and selectivities are given in figure 1(27 ) ) from a clinical standpoint . nonetheless , these simplified molecules are not without fault ; their isoform selectivities are lower , 3 suffers from poor caco-2 permeability , and 4 is much less potent in cell - based assays than against isolated enzymes , both likely the result , in part , of the amidine moiety , which will be charged at physiological ph . one avenue that we wished to explore for the generation of simpler nnos inhibitor scaffolds was the replacement of the amidine group of a molecule such as 3 with another arginine isostere . such a group should be stable , weakly basic ( pka between 6 and 8) , and possess as few hydrogen - bond donors as possible . one such moiety is the 2-aminoquinoline group ( pka of 7.3 ) , which is desirable because of both its resemblance to the aminopyridines of 1 and 2 ( in both structure and pka , which is 7.1 for 2-aminopyridine ) and its considerably higher clogp . we were also encouraged by the reported use of dihydroaminoquinolines as nnos inhibitors by jaroch et al . additionally , a recent report indicates that 2-aminoquinoline - based bace-1 inhibitors have high cellular activity and good blood brain barrier permeability in a rat model . to this end , 2-aminoquinoline and the structure of 3 were effectively hybridized to produce compound 5 , a simple compound with good calculated physicochemical properties . there is a hydrophobic pocket at the far end of the substrate access channel of nnos ; contact between an inhibitor and the residues lining this pocket is implicated in high selectivity for nnos over the other two isoforms . preliminary docking studies and crystallography indicated that elongation of the chain between the aminoquinoline system and the distal fluorophenyl ring of 5 , moving the position of the secondary amine , or a combination of both , might provide the right length and orientation to reach this hydrophobic pocket , and a series of analogues investigating chain length ( 69 ) and nitrogen position was , therefore , prepared ( figure 2 ) . additionally , on the basis of computer modeling , we hypothesized that placement of the tail of the inhibitor at position 6 of the aminoquinoline system ( instead of position 7 ) could also be effective ; to this end , compounds 1013 were prepared ( figure 2 ) . finally , several literature studies indicate that the use of other halogens and substitution patterns on the noncoordinating aryl ring could be beneficial for enhancing potency and selectivity , so a small series of 7-substituted compounds ( 1416 ) with different halogens and substitution patterns was prepared ( figure 2 ) . all compounds were assayed against purified rat nnos , and select compounds were assayed against enos , inos , and human nnos , and for cellular permeability in a caco-2 model . 6- and 7-substituted 2-aminoquinolines were prepared by methods originally reported by manimaran et al . and johnson et al . in the present study , 7-substituted aminoquinolines ( 59 and 1416 ) were prepared by a versatile , late - divergent route that began with the preparation of 3-methylcinnamanilide ( 17 ) by literature procedures . compound 17 was subsequently treated with an excess of aluminum chloride in chlorobenzene to affect cyclization and concomitant cleavage of the c - aryl bond to yield the carbostyril 18 as a mixture of the 7-isomer 18a ( major ) and 5-isomer 18b ( minor ) . the isomers were not separated at this stage but were converted into the 2-chloroquinolines 19a and 19b ; the unwanted 5-isomer 19b was removed by fractional crystallization . pure 19a was converted into 2-acetamidoquinoline 20 by the method of krdi , and free - radical bromination afforded versatile intermediate 21 ( scheme 1 ) . to prepare aminoquinoline analogues with one methylene unit between the quinoline system and the secondary amine ( 5 and 9 , schemes 2 and 3 ) , compound 21 was treated with 3-fluorophenethylamine ( 22 , scheme 2 ) or 3-fluoro-1-phenylpropanamine ( 25 , scheme 3 , prepared by hydrogenation of 3-fluorophenethyl cyanide [ 24 , prepared from 23 ] ) to afford amines 26 and 27 , respectively . deacetylation afforded the final compounds as free - bases , which were readily converted to the water - soluble dihydrochloride salts 5 and 9 . aminoquinolines possessing two methylene units between the quinoline system and secondary amine ( 6 , 7 , 8 , and 1416 , scheme 4 ) were likewise prepared from bromide 21 by homologation with cyanide ion to afford nitrile 28 . this compound was reduced to the polar quinolinyl - ethanamine ( 29 ) using hydrogen and raney nickel ; 29 was used crude in the next step . from this amine indirect reductive amination , where 29 was treated with 3-fluorobenzaldehyde ( 30 ) under mildly acidic , dehydrating conditions . when the aldehyde was consumed ( as measured by tlc ) , subsequent deacetylation , workup , and acidification afforded 6 . to prepare phenethyl analogues 7 , 14 , 15 , and 16 ( see scheme 4 ) , requisite phenylacetaldehydes 3538 were prepared by dess - martin oxidation of commercially available phenethyl alcohols 3134 , respectively . a direct reductive amination using 29 and the desired aldehyde was used to assemble the cores of the final analogues . yields were low because of dialkylation and aldehyde condensation byproducts ; the use of other solvents , dehydrating agents , and reductants failed to alleviate these problems ; the aldehydes may be light - and acid - sensitive as well . for these analogues , the intermediate acetamides were immediately deprotected after isolation ( because of some concerns about their stability ) to yield 7 , 14 , 15 , and 16 and converted into dihydrochloride salts , which could be easily purified by crystallization , trituration , or preparative hplc . finally , the preparation of propyl analogue 8 began with 3-fluorophenylpropionic acid ( 39 ) . reduction to phenylpropanol 40 , followed by swern oxidation , afforded sensitive aldehyde 41 . reductive amination using amine 29 , deacetylation , workup , and acidification afforded 8 , as also shown in scheme 4 . 6-substituted 2-aminoquinolines were prepared similarly to those described above , beginning instead with 4-methylcinnamanilide ( 42 , scheme 5 ) . using the cyclization compound 46 was treated with 22 , and the resulting acetamide was deacetylated , isolated , and acidified as before to yield 10 . likewise , as shown in scheme 6 , homologation of 46 with cyanide ion afforded 47 , which was readily reduced to ethanamine 48 . the indirect reductive amination procedure ( using 30 ) afforded 11 after deacetylation , isolation , and acidification . the direct reductive amination employing aldehyde 35 similarly afforded 12 after deacetylation / acidification , while the same reductive amination procedure using 41 instead yielded 13 after deprotection and salt formation . compounds 516 were assayed against purified rat nnos , bovine enos , and murine macrophage inos using the hemoglobin capture assay , as previously described . the apparent ki values and isoform selectivities are summarized in table 1 , and values for compounds 1 , 2 , and 3 are included for comparative purposes ; the ic50 values and selectivities for 4 are given in figure 1 . the compounds were assayed for in vitro inhibition against three purified nos isoforms : rat nnos , bovine enos , and murine inos , using known literature methods ( see experimental section for details ) , and ki values are calculated directly from ic50 values . ic50 values are the average of at least two replicates from nine data points ; all experimental standard error values are less than 14% , and all correlation coefficients are > 0.81 . the lead 7-substituted 2-aminoquinoline , compound 5 , has potent nnos inhibitory activity ( 74 nm ) and high n / i selectivity ( 124-fold ) , yet it is only weakly selective for nnos over enos ( around 6-fold ) . the crystal structures of 5 bound to both nnos and enos ( figure 3a and b , respectively ) indicate that the bound conformation of 5 is virtually identical in both isoforms . in both cases , the aminoquinoline acts as an arginine mimic and interacts with the active site glutamate as arginine does ( glu592 in nnos ( 1om4 ) ; glu363 in enos ( 2nse ) ) , while the secondary amine sits between the heme propionates , making h - bonds to both . to simultaneously establish hydrogen bonds between the aminoquinoline and glu592 as well as between the secondary amine and both heme propionates , the rigid quinoline plane must tilt significantly from the heme plane . the fluorophenethyl moiety , as predicted from the short linker length , does not quite reach the hydrophobic pocket ( figure 3 ) consisting of tyr706 , leu337 , met336 , and trp306 in nnos ( tyr477 , leu107 , val106 , and trp76 in enos ) . these contacts are absent in 5 , thus resulting in poor selectivity for nnos over enos ; this observation is similar to that of the crystal structure of 3 . active site structures of lead 5 bound to rat nnos ( a ) and bovine enos ( b ) . the omit fo fc density map for the inhibitor is shown at the 2.5 contour level . major hydrogen bonds are shown as dashed lines . in each panel , the four residues that line a hydrophobic pocket are highlighted by a dot surface representation . while residues in chain a of nnos ( a ) and enos ( b ) are colored green and cyan , respectively , the residue from chain b ( second monomer in the homodimeric structure ) is distinguished by a different color . we sought to improve potency and isoform selectivity by elongating the chain between the aminoquinoline and the noncoordinating aryl ring ( figure 2 ) . to this end , extra methylene groups were inserted between the secondary amine and fluorophenyl group ( 9 ) or between the quinoline and the secondary amine ( 7 , 8) . we reasoned that moving the amine farther from the quinoline could also have the advantage of relaxing the constraints on the quinoline ring orientation but still allow the amine to interact with the heme propionates , thus , in turn , anchoring the tail in a favorable orientation to make hydrophobic contacts . there are two factors that affect the comparative inhibitor potency in this series of aminoquinoline compounds : the linker length and the position of the amine group . contrary to our prediction regarding amine position , the structure of nnos with 6 bound ( figure 4a ) reveals that placing two carbons between the quinoline and the amine actually diminishes the interaction with the heme propionates ( more than 3.6 distance ) , leading to increased flexibility as evidenced by the disordered fluorophenethyl tail in the structure of 6 and decreased potency relative to 5 . superimposition of these two nnos structures ( 5 and 6 , figure 4b ) reveals that the loose interaction between the amine of 6 and the heme propionates ( lacking of h - bonds ) allows the quinoline to assume a more parallel orientation ( relative to the heme ) than observed in the structure of 5 . however , as the 4-atom linker ( of 5 and 6 ) is not long enough to bring the fluorophenyl ring in contact with the aforementioned hydrophobic pocket ( as in figure 3 ) , the majority of the stabilization results from the hydrogen bonds from the aminoquinoline and the linker amine . therefore , 5 , with an extra hydrogen bond , is a stronger inhibitor than 6 . fc density map for the inhibitor is shown at the 2.5 contour level . ( b ) overlay of 5 ( cyan ) and 6 ( yellow ) in nnos . the different tilt angles of the aminoquinoline ring relative to the heme plane is in part related to whether hydrogen bonds ( dashed lines ) from the heme propionates to the linker amine are present ( compound 5 ) or absent ( compound 6 ) . in general , compounds with shorter linkers ( 5 and 6 ) have lower nnos inhibitory activity than compounds with longer linkers ( 7 and 9 , table 1 ) . the ideal chain length appears to be five atoms between the quinoline and fluorophenyl groups . nnos inhibitory activity is very similar between 7 and 9 ( table 1 ) with the nitrogen placement not drastically affecting potency ; it appears that the influence of the amine position is weakened in these inhibitors with longer linker lengths . the omit electron density map reveals that 7 ( figure 5a ) , which lacks a strong secondary amine heme propionate interaction , is more flexible / disordered in the fluorophenyl tail region relative to the structure of 9 ( which does show the amine nonetheless , the similar potencies of 7 and 9 indicate that the nitrogen position is not crucial for these compounds with longer linkers , likely because additional stabilization results from contact with the hydrophobic pocket . indeed , the structure of 9 ( figure 5b ) shows numerous favorable hydrophobic contacts between the fluorophenyl group and the nonpolar residues at the far end of the substrate access channel , tyr706 , leu337 , met336 , and trp306 ( of the other monomer of the nnos homodimer ) . although the tail of 7 ( figure 5a ) is more disordered in structure than that of 9 ( figure 5b ) , these hydrophobic contacts exist with 7 as well . therefore , when the linker is long enough to allow contact between the fluorophenyl ring and the hydrophobic pocket , the strong combined stabilization from both the hydrophobic interactions and the aminoquinoline glu592 interaction may effectively outweigh any lack of interaction between the secondary amine and heme propionates . active site structure of 7 ( a ) or 9 ( b ) bound to nnos . the omit fo fc density map for the inhibitor is shown at the 2.5 contour level . chain lengths that are longer than the ideal ( e.g. , compound 8 , with six atoms between the quinoline and fluorophenyl group ) result in a drop in potency when compared with 7 or 9 . the crystal structure with 8 ( figure 6a ) shows that the fluorophenyl ring of 8 makes hydrophobic contacts similar to those of 7 and 9 . nonetheless , to make these contacts , the flexible chain has to assume a kinked conformation , in contrast to the fully extended linker conformation seen in 9 ( figure 5b ) . the kinked conformation of 8 may result in unfavorable torsional strain in the linker region upon binding . active site structure of 8 ( a ) or 15 ( b ) bound to nnos . the omit fo fc density map for the inhibitor is shown at the 2.5 contour level . it is worth noting that 7-substituted 2-aminoquinolines are , as a whole , very poor inos inhibitors ; an earlier report also describes the parent compound , 2-aminoquinoline , as having only weak inos inhibitory activity ( 1.7 m ) . compounds 7 , 9 , and 15 have ki values of 44 m , 32.3 m , and 28.4 m , respectively , and 7 has nearly 900-fold selectivity for nnos over inos , a value which is significantly higher than those of 14 , and is among the highest selectivity reported for nnos over inos for nonpeptidic inhibitors . contact with the substrate - channel hydrophobic pocket ( vide supra ) could be in part responsible for this high n / i selectivity . murine inos contains a polar asparagine residue ( asn115 ) in this pocket ( at the position of leu337 of nnos ) that would strongly disfavor binding by a hydrophobic group . nonetheless , even the short - chain inhibitors ( 5 and 6 ) still possess good n / i selectivity , despite not reaching this distal pocket , indicating that interactions with residues at this end of the binding site are not the full determinant of this poor inos inhibition . it is reported that the heme - binding sites themselves differ between inos and nnos isoforms , with the former possessing a smaller and more rigid active site that may not tolerate the bulky and inflexible aminoquinoline as well . interestingly , the selectivity patterns ( higher n / i selectivity ) contrast with many aminopyridine - based inhibitors , which have higher n / e selectivity . in some cases ( such as the r , r - enantiomer of 1 ) , this high n / e selectivity can be explained by water - mediated contacts made between the center pyrrolidine ring and asp597 , a residue that exists in both nnos and inos but is asn369 in enos . this aspartate residue can provide considerable electrostatic or hydrogen - bonding stabilization in nnos versus enos ; this stabilization also manifests itself in the high n / e selectivity of dipeptide - based inhibitors , but no contacts with asp597 are observed in the aminoquinoline crystal structures . in other cases , high n / e selectivity is rationalized by the tighter -stacking with tyr706 of nnos than with the analogous tyr477 of enos , leading to greater nonbonded contacts and better desolvation . while no clear -stacking interactions are visible in the nnos crystal structures of 6 , 7 , 8 , or 9 , hydrophobic contacts and desolvation may still play a substantial role in n / e selectivity for aminoquinolines . the binding mode of the aminoquinoline portion is identical in the structure of 7 bound to nnos ( figure 5a ) or enos ( figure 7 ) and does not contribute to isoform selectivity . however , the length of the linker in 7 enables the fluorophenyl ring to make good hydrophobic contacts with the residues met336 , leu337 , tyr706 , and trp306 ( from the other monomer of the nnos homodimer ) . the bulky and flexible met336 side chain makes extensive contacts with the fluorophenyl group of 7 , whereas the analogous residue , val106 in enos , with a smaller surface area , can not make that many contacts . additionally , the side chain of tyr706 in nnos rotates by about 60 in order to make better contacts with the tail of 7 , while in the enos structure ( figure 7 ) , tyr477 remains in its original side chain orientation . overall , these differences are fairly subtle but still contribute to the slightly tighter binding of 7 to nnos over enos . small changes in these hydrophobic contacts could also explain why 7 is more selective than 9 . the omit fo fc density map for the inhibitor is shown at the 2.5 contour level . major hydrogen bonds are shown as dashed lines . while 7-substituted aminoquinolines ( 59 and 1416 ) are all highly potent against nnos , the analogous 6-substituted aminoquinolines 1013 have low potency , regardless of chain length or nitrogen position . this disparity is also explained by the crystal structures of the bound 2-aminoquinolines . in the heme - binding pocket , the aminoquinoline system does not stack parallel to the heme but rather tilts down slightly toward the back wall of this pocket ( figure 4a and b ) . in cases where an h - bond is formed between the secondary amine and heme propionates , the angle between the planes of the aminoquinoline and heme can be as large as 45 , held in this conformation by the h - bond . even when no hydrogen bond is present , the aminoquinoline still tilts to avoid unfavorable contact with val567 and phe584 , bulky residues that project downward from the roof of this pocket . a large or flexible substituent located at position 6 , in any case , would clash directly with these bulky residues or the heme propionates , or force the rigid aminoquinoline system into a position where it can no longer be accommodated in the heme - binding pocket . this also explains why 11 has low nnos inhibitory activity despite sharing a similar overall structure with 4 ( the same number of atoms and a similar orientation between the arginine - mimic portions and the noncoordinating aryl rings ) . the flexibility ( more rotatable bonds ) of 4 allows it to be accommodated more easily in the heme - binding pocket , whereas 11 is too rigid to fit . it also was reported in the literature that rigid fused 2-aminodihydroquinoline - based nnos inhibitors show a similar sar regarding substituent placement ; large amine - containing tails can be easily placed in the region analogous to the 7-position , whereas the area occupied by the 6-position can only fit small substituents , such as fluorine . interestingly , the replacement of the fluorine in the 3-fluorophenyl group of 7 with a bulkier chlorine ( compound 15 ) does not significantly decrease the nnos inhibitory potency of 15 and is only modestly detrimental to isoform selectivity , which remains 431-fold and 110-fold for inos and enos , respectively . as shown in figure 6b , 15 binds to nnos in a manner very similar to that of 7 ( figure 5a ) . without a strong interaction between the amine and the heme propionates , the chlorophenethyl tail is partially disordered but can still be located based on the partial density contoured at 0.5 . in this model , the chlorine atom is not pointing directly into the hydrophobic pocket , so the switch between chlorine and fluorine should not significantly alter contacts with the enzyme . placement of the fluorine ( or chlorine ) at the 4-position , however , is a disfavored modification ( compare 7 to 14 or 15 to 16 ) . this drop in potency could arise from unfavorable steric clashes between the 4-position substituent ( which would face directly toward the back of the hydrophobic pocket ) and any hydrophobic pocket residue , especially met336 and leu337 . encouraged by the high potency and selectivity of 7 and 15 , we assayed these compounds ( and lead 5 ) against purified human nnos ( table 2 ) . the human isoform has an active site that is nearly identical to that in the rat enzyme , with the exception of the hydrophobic pocket , where leu337 is replaced by a histidine ( his341 ) . this pocket is smaller and more polar , and may prefer to bind inhibitors with less bulky and more hydrophilic tails . previously , aminopyridine - based inhibitors showed lower potency against the human enzyme when compared to the rat enzyme , and the same trend is observed for the aminoquinolines , although 5 , 7 , and 15 still display good human nnos inhibition . because of the very similar selectivities ( ki - human / ki - rat ) among these three compounds , it can be concluded that the modifications that are well tolerated by the rat isoform ( chain elongation and replacement of fluorine with chlorine ) are likewise tolerated similarly by human nnos , including the introduction of the bulkier chlorine . ic50 values are the average of at least two replicates from nine data points ; all experimental standard error values are less than 10% , and all correlation coefficients are > 0.90 . finally , compounds 7 and 15 were assayed in a caco-2 monolayer permeability assay ( table 3 ) . this assay is an approximation of both a compound s ability to penetrate the epithelium of the gi tract as well as the blood brain barrier ; ideally , an orally bioavailable nnos inhibitor should show high permeability in this assay . an efflux ratio ( ratio of membrane permeability ( ab ) to efflux ( ba ) ) < 3 is considered favorable . pleasingly , both 7 and 15 display good membrane permeability in the apical to basolateral ( ab ) direction and high compound recovery values . compound 15 even shows improved membrane permeation relative to compound 4 , and both 7 and 15 display relatively low efflux ratios , diminishing the possibility that p - gp or other active transport mechanisms are significantly acting on these compounds ( especially on 15 ) . interestingly , compound 15 is more membrane - permeable than 7 despite their nearly identical structures ; this could be the result of the higher clogp of 15 ( 3.8 ) relative to 7 ( 3.2 ) or to variability in the assay . in summary , we have prepared a series of novel simplified 2-aminoquinolines based on the rationale that they might bind to and inhibit nnos in a manner similar to that of aminopyridines , while being less polar , less basic , more lipophilic , and , therefore , more bioavailable . compounds were assayed with purified nos enzymes , and it was revealed that 7-substituted 2-aminoquinolines are highly potent inhibitors of nnos and that subtle modifications ( such as increasing the chain length between the aminoquinoline and a noncoordinating aryl ring ) can enhance potency and greatly improve isoform selectivity to > 200-fold over both inos and enos . crystal structures indicate that these compounds act as competitive arginine mimics , where the aminoquinoline moiety makes hydrogen bonds with the active - site glutamate residue , and that the noncoordinating aryl rings are stabilized in a hydrophobic pocket on the far end of the substrate access channel . enhanced hydrophobic contacts with 7 in this pocket in nnos , relative to that of enos , may also , in part , dictate the high isoform selectivity . most promisingly , two of these highly effective compounds , 7 and 15 , show good permeability in a caco-2 assay . these results indicate that these compounds have high potential for oral bioavailability and brain penetration and that the 7-substituted 2-aminoquinoline cores offer very promising leads for further nnos inhibitor development . anhydrous solvents ( thf , ch2cl2 , and dmf ) were distilled prior to use . the remaining solvents , reactants , and reagents were purchased from commercial vendors and were used without further purification , with the exception of acetamide , which was heated to 80 c and dried under vacuum before use . melting points were determined in capillary tubes using a buchi melting point b-540 apparatus and are uncorrected . h nmr spectra were recorded at 500 mhz , using a bruker avance iii 500 ( direct cryoprobe ) , and c nmr spectra were obtained at 126 mhz using the same instrument . high - resolution mass spectral data were obtained at the integrated molecular structure education and research facility ( northwestern university ) on an agilent 6210a tof mass spectrometer in positive ion mode using electrospray ionization , with an agilent g1312a hplc pump and an agilent g1367b autoinjector . flash column chromatography was performed using an agilent 971-fp automated flash purification system , using a varian column station with silicycle cartridges ( 880 g ) , or manually in glass columns using silicycle siliaflash p60 4063 m silica gel . analytical hplc was performed either using a beckman system gold 125 solvent module and 166 detector or an agilent infinity 1260 system and an injection volume of 10 l . a phenomenex gemini c18 5 m , 110 reverse - phase column , gemini nx 5 m , 100 column ( both with dimensions of 250 mm 4.6 mm ) , or phenomenex synergi 5 m , polar rp column ( 4.6 50 mm ) was used for all hplc experiments . the purity of all final target compounds was found to be 95% by hplc , using either isocratic elution at 70% meoh in h2o ( with 0.1% tfa ) or a gradient of 6595% meoh in h2o ( with 0.1% tfa ) at 0.8 ml / min . when the polar rp column was used , elution was isocratic at either 50% acetonitrile in h2o or 35% acetonitrile in h2o at 1.5 ml / min . preparative hplc was performed at the northwestern university center for molecular innovation and drug discovery chemcore lab , using an agilent 1200 series hplc and agilent 6120 quadrupole mass spectrometer ( api - ms mode ) and a phenomenex gemini - nx 5 m c18 column ( 150 21.2 mm ) . analytical thin - layer chromatography was performed on silicycle extra hard 250 m tlc plates . compounds were visualized with short - wavelength uv light , ninhydrin , and kmno4 stain , where relevant . compounds 17 , 35 , 36 , and 42 were prepared by literature procedures , and their spectral data are consistent with those data reported for the same . compound 26 ( 0.187 g , 0.554 mmol ) was diluted in meoh ( 8 ml ) , and k2co3 ( 0.077 g , 0.554 mmol ) was added . the mixture was heated at 50 c for 2 h and then at reflux for an additional 1 h. the mixture was cooled and concentrated , and the residue was diluted in etoac ( 50 ml ) , washed with h2o ( 2 50 ml ) , and dried over anhydrous sodium sulfate . the solution was concentrated , the residue was diluted in methanolic hcl ( 1.4 m , 12 ml ) , and the mixture was heated for 3 h at 50 c , upon which a white crystalline precipitate formed . the mixture was cooled and filtered , and additional product was obtained upon concentration of the filtrate and recrystallization of the residue from meoh . a total of 0.140 g of product ( 69% ) was obtained : mp 283285 c ( dec . ) . h nmr ( 500 mhz ; dmso - d6 ) : 14.47 ( s , 1 h ) , 9.65 ( br s , 2 h ) , 9.31 ( br m , 1 h ) , 8.39 ( d , j = 9.3 hz , 1 h ) , 8.30 ( br s , 1 h ) , 7.99 ( d , j = 8.2 hz , 1 h ) , 7.87 ( s , 1 h ) , 7.68 ( d , j = 8.5 hz , 1 h ) , 7.40 ( td , j = 7.8 , 6.4 hz , 1 h ) , 7.167.09 ( m , 4 h ) , 4.364.35 ( m , 2 h ) , 3.233.22 ( m , 2 h ) , 3.06 ( t , j = 8.1 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) : ( 163.2 + 161.3 , 1 c ) , 154.7 ( 1 c ) , 142.6 ( 1 c ) , ( 140.09 + 140.03 , 1 c ) , 136.5 ( 1 c ) , 135.5 ( 1 c ) , ( 130.60 + 130.53 , 1 c ) , 129.1 ( 1 c ) , 126.5 ( 1 c ) , ( 124.82 + 124.81 , 1 c ) , 120.9 ( 1 c ) , 118.8 ( 1 c ) , ( 115.53 + 115.36 , 1 c ) , 114.5 ( 1 c ) , ( 113.70 + 113.54 , 1 c ) , 49.5 ( 1 c ) , 47.3 ( 1 c ) , 31.0 ( 1 c ) . hrms calcd for c18h18fn3 , 295.1485 ; found , 295.1487 . to a solution of 29 ( 0.062 g , 0.266 mmol ) in 5:1 chcl3/meoh ( 6 ml ) was added aldehyde 30 ( 0.033 g , 0.319 mmol ) and anhydrous sodium sulfate ( approximately 0.5 g ) . the mixture was stirred rapidly for 90 min , and additional na2so4 ( 0.3 g ) and a catalytic amount of glacial acoh ( approximately 10 l ) were added . after a total of 3 h , extra na2so4 ( 0.3 g ) was added . after 4 h , tlc indicated the consumption of amine 29 , the mixture was filtered to remove the na2so4 , and the filter cake was washed with 10 ml of chcl3 . the mixture was concentrated , the oily residue was diluted in meoh ( 5 ml ) , then nabh4 ( 0.015 g , 0.4 mmol ) was added . after being stirred for 20 min at room temperature , the solution was concentrated , and the residue was partitioned between etoac and h2o ( 20 ml each ) . the layers were separated , and the aqueous layer was extracted with etoac ( 20 ml ) . concentration afforded an oily residue that was purified by flash column chromatography ( sio2 ) , eluting with a gradient of etoac to 10% meoh in etoac to yield the intermediate acetamide ( 0.055 g , 75% , confirmed by ms ) , which was immediately dissolved in meoh ( 6 ml ) . k2co3 ( 0.023 g , 0.167 mmol ) was added , and the mixture was heated to vigorous reflux for 1 h 45 min . the mixture was cooled and concentrated , and the residue was partitioned between etoac and 1:1 h2o / sat . the layers were separated , and the aqueous layer was extracted with etoac ( 5 ml ) . the combined organic layers were dried over anhydrous sodium sulfate and concentrated to yield a sticky residue that was diluted with ch2cl2 ( 5 ml ) and filtered to remove particulate matter . methanolic hcl ( 1.4 m , 2 ml ) was added , the mixture was stirred for 10 min , and ether ( 25 ml ) was added slowly until a whitish precipitate formed . this solid was collected and dried to afford the title compound as a cream - colored amorphous solid ( 0.052 g , 65% based on 29 ) : mp 278279 c . h nmr ( 500 mhz ; dmso - d6 ) : 14.36 ( s , 1 h ) , 9.65 ( s , 2 h ) , 9.20 ( br s , 1 h ) , 8.36 ( d , j = 9.3 hz , 1 h ) , 8.25 ( br s , 1 h ) , 7.91 ( d , j = 8.2 hz , 1 h ) , 7.59 ( s , 1 h ) , 7.51 ( m , j = 5.0 hz , 2 h ) , 7.447.39 ( m , 2 h ) , 7.307.26 ( m , 1 h ) , 7.09 ( d , j = 9.3 hz , 1 h ) , 4.22 ( s , 2 h ) , 3.22 ( br s , 4 h ) . c nmr ( 126 mhz ; dmso - d6 ) : ( 162.9 + 160.9 , 1 c ) , 154.3 ( 1 c ) , 142.8 ( 1 c ) , 142.4 ( 1 c ) , 135.9 ( 1 c ) , ( 134.63 + 134.57 , 1 c ) , ( 130.76 + 130.70 , 1 c ) , 129.1 ( 1 c ) , ( 126.24 + 126.22 , 1 c ) , 125.8 ( 1 c ) , 119.8 ( 1 c ) , 117 ( 1 c ) , ( 116.96 + 116.79 , 1 c ) , ( 115.90 + 115.73 , 1 c ) , 113.4 ( 1 c ) , 49.2 ( 1 c ) , 47.1 ( 1 c ) , 31.6 ( 1 c ) . hrms calcd for c18h18fn3 , 295.1485 ; found , 295.1487 . to a solution of 29 ( 0.74 g , 0.321 mmol ) in 7:1 chcl3/meoh ( 8 ml ) , aldehyde 35 ( 0.052 g , 0.375 mmol ) was added , followed by glacial acoh ( 7 l ) and anhydrous mgso4 ( approximately 0.5 g ) . the mixture was stirred at room temperature for 30 min and then cooled to 0 c . sodium triacetoxyborohydride ( 0.079 g , 0.375 mmol ) was added in one portion , and the mixture was slowly warmed to room temperature over 45 min , stirred 15 min at room temperature , and diluted with chcl3 ( 30 ml ) . nahco3 ( 10 ml ) , and the aqueous layer was extracted with chcl3 ( 5 ml ) . the solution was concentrated , and the residue was purified by flash column chromatography ( sio2 ) , eluting with a gradient of etoac to 18% meoh in etoac to yield the intermediate acetamide as a sticky syrup ( 0.030 g , 25% ) , which was immediately dissolved in meoh ( 4 ml ) . k2co3 ( 0.023 g , 0.163 mmol ) was added , and the mixture was heated to vigorous reflux for 2 h. the mixture was cooled and concentrated , and the residue was partitioned between etoac and 3:2 h2o / sat . the layers were separated , and the aqueous layer was extracted with etoac ( 2 ml ) . nacl ( 4 ml ) , dried over anhydrous sodium sulfate , concentrated to yield a sticky residue that was diluted with ch2cl2 ( 4 ml ) , and filtered to remove particulate matter . ( 1.4 m , 2 ml ) was added , the mixture was stirred for 10 min , ether ( 20 ml ) was added slowly , and the mixture was sonicated until a whitish precipitate formed . this solid was collected and dried to afford the title compound as a hygroscopic , cream - colored amorphous solid ( 0.023 g , 18% based on 29 ) : mp 247249 c ( dec ) . h nmr ( 500 mhz ; dmso - d6 ) : 14.34 ( s , 1 h ) , 9.21 ( br s , 3 h ) , 8.37 ( d , j = 9.3 hz , 1 h ) , 8.27 ( br s , 1 h ) , 7.92 ( d , j = 8.2 hz , 1 h ) , 7.60 ( s , 1 h ) , 7.437.38 ( m , 2 h ) , 7.187.08 ( m , 4 h ) , 3.253.16 ( m , 6 h ) , 3.02 ( t , j = 8.1 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) : ( 163.7 + 161.8 , 1 c ) , 154.8 ( 1 c ) , 143.3 ( 1 c ) , 142.9 ( 1 c ) , ( 140.55 + 140.49 , 1 c ) , 136.4 ( 1 c ) , ( 131.07 + 131.00 , 1 c ) , 129.6 ( 1 c ) , 126.3 ( 1 c ) , ( 125.35 + 125.33 , 1 c ) , 120.2 ( 1 c ) , 117.5 ( 1 c ) , ( 116.03 + 115.86 1 c ) , ( 114.19 + 114.02 , 1 c ) , 113.86 ( 1 c ) , 47.7 ( 1 c ) , 32.1 ( 1 c ) , 31.6 ( 1 c ) . esims m / z ( rel . compound 29 ( 0.064 g , 0.280 mmol ) was diluted in 7:1 chcl3/meoh ( 7 ml ) , and aldehyde 41 ( 0.049 g , 0.322 mmol ) was added , followed by glacial acetic acid ( 6 l ) and anhydrous mgso4 ( 0.5 g ) . the mixture was stirred for 30 min , cooled to 0 c , and sodium triacetoxyborohydride ( 0.070 g , 0.333 mmol ) was added in one portion . the mixture was allowed to warm to room temperature slowly over 50 min and was diluted with chcl3 ( to a volume of approximately 50 ml ) and filtered . nahco3 ( 8 ml ) , and the aqueous layer was extracted with chcl3 ( 5 ml ) . the organic phase was washed with sat . nacl ( 10 ml ) , dried over anhydrous sodium sulfate , and concentrated . the resulting residue was purified by flash column chromatography ( sio2 ) eluting with a gradient of etoac to 17% meoh in etoac to yield the intermediate acetamide ( 0.045 g , 44% ) as a sticky semisolid . this substance was immediately diluted with anhydrous meoh ( 6 ml ) , and k2co3 ( 0.034 g , 0.246 mmol ) was added . the mixture was heated at reflux for 1 h 50 min , cooled , and concentrated . the residue was diluted with etoac ( 10 ml ) , and the solution was washed with h2o / sat . nacl ( 5 ml ) , dried over anhydrous sodium sulfate , and concentrated . the resulting syrup was diluted in ch2cl2 ( 3 ml ) and filtered to remove particulate matter , and methanolic hcl was added ( 1.4 m , 3 ml ) and the mixture stirred at room temperature for 10 min . ether ( 30 ml ) was added , the mixture was sonicated , concentrated , and the residue was washed twice with ether ( 2 ml each ) to afford the product as a cream - colored hygroscopic solid ( 0.042 g , 37% from 29 ) : mp 8183 c ( softens ) , 210 c ( dec ) . h nmr ( 500 mhz ; dmso - d6 ) : 14.31 ( s , 1 h ) , 9.219.13 ( m , 3 h ) , 8.37 ( d , j = 9.3 hz , 1 h ) , 8.25 ( br s , 1 h ) , 7.91 ( d , j = 8.2 hz , 1 h ) , 7.59 ( s , 1 h ) , 7.42 ( dd , j = 8.2 , 1.3 hz , 1 h ) , 7.387.34 ( m , 1 h ) , 7.127.03 ( m , 4 h ) , 3.213.14 ( m , 4 h ) , 2.952.90 ( m , 2 h ) , 2.71 ( t , j = 7.6 hz , 2 h ) , 1.96 ( dt , j = 15.3 , 7.7 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) : ( 163.2 + 161.3 , 1 c ) , 154.3 ( 1 c ) , ( 143.71 + 143.65 , 1 c ) , 142.8 ( 1 c ) , 142.5 ( 1 c ) , 135.8 ( 1 c ) , ( 130.32 + 130.25 , 1 c ) , 129.1 ( 1 c ) , 125.8 ( 1 c ) , ( 124.47 + 124.45 , 1 c ) , 119.7 ( 1 c ) , 117.0 ( 1 c ) , ( 115.09 + 114.92 , 1 c ) , 113.4 ( 1 c ) , ( 112.95 + 112.79 , 1 c ) , 47.1 ( 1 c ) , 46.1 ( 1 c ) , 31.73 ( 1 c ) , 31.54 ( 1 c ) , 26.8 ( 1 c ) . anhydrous cs2co3 ( 0.105 g , 0.322 mmol ) was diluted with anhydrous dmf ( 3 ml ) , 25 ( 0.050 g , 0.322 mmol ) was added as a solution in dmf ( 2 ml ) , and the mixture was stirred for 30 min at room temperature . compound 21 ( 0.075 g , 0.269 mmol ) was then added as a solution in dmf ( 1.3 ml ) over several minutes . the residue was partitioned between etoac and h2o ( 10 ml each ) , the layers were separated , and the aqueous layer was saturated with nacl and extracted with etoac ( 2 5 ml ) . nacl ( 10 ml ) , dried over anhydrous sodium sulfate , and concentrated . the residue was purified by flash column chromatography ( sio2 ) , eluting with a gradient of etoac to 15% meoh in etoac to yield 27 as a yellow syrup ( 0.039 g , 41% ) , which was used without further characterization . this compound was diluted with anhydrous meoh ( 6 ml ) , and anhydrous k2co3 ( 0.031 g , 0.022 mmol ) was added . the mixture was heated at reflux for 2 h 15 min , cooled , and concentrated . the residue was diluted with etoac ( 10 ml ) , and 3 ml each of h2o and sat . the layers were separated , the aqueous layer was extracted with etoac ( 3 4 ml ) , and the combined organic layers were washed with sat . nacl ( 4 ml ) , dried over anhydrous sodium sulfate , and concentrated . the residue was diluted with ch2cl2 ( 5 ml ) , filtered to remove particulate matter , and reconcentrated . methanolic hcl ( 1.4 m , 3 ml ) was added , the mixture was stirred for 5 min , and ether ( 30 ml ) was added slowly until a white precipitate formed . this solid was collected and dried to afford the title compound as a white microcrystalline solid ( 0.029 g , 28% based on 21 ) after drying in vacuo : mp 250252 c ( dec ) . h nmr ( 500 mhz ; dmso - d6 ) : 14.44 ( s , 1 h ) , 9.50 ( s , 2 h ) , 9.30 ( br s , 1 h ) , 8.39 ( d , j = 9.2 hz , 1 h ) , 8.30 ( br s , 1 h ) , 7.98 ( d , j = 8.2 hz , 1 h ) , 7.86 ( s , 1 h ) , 7.66 ( d , j = 8.3 hz , 1 h ) , 7.35 ( td , j = 8.0 , 6.4 hz , 1 h ) , 7.15 ( d , j = 9.3 hz , 1 h ) , 7.107.02 ( m , 3 h ) , 4.32 ( t , j = 5.5 hz , 2 h ) , 2.952.90 ( m , 2 h ) , 2.70 ( t , j = 7.6 hz , 2 h ) , 2.00 ( quintet , j = 7.7 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) : ( 163.2 + 161.3 , 1 c ) , 154.6 ( 1 c ) , ( 143.67 + 143.61 , 1 c ) , 142.6 ( 1 c ) , 136.6 ( 1 c ) , ( 130.32 + 130.25 , 1 c ) , 129.0 ( 1 c ) , 126.4 ( 1 c ) , ( 124.47 + 124.45 , 1 c ) , 120.9 ( 1 c ) , 118.7 ( 1 c ) , ( 115.08 + 114.91 , 1 c ) , 114.5 ( 1 c ) , ( 112.95 + 112.78 , 1 c ) , 49.3 ( 1 c ) , 46.0 ( 1 c ) , 31.5 ( 1 c ) , 26.7 ( 1 c ) ; one of the aminoquinoline carbons is not visible due to baseline broadening ; esims m / z ( rel . anhydrous cs2co3 ( 0.090 g , 0.288 mmol ) was diluted in anhydrous dmf ( 5 ml ) , and amine 22 ( 0.040 g , 0.288 mmol ) was added . the mixture was stirred for 30 min at room temperature before compound 46 ( 0.070 g , 0.250 mmol ) was added dropwise as a solution in anhydrous dmf ( 2 ml ) . the resultant suspension was stirred for 16 h at room temperature and concentrated , the residue was partitioned between etoac and h2o ( 5 ml each ) , and the layers were separated . the aqueous layer was extracted with etoac ( 2 5 ml ) , and the organic layers were washed with sat . nacl ( 5 ml ) , dried over anhydrous sodium sulfate , and concentrated . the residue was purified by flash column chromatography ( sio2 ) , eluting with a gradient of etoac to 10% meoh in etoac to yield the intermediate acetamide as a yellow syrup ( 0.040 g , 47% , confirmed by ms ) . this syrup was dissolved in meoh ( 5 ml ) , and k2co3 ( 0.026 g , 0.148 mmol ) was added . the mixture was heated at reflux for 2 h , cooled to room temperature , and concentrated . the layers were separated , and the aqueous layer was extracted with etoac ( 2 5 ml ) . the combined organic phase was washed with sat aq . nacl ( 4 ml ) , dried over anhydrous sodium sulfate , and concentrated . the resulting residue was diluted in ch2cl2 ( 5 ml ) and filtered to remove particulate matter , and methanolic hcl ( 1.4 m , 3 ml ) was added . after 10 min , ether ( 20 ml ) was added , and a precipitate formed . this was collected and dried to yield the title compound as a cream - colored powder ( 0.036 g , 38% from 46 : mp 277278 c ) . h nmr ( 500 mhz ; dmso - d6 ) : 14.35 ( s , 1 h ) , 9.58 ( s , 2 h ) , 9.28 ( br s , 1 h ) , 8.37 ( d , j = 9.5 hz , 1 h ) , 8.32 ( br s , 1 h ) , 8.05 ( s , 1 h ) , 7.96 ( d , j = 8.6 hz , 1 h ) , 7.78 ( d , j = 8.5 hz , 1 h ) , 7.39 ( td , j = 7.8 , 6.4 hz , 1 h ) , 7.167.09 ( m , 4 h ) , 4.29 ( s , 2 h ) , 3.203.19 ( m , 2 h ) , 3.05 ( t , j = 8.1 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) : ( 163.2 + 161.2 , 1 c ) , 154.4 ( 1 c ) , 142.8 ( 1 c ) , ( 140.11 + 140.05 , 1 c ) , 135.9 ( 1 c ) , 134.2 ( 1 c ) , 130.59 ( 1 c ) , ( 130.58 + 130.52 , 1 c ) , 128.6 ( 1 c ) , ( 124.83 + 124.81 , 1 c ) , 120.5 ( 1 c ) , 117.6 ( 1 c ) , ( 115.53 + 115.36 , 1 c ) 114.5 ( 1 c ) , ( 113.69 + 113.52 , 1 c ) , 49.2 ( 1 c ) , 47.0 ( 1 c ) , 31.0 ( 1 c ) . amine 48 ( 0.050 g , 0.218 mmol ) was dissolved in anhydrous chcl3 ( 3 ml ) , and aldehyde 30 ( 0.034 g , 0.274 mmol ) was added , followed by 3 ml of a 2:1 mixture of chcl3/meoh and anhydrous sodium sulfate ( 0.5 g ) . the mixture was stirred rapidly at room temperature for 90 min , after which glacial acetic acid ( 10 l ) was added . after a total of 4 h , the amine appeared to be consumed by tlc , and the mixture was filtered to remove the sodium sulfate . the filtrate was concentrated , and the oily residue was diluted in meoh ( 5 ml ) . nabh4 ( 0.020 g , 0.523 mmol ) was added , and the mixture was stirred for 40 min at room temperature . the mixture was concentrated and partitioned between etoac and h2o ( 10 ml of each ) . the layers were separated , and the aqueous layer was extracted with etoac ( 10 ml ) . the organic layer was washed with h2o and sat aq . nacl ( 20 ml each ) , dried over anhydrous sodium sulfate , and concentrated . the residue was purified by flash column chromatography ( sio2 ) , eluting with a gradient of etoac to 20% meoh in etoac to yield the intermediate acetamide as a yellow syrup ( 0.052 g , 72% , confirmed by ms ) . this compound was immediately diluted in meoh ( 5 ml ) , and k2co3 ( 0.021 g , 0.154 mmol ) was added . the mixture was heated at vigorous reflux for 2 h , cooled , and concentrated , and the resulting residue was diluted with etoac ( 20 ml ) and washed with h2o ( 10 ml ) . the aqueous layer was extracted with etoac ( 2 10 ml ) , and the combined organic layers were washed with sat . concentration afforded a white solid , which was diluted with methanolic hcl ( 1.4 m , 3 ml ) and stirred for 10 min . the addition of ether ( 50 ml ) resulted in the precipitation of a solid that was collected , washed with ether ( 20 ml ) and dried in vacuo to yield the title compound as a white solid ( 0.043 g , 54% from 48 ) : mp 282284 c . h nmr ( 500 mhz ; dmso - d6 ) : 14.24 ( s , 1 h ) , 9.62 ( s , 2 h ) , 9.16 ( br s , 1 h ) , 8.35 ( d , j = 9.4 hz , 1 h ) , 8.19 ( br s , 1 h ) , 7.80 ( d , j = 0.7 hz , 1 h ) , 7.717.67 ( m , 2 h ) , 7.527.48 ( m , 2 h ) , 7.42 ( d , j = 7.7 hz , 1 h ) , 7.307.26 ( m , 1 h ) , 7.12 ( d , j = 9.3 hz , 1 h ) , 4.22 ( s , 2 h ) , 3.173.14 ( m , 4 h ) . c nmr ( 126 mhz ; dmso - d6 ) : ( 162.9 + 160.9 , 1 c ) , 154.0 ( 1 c ) , 142.8 ( 1 c ) , ( 134.62 + 134.56 , 1 c ) , 134.0 ( 1 c ) , 133.3 ( 1 c ) , ( 130.76 + 130.70 , 1 c ) , 128.3 ( 1 c ) , ( 126.24 + 126.22 , 1 c ) , 121.0 ( 1 c ) , 117.7 ( 1 c ) , ( 116.96 + 116.79 , 1 c ) , ( 115.90 + 115.74 , 1 c ) , 114.0 ( 1 c ) , 49.2 ( 1 c ) , 47.2 ( 1 c ) , 30.8 ( 1 c ) ; one of the aminoquinoline carbons is not visible because of baseline broadening ; esims m / z ( rel . hrms calcd for c18h18fn3 , 295.1485 ; found , 295.1486 . to a solution of 48 ( 0.074 g , 0.321 mmol ) in 7:1 chcl3/meoh ( 8 ml ) , aldehyde 35 ( 0.049 g , 0.353 mmol ) was added , followed by glacial acoh ( 7 l ) and anhydrous mgso4 ( approx 0.5 g ) . the mixture was stirred at room temperature for 20 min and then cooled to 0 c . sodium triacetoxyborohydride ( 0.082 g , 0.385 mmol ) was added in one portion , and the mixture was slowly warmed to room temperature over 50 min , then diluted with ch2cl2 ( 10 ml ) . nahco3 ( 2 20 ml ) , and the aqueous layer was extracted with ch2cl2 ( 2 10 ml ) . the solution was concentrated , and the residue was purified by flash column chromatography ( sio2 ) , eluting with a gradient of etoac to 20% meoh in etoac to yield the intermediate acetamide as a sticky syrup ( 0.039 g , 34% ) that was immediately dissolved in meoh ( 3 ml ) . k2co3 ( 0.023 g , 0.167 mmol ) was added , and the mixture was heated to vigorous reflux for 1 h 50 min . the mixture was cooled and concentrated , and the residue was partitioned between etoac ( 10 ml ) and 1:1 h2o / sat . the layers were separated , and the aqueous layer was extracted with etoac ( 2 4 ml ) . nacl ( 4 ml ) , dried over anhydrous sodium sulfate , and concentrated to yield a sticky residue that was diluted with ch2cl2 ( 3 ml ) and filtered to remove particulate matter . methanolic hcl ( 1.4 m , 2 ml ) was added , the mixture was stirred for 10 min and concentrated , and the residue was recrystallized from 1:1 meoh / ether ( 1 ml ) to yield the product as a pale tan hygroscopic solid ( 0.025 g , 21% based on 48 ) : mp 223226 c ( dec ) . h nmr ( 500 mhz ; dmso - d6 ) : 14.37 ( s , 1 h ) , 9.359.23 ( m , 3 h ) , 8.36 ( d , j = 9.4 hz , 1 h ) , 8.30 ( br s , 1 h ) , 7.83 ( s , 1 h ) , 7.737.69 ( m , 2 h ) , 7.427.38 ( m , 1 h ) , 7.187.09 ( m , 4 h ) , 3.223.19 ( m , 4 h ) , 3.14 ( t , j = 7.9 hz , 2 h ) , 3.04 ( t , j = 8.1 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) : ( 163.2 + 161.3 , 1 c ) , 154.0 ( 1 c ) , 142.8 ( 1 c ) , ( 140.14 + 140.08 , 1 c ) , 134.6 ( 1 c ) , 134.1 ( 1 c ) , 133.3 ( 1 c ) , ( 130.58 + 130.51 , 1 c ) , 128.3 ( 1 c ) , ( 124.86 + 124.84 , 1 c ) , 120.9 ( 1 c ) , 117.5 ( 1 c ) , ( 115.54 + 115.37 , 1 c ) , 114.0 ( 1 c ) , ( 113.68 + 113.52 , 1 c ) , 47.35 ( 1 c ) , 47.20 ( 1 c ) , 31.0 ( 1 c ) , 30.8 ( 1 c ) . esims m / z ( rel . hrms calcd for c19h20fn3 , 309.1641 ; found , 309.1647 . to a solution of 48 ( 0.060 g , 0.261 mmol ) in 10:1 chcl3/meoh ( 5 ml ) , aldehyde 41 ( 0.047 g , 0.313 mmol ) was added , followed by glacial acoh ( 6 l ) and anhydrous mgso4 ( approx 0.5 g ) . the mixture was stirred at room temperature for 25 min and then cooled to 0 c . sodium triacetoxyborohydride ( 0.070 g , 0.332 mmol ) was added in one portion , and the mixture was slowly warmed to room temperature over 30 min , filtered , and concentrated . the residue was purified by flash column chromatography ( sio2 ) , eluting with a gradient of etoac to 20% meoh in etoac to yield a sticky yellow solid ( 0.036 g , 27% ) . this substance was immediately dissolved in meoh ( 3 ml ) , k2co3 ( 0.030 g , 0.217 mmol ) was added , and the mixture was heated to vigorous reflux for 2 h. the mixture was cooled and concentrated , and the residue was partitioned between etoac ( 6 ml ) and 1:1 h2o / sat . the layers were separated , and the aqueous layer was extracted with etoac ( 2 3 ml ) . nacl ( 3 ml ) , dried over anhydrous sodium sulfate , and concentrated to yield a sticky residue that was diluted with ch2cl2 ( 3 ml ) and filtered to remove particulate matter . methanolic hcl ( 1.4 m , 2 ml ) was added , the mixture was stirred for 10 min , concentrated , and the residue was washed with 1:1 ch2cl2/ether ( 3 ml ) to yield the product as a yellow - green hygroscopic solid ( 0.033 g , 32% based on 48 ) : mp 70 c ( softens ) , 211213 c ( dec ) . h nmr ( 500 mhz ; dmso - d6 ) : 14.31 ( s , 1 h ) , 9.20 ( br s , 3 h ) , 8.34 ( d , j = 9.4 hz , 1 h ) , 8.22 ( br s , 1 h ) , 7.82 ( s , 1 h ) , 7.70 ( s , 2 h ) , 7.387.33 ( m , 1 h ) , 7.147.03 ( m , 4 h ) , 3.233.17 ( m , 2 h ) , 3.11 ( t , j = 7.8 hz , 2 h ) , 2.942.89 ( m , 2 h ) , 2.71 ( t , j = 7.6 hz , 2 h ) , 1.97 ( quintet , j = 7.6 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) : ( 163.2 + 161.3 , 1 c ) , 154.0 ( 1 c ) , ( 143.72 + 143.66 , 1 c ) , 142.8 ( 1 c ) , 134.6 ( 1 c ) , 134.1 ( 1 c ) , 133.3 ( 1 c ) , ( 130.31 + 130.25 , 1 c ) , 128.2 ( 1 c ) , ( 124.47 + 124.45 , 1 c ) , 120.9 ( 1 c ) , 117.5 ( 1 c ) , ( 115.09 + 114.93 , 1 c ) , 114.0 , ( 112.95 + 112.78 , 1 c ) , 47.3 ( 1 c ) , 46.1 ( 1 c ) , 31.5 ( 1 c ) , 30.9 ( 1 c ) , 26.8 ( 1 c ) . esims m / z ( rel . compound 29 ( 0.076 g , 0.333 mmol ) was diluted in 7:1 chcl3/meoh ( 7 ml ) , and aldehyde 36 ( 0.045 g , 0.327 mmol ) was added as a solution in 1 ml of chcl3 , followed by glacial acetic acid ( 7 l ) and anhydrous mgso4 ( 0.5 g ) . the flask was sheathed with aluminum foil , and the mixture was stirred for 45 min and cooled to 0 c , and sodium triacetoxyborohydride ( 0.085 g , 0.401 mmol ) was added in one portion . the mixture was allowed to warm to room temperature slowly over 1 h and 15 min and was diluted with chcl3 ( to a volume of approximately 50 ml ) and filtered . nahco3 ( 10 ml ) , and the aqueous layer was extracted with chcl3 ( 2 5 ml ) . nacl ( 20 ml ) , dried over anhydrous sodium sulfate , and concentrated . the resulting residue was purified by flash column chromatography ( sio2 ) eluting with a gradient of etoac to 14% meoh in etoac to yield the intermediate acetamide ( 0.051 g , 44% ) as an oil that began to solidify on standing . this substance was immediately diluted with anhydrous meoh ( 8 ml ) , and k2co3 ( 0.030 g , 0.217 mmol ) was added . the mixture was heated at reflux for 2 h , cooled , and concentrated . the residue was diluted with etoac ( 10 ml ) , and the solution was washed with h2o / sat . the aqueous layer was extracted with etoac ( 3 6 ml ) , and the combined organic layers were washed with sat . nacl ( 6 ml ) , dried over anhydrous sodium sulfate , and concentrated . the resulting syrup was diluted in ch2cl2 ( 5 ml ) , filtered to remove particulate matter , and reconcentrated . to the residue was added methanolic hcl ( 1.4 m , 1 ml ) , and the mixture was stirred at room temperature for 1 h , upon which a white crystalline solid formed . the mixture was cooled to 30 c and filtered to yield the title compound as white flocculent crystals ( 0.021 g , 16% from 29 ) : mp 279281 c . h nmr ( 500 mhz ; dmso - d6 ) : 14.34 ( s , 1 h ) , 9.20 ( s , 3 h ) , 8.37 ( d , j = 9.3 hz , 1 h ) , 8.268.24 ( br s , 1 h ) , 7.92 ( d , j = 8.2 hz , 1 h ) , 7.60 ( s , 1 h ) , 7.42 ( dd , j = 8.2 , 1.2 hz , 1 h ) , 7.33 ( td , j = 6.1 , 2.5 hz , 2 h ) , 7.217.17 ( m , 2 h ) , 7.09 ( d , j = 9.3 hz , 1 h ) , 3.263.16 ( m , 6 h ) , 2.98 ( t , j = 8.1 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) : ( 162.1 + 160.2 1 c ) , 154.3 ( 1 c ) , 142.8 ( 1 c ) , 142.4 ( 1c ) , 135.9 ( 1 c ) , ( 133.34 + 133.32 , 1 c ) , ( 130.59 + 130.53 , 1 c ) , 129.1 ( 1 c ) , 125.8 ( 1 c ) , 119.7 ( 1 c ) , 117.0 ( 1 c ) , ( 115.45 + 115.28 , 1 c ) , 113.4 ( 1 c ) , 47.7 ( 1 c ) , 47.2 ( 1 c ) , 31.7 ( 1 c ) , 30.7 ( 1 c ) . esims m / z ( rel . compound 29 ( 0.076 g , 0.333 mmol ) was diluted in 8:1 chcl3/meoh ( 7 ml ) , and aldehyde 37 ( 0.051 g , 0.330 mmol ) was added as a solution in 1 ml of chcl3 , followed by glacial acetic acid ( 7 l ) and anhydrous mgso4 ( 0.5 g ) . the mixture was stirred for 30 min , and cooled to 0 c , and sodium triacetoxyborohydride ( 0.085 g , 0.401 mmol ) was added in one portion . the mixture was allowed to warm to room temperature slowly over 1 h and was diluted with chcl3 ( to a volume of approximately 50 ml ) and filtered . nahco3 ( 10 ml ) , and the aqueous layer was extracted with chcl3 ( 10 ml ) . nacl ( 20 ml ) , dried over anhydrous sodium sulfate , and concentrated . the resulting residue was purified by flash column chromatography ( sio2 ) eluting with a gradient of etoac to 13% meoh in etoac to yield the intermediate acetamide ( 0.039 g , 32% ) as a semisolid . this substance was diluted with anhydrous meoh ( 6 ml ) , and k2co3 ( 0.029 g , 0.210 mmol ) was added . the mixture was heated at reflux for 2 h , cooled , and concentrated . the residue was immediately diluted with etoac ( 10 ml ) , and the solution was washed with h2o / sat . nacl ( 5 ml ) , dried over anhydrous sodium sulfate , and concentrated . the resulting syrup was diluted in ch2cl2 ( 5 ml ) , filtered to remove particulate matter , and reconcentrated . to the residue was added methanolic hcl ( 1.4 m , 3 ml ) , the mixture was stirred at room temperature for 5 min , and ether ( 20 ml ) was added , upon which an off - white solid ( 0.030 g , 23% ) was collected . an analytically pure sample for assay was prepared by preparative lc - ms , using the instrument and column detailed in the general procedures section , eluting with a gradient of 95% h2o + 0.1%/formic acid 5% mecn + 0.1% formic acid for 2 min , to 70% h2o at 27 min , then to 0% h2o at 32 min . evaporation and retreatment of the residue with methanolic hcl ( 1 ml ) and ether ( 1 ml ) afforded the pure compound as a white flocculent solid ( 0.014 g , 11% from 29 ) : mp 281282 c . h nmr ( 500 mhz ; dmso - d6 ) : 14.29 ( s , 1 h ) , 9.17 ( br s , 3 h ) , 8.37 ( d , j = 9.3 hz , 1 h ) , 8.25 ( br s , 1 h ) , 7.92 ( d , j = 8.2 hz , 1 h ) , 7.60 ( s , 1 h ) , 7.447.34 ( m , 4 h ) , 7.27 ( d , j = 7.4 hz , 1 h ) , 7.09 ( d , j = 9.3 hz , 1 h ) , 3.253.20 ( m , 4 h ) , 3.203.16 ( m , 2 h ) , 3.00 ( t , j = 8.1 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) : 154.3 ( 1 c ) , 142.8 ( 1 c ) , 142.5 ( 1 c ) , 139.8 ( 1 c ) , 135.8 ( 1 c ) , 133.2 ( 1 c ) , 130.5 ( 1 c ) , 129.1 ( 1 c ) , 128.6 ( 1 c ) , 127.5 ( 1 c ) , 126.8 ( 1 c ) , 125.8 ( 1 c ) , 119.7 ( 1 c ) , 116.9 ( 1 c ) , 113.4 ( 1 c ) , 47.2 ( 1 c ) , 31.6 ( 1 c ) , 31.0 ( 1 c ) . esims m / z ( rel . compound 29 ( 0.076 g , 0.333 mmol ) was diluted in 7:1 chcl3/meoh ( 7 ml ) , and aldehyde 38 ( 0.051 g , 0.330 mmol ) was added as a solution in 1 ml of chcl3 , followed by glacial acetic acid ( 7 l ) and anhydrous mgso4 ( 0.5 g ) . the mixture was stirred for 30 min and cooled to 0 c , and sodium triacetoxyborohydride ( 0.085 g , 0.401 mmol ) was added in one portion . the mixture was allowed to warm to room temperature slowly over 1 h 15 min and was diluted with chcl3 ( to a volume of approximately 50 ml ) and filtered . nahco3 ( 10 ml ) , and the aqueous layer was extracted with chcl3 ( 2 5 ml ) . the organic phase was washed with sat . nacl ( 20 ml ) , dried over anhydrous sodium sulfate , and concentrated . the resulting residue was purified by flash column chromatography ( sio2 ) eluting with a gradient of etoac to 13% meoh in etoac ) to yield the intermediate acetamide ( 0.032 g , 26% ) as a white semisolid . this substance was immediately diluted with anhydrous meoh ( 7 ml ) , and k2co3 ( 0.024 g , 0.174 mmol ) was added . the mixture was heated at reflux for 2 h , cooled , and concentrated . the residue was diluted with etoac ( 10 ml ) , and the solution was washed with h2o / sat . nacl ( 5 ml ) , dried over anhydrous sodium sulfate , and concentrated . the resulting syrup was diluted in ch2cl2 ( 5 ml ) , filtered to remove particulate matter , and reconcentrated . to the residue was added methanolic hcl ( 1.4 m , 3 ml ) , the mixture was stirred at room temperature for 5 min , and ether ( 20 ml ) was added , upon which an off - white solid ( 0.027 g , 20% ) was collected . an analytically pure sample for assay was prepared by preparative lc - ms , using the instrument and column detailed in the general procedures section , eluting with a gradient of 95% h2o + 0.1% formic acid/5% mecn + 0.1% formic acid for 5 min , to 93% h2o in 30 min , then to 0% h2o at 32 min . evaporation and retreatment of the residue with methanolic hcl ( 1 ml ) and ether ( 1 ml ) afforded the pure compound as a white flocculent solid ( 0.0094 g , 7.4% from 29 ) : mp 288290 c ( dec ) . h nmr ( 500 mhz ; dmso - d6 ) : 14.20 ( s , 1 h ) , 9.07 ( s , 3 h ) , 8.36 ( dd , j = 9.2 , 0.5 hz , 1 h ) , 8.22 ( br s , 1 h ) , 7.91 ( d , j = 8.1 hz , 1 h ) , 7.58 ( s , 1 h ) , 7.447.41 ( m , 3 h ) , 7.32 ( d , j = 8.4 hz , 2 h ) , 7.07 ( d , j = 9.2 hz , 1 h ) , 3.283.14 ( m , 6 h ) , 2.97 ( t , j = 8.1 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) : 154.4 ( 1 c ) , 142.8 ( 1 c ) , 142.4 ( 1 c ) , 136.2 ( 1 c ) , 131.4 ( 1 c ) , 130.6 ( 1 c ) , 129.1 ( 1 c ) , 128.6 ( 1 c ) , 125.8 ( 1 c ) , 119.8 ( 1 c ) , 117.0 ( 1 c ) , 113.4 ( 1 c ) , 47.40 ( 1 c ) , 47.22 ( 1 c ) , 31.7 ( 1 c ) , 30.8 ( 1 c ) ; one of the aminoquinoline carbons is not visible due to line - broadening . compound 17 ( 4.26 g , 18.0 mmol ) was diluted in chlorobenzene ( 45 ml ) , and anhydrous alcl3 ( 12.0 g , 5.00 mmol ) was added . the mixture was heated to 90 c for 2 h , upon which the solution became black . h2o ( 300 ml ) , which was extracted with etoac ( 2 300 ml ) . the organic phase was washed with h2o ( 200 ml ) , and the aqueous layer was extracted with etoac ( 100 ml ) . the orange solution was filtered through celite and concentrated to afford the mixture of products as a beige solid ( 2.53 g , 88% ) after washing with hexanes and drying . h nmr spectra indicated that 18a and 18b were present as a 70:30 mixture ( consistent with prior reports ) , which was used without any further purification . a mixture of 18a and 18b ( 2.53 g , 15.9 mmol ) was diluted in pocl3 ( 35 ml ) , and the mixture was heated at reflux for 70 min , before the clear orange solution was cooled to room temperature and poured into ice the beaker was immersed in ice and cooled to 0 c with stirring , and solid naoh was added until the ph of the mixture was approximately 7 . the resultant cloudy suspension was extracted with etoac ( 300 ml ) , and the organic layers were washed with h2o ( 100 ml ) and sat . the organic layer was dried over anhydrous sodium sulfate and concentrated , and the residue was purified by flash column chromatography ( sio2 ) , eluting with a gradient of hexanes to 12% ethyl acetate in hexanes to afford orange crystals . fractional crystallization from hot isopropanol yielded pure 19a ( 0.850 g , 30% ) as light orange iridescent crystals ; the analytical data for this compound are identical to those in prior literature reports.h nmr ( 500 mhz ; cdcl3 ) : 8.05 ( d , j = 8.5 hz , 1 h ) , 8.00 ( m , 1 h ) , 7.71 ( d , j = 8.3 hz , 1 h ) , 7.39 ( dd , j = 8.3 , 1.5 hz , 1 h ) , 7.32 ( d , j = 8.5 hz , 1 h ) , 2.56 ( s , 3 h ) . chloride 19a ( 0.300 g , 1.69 mmol ) was diluted with molten anhydrous acetamide ( 8 g , 135 mmol ) , and k2co3 ( 1.17 g , 8.45 mmol ) was added . the mixture was heated in a sand bath , at reflux ( 230 c ) for 17 h. the mixture was cooled , poured into h2o ( 120 ml ) , and extracted with etoac ( 4 30 ml ) . the organic layers were washed with h2o ( 3 100 ml ) and sat . nacl ( 50 ml ) , dried over anhydrous sodium sulfate , and concentrated . purification of the residue by flash column chromatography ( sio2 , 15% etoac in ch2cl2 ) afforded the desired compound as a white solid ( 0.265 g , 78% ) . h nmr chemical shifts for this compound are consistent with those reported by inglis et al . for the 7-isomer.h nmr ( 500 mhz ; cdcl3 ) : 9.899.88 ( br s , 1 h ) , 8.40 ( d , j = 8.9 hz , 1 h ) , 8.15 ( d , j = 9.0 hz , 1 h ) , 7.67 ( d , j = 8.3 hz , 1 h ) , 7.58 ( d , j = 0.6 hz , 1 h ) , 7.29 ( dd , j = 8.3 , 1.4 hz , 1 h ) , 2.54 ( s , 3 h ) , 2.27 ( s , 3 h ) . compound 20 ( 0.265 g , 1.32 mmol ) was diluted in anhydrous benzene ( 10 ml ) . n - bromosuccinimide ( 0.247 g , 1.39 mmol ) and a catalytic amount ( 0.020 g ) of benzoyl peroxide were added , and the mixture was heated to reflux under argon until an orange tint was no longer visible in the solution refluxing in the condenser ( typically 4 h ) . the mixture was cooled , concentrated , and purified by flash column chromatography ( sio2 ) , eluting with a gradient of 7% to 14% etoac in ch2cl2 , to yield the product ( 0.236 g , 64% ) as a flocculent yellow solid . h nmr chemical shifts for this compound are consistent with those reported by inglis et al . for the 7-isomer.h nmr ( 500 mhz ; cdcl3 ) : 8.438.41 ( m , 2 h ) , 8.16 ( d , j = 8.9 hz , 1 h ) , 7.797.77 ( m , 2 h ) , 7.49 ( dd , j = 8.4 , 1.7 hz , 1 h ) , 4.65 ( s , 2 h ) , 2.27 ( s , 3 h ) . 3-fluorophenethyl bromide ( 23 , 1.00 g , 12.3 mmol ) was diluted in dry dmf ( 25 ml ) . sodium cyanide ( 1.06 g , 61.6 mmol ) was added in one portion , and the mixture was heated to 60 c under argon for 16 h. the mixture was cooled and concentrated , and the residue was partitioned between etoac and h2o ( 50 ml each ) . the layers were separated , and the aqueous phase was extracted with etoac ( 2 20 ml ) . nacl ( 50 ml each ) , dried over anhydrous sodium sulfate , and concentrated . the resulting oil was purified by flash column chromatography ( sio2 ) , eluting with a gradient of 5% etoac in hexanes to 30% etoac in hexanes to yield the desired product as a colorless oil ( 0.638 g , 87% ) . h nmr ( 500 mhz ; cdcl3 ) : 7.31 ( td , j = 7.9 , 6.0 hz , 1 h ) , 7.036.93 ( m , 3 h ) , 2.96 ( t , j = 7.4 hz , 2 h ) , 2.63 ( t , j = 7.4 hz , 2 h ) . compound 24 ( 0.180 g , 1.21 mmol ) was diluted in etoh ( 3 ml ) , and methanolic ammonia ( 7 m , 6 ml ) and raney nickel ( 1 g ) were added . the mixture was filtered through a pall 0.2 m syringe filter and concentrated to yield a sticky green syrup ( 0.083 g , 45% ) . the presence of amine was confirmed by h nmr spectrometry , tlc , and ninhydrin staining , and this material was used crude without any further purification . anhydrous cs2co3 ( 0.295 g , 0.906 mmol ) was diluted in anhydrous dmf ( 10 ml ) . 3-fluorophenethylamine ( 22 , 0.126 g , 0.906 mmol ) was added , and the mixture was stirred at room temperature for 30 min before a solution of 21 ( 0.220 g , 0.788 mmol ) in dmf ( 4 ml ) was added slowly over 5 min . the cloudy yellow mixture was stirred at room temperature for 16 h and concentrated . the residue was diluted with etoac ( 50 ml ) and washed with h2o ( 2 50 ml ) and sat . the organic phase was dried over anhydrous sodium sulfate , concentrated , and purified by flash column chromatography ( sio2 ) eluting with 10% meoh in etoac to yield the product as a clear yellow oil ( 0.187 g , 70% ) . h nmr ( 500 mhz ; cdcl3 ) : 8.95 ( s , 1 h ) , 8.40 ( br d , j = 8.2 hz , 1 h ) , 8.15 ( d , j = 8.9 hz , 1 h ) , 7.74 ( d , j = 8.3 hz , 1 h ) , 7.72 ( s , 1 h ) , 7.41 ( dd , j = 8.3 , 1.5 hz , 1 h ) , 7.267.22 ( m , 1 h ) , 6.98 ( d , j = 7.7 hz , 1 h ) , 6.936.88 ( m , 2 h ) , 4.00 ( s , 2 h ) , 2.95 ( t , j = 7.0 hz , 2 h ) , 2.85 ( t , j = 7.0 hz , 2 h ) , 2.23 ( s , 3 h ) . c nmr ( 126 mhz ; cdcl3 ) : 169.3 ( 1 c ) , ( 163.9 + 162.0 , 1 c ) , 151.3 ( 1 c ) , 146.5 ( 1 c ) , ( 142.47 + 142.41 , 1 c ) , 142.37 ( 1 c ) , 138.5 ( 1c ) , ( 129.95 + 129.88 , 1 c ) , 127.8 ( 1 c ) , 125.76 ( 1 c ) , 125.71 ( 1 c ) , 125.4 ( 1 c ) , ( 124.42 + 124.40 , 1 c ) , ( 115.63 + 115.46 , 1 c ) , 114.0 ( 1 c ) , ( 113.23 + 113.06 , 1 c ) , 53.7 ( 1 c ) , 50.1 ( 1 c ) , 36.1 ( 1 c ) , 24.9 ( 1 c ) . esims m / z ( rel . compound 21 ( 0.216 g , 0.773 mmol ) was diluted with anhydrous dmf ( 10 ml ) , and nacn ( 0.190 g , 3.87 mmol ) was added . the orange mixture was stirred at room temperature for 17 h. the mixture was concentrated and partitioned between etoac and h2o ( 50 ml each ) , and the layers were separated . the aqueous phase was extracted with etoac ( 2 50 ml ) , and the combined organic layers were washed with h2o ( 2 80 ml ) and sat . the residue was purified by flash column chromatography ( sio2 ) , eluting with a gradient of 15% etoac in ch2cl2 to 25% etoac in ch2cl2 to yield the title compound as a white solid ( 0.109 g , 63% ) : mp 180182 c . h nmr ( 500 mhz ; cdcl3 ) : 8.44 ( dd , j = 8.3 , 0.4 hz , 1 h ) , 8.278.22 ( m , 1 h ) , 8.18 ( d , j = 9.0 hz , 1 h ) , 7.81 ( d , j = 8.4 hz , 1 h ) , 7.79 ( s , 1 h ) , 7.40 ( dd , j = 8.3 , 1.7 hz , 1 h ) , 3.94 ( s , 2 h ) , 2.28 ( s , 3 h ) . c nmr ( 126 mhz ; cdcl3 ) : 169.2 ( 1 c ) , 151.5 ( 1 c ) , 138.8 ( 1 c ) , 132.1 ( 1 c ) , 128.7 ( 1 c ) , 126.2 ( 1 c ) , 125.6 ( 1 c ) , 124.9 ( 1 c ) , 117.3 ( 1 c ) , 114.7 ( 1 c ) , 25.1 ( 1 c ) , 24.0 ( 1 c ) . esims m / z ( rel . compound 28 ( 0.060 g , 0.266 mmol ) was diluted in absolute etoh ( 7 ml ) , and methanolic ammonia ( 7 n , 7 ml ) was added . raney nickel ( 1.5 g , washed with h2o and meoh ) was added , and the mixture was degassed and hydrogenated with a h2-filled balloon at room temperature for 30 min while stirring rapidly . the clear solution was decanted from the nickel and was filtered through a pall 0.2 m syringe filter to remove fine particulate matter . the solution was concentrated and dried in vacuo to yield an off - white semisolid ( 0.062 g , 100% ) . conversion to this amine was confirmed by tlc and ninhydrin staining , and it was used without any further characterization or purification . dess - martin periodinane ( 1.02 g , 2.4 mmol ) was diluted in anhydrous ch2cl2 ( 25 ml ) under argon , and when solution was affected , 3-chlorophenethyl alcohol ( 33 , 0.313 g , 2.00 mmol ) was added dropwise . the mixture was stirred for 2 h and 15 min at room temperature and was then quenched by the addition of 20 ml of sat . after stirring at room temperature for 15 min , the layers were separated , and the aqueous layer was extracted with ch2cl2 ( 2 50 ml ) . the organic layer was washed with h2o and sat . nacl ( 50 ml each ) and was dried over anhydrous sodium sulfate and concentrated . the resulting semisolid residue was triturated with 10% etoac in hexanes , and the solid was filtered out and discarded . the filtrate was concentrated , and the oily residue was purified by flash column chromatography ( sio2 ) , eluting with a gradient of hexanes to 10% etoac in hexanes to afford the title compound as a clear yellow volatile oil ( 0.241 g , 78% ) . h nmr ( 500 mhz ; cdcl3 ) : 9.75 ( t , j = 2.1 hz , 1 h ) , 7.317.23 ( m , 3 h ) , 7.117.09 ( m , 1 h ) , 3.69 ( d , j = 2.1 hz , 2 h ) . dess - martin periodinane ( 1.02 g , 2.4 mmol ) was diluted in anhydrous ch2cl2 ( 25 ml ) under argon , and when the solution was affected , 4-chlorophenethyl alcohol ( 33 , 0.313 g , 2 mmol ) was added dropwise . the mixture was stirred for 2 h and 15 min at room temperature and was then quenched by the addition of 20 ml of sat . after stirring at room temperature for 15 min , the layers were separated , and the aqueous layer was extracted with ch2cl2 ( 2 50 ml ) . the organic layer was washed with h2o and sat . nacl ( 50 ml each ) and was dried over anhydrous sodium sulfate and concentrated . the resulting semisolid residue was triturated with 10% etoac in hexanes , and the solid was filtered and discarded . the filtrate was concentrated , and the oily residue was purified by flash column chromatography ( sio2 ) , eluting with a gradient of hexanes to 15% etoac in hexanes to afford the title compound as a clear yellow volatile oil ( 0.211 g , 88% ) . h nmr ( 500 mhz ; cdcl3 ) : 9.75 ( t , j = 2.1 hz , 1 h ) , 7.34 ( d , j = 8.3 hz , 2 h ) , 7.15 ( d , j = 8.2 hz , 2 h ) , 3.69 ( d , j = 2.0 hz , 2 h ) . 3-fluorophenylpropionic acid ( 39 , 0.500 g , 2.97 mmol ) was diluted in anhydrous thf ( 2 ml ) under argon and cooled to 0 c . borane - thf ( 1 m , 4.16 ml , 4.16 mmol ) was added dropwise , and the mixture was allowed to warm to room temperature and stirred for 18 h. the reaction was quenched by the addition of 1:1 thf / h2o ( 5 ml ) . when gas evolution ceased , solid k2co3 was added until the mixture separated into two layers , which were separated . the aqueous layer was extracted with etoac ( 2 5 ml ) , and the combined organic layers were washed with h2o ( 15 ml ) and sat . nacl ( 15 ml ) , dried over anhydrous sodium sulfate , and concentrated to yield the product as a clear oil ( 0.446 g , 97% ) after drying in vacuo . h nmr ( 500 mhz ; cdcl3 ) : 7.297.25 ( m , 1 h ) , 7.00 ( d , j = 7.6 hz , 1 h ) , 6.956.89 ( m , 2 h ) , 3.71 ( t , j = 6.4 hz , 2 h ) , 2.74 ( t , j = 7.7 hz , 2 h ) , 1.951.89 ( m , 2 h ) , 1.39 ( s , 1 h ) . anhydrous ch2cl2 ( 10 ml ) was cooled to 78 c , and anhydrous dmso ( 0.546 g , 7.00 mmol ) was added , followed , dropwise , by oxalyl chloride ( 0.380 g , 3.00 mmol ) . once gas evolution ceased , compound 40 ( 0.308 g , 2 mmol ) was added dropwise , and the resulting milky solution was stirred for 15 min . et3n ( 1.17 ml , 8.4 mmol ) was added slowly , and the mixture was stirred for 15 min at 78 c and then warmed to room temperature and stirred for 1 h. the yellow mixture was diluted with h2o ( 30 ml ) , and the layers were separated . the aqueous layer was extracted with ch2cl2 ( 2 15 ml ) , and the organic layers were washed with h2o and sat . the solution was dried over anhydrous sodium sulfate , concentrated , and the resulting residue was purified by flash column chromatography ( sio2 ) , eluting with a gradient of hexanes to 10% etoac in hexanes to yield the title aldehyde as a colorless volatile oil ( 0.220 g , 72% ) . h nmr ( 500 mhz ; cdcl3 ) : 9.82 ( s , 1 h ) , 7.277.23 ( m , 1 h ) , 6.97 ( d , j = 7.6 hz , 1 h ) , 6.926.89 ( m , 2 h ) , 2.96 ( t , j = 7.5 hz , 2 h ) , 2.812.78 ( m , 2 h ) . compound 43 ( 3.75 g , 15.8 mmol ) was diluted in chlorobenzene ( 40 ml ) , and aluminum chloride ( 10.5 g , 75.0 mmol ) was added . the mixture was heated to 90 c under nitrogen for 2 h , upon which the mixture became black , was subsequently cooled , and poured into ice the resulting suspension was extracted with etoac ( 700 ml ) , and the organic layer was washed with h2o ( 300 ml ) and dried over anhydrous sodium sulfate . concentration afforded an orange solid that was recrystallized from hot meoh ( 60 ml ) to yield an orange iridescent solid ( 1.95 g , 77% ) . this was not characterized but was instead diluted in pocl3 ( 30 ml ) and heated at reflux for 70 min before cooling and pouring into ice the beaker was immersed in a cooler of ice , with stirring , and solid naoh was added until the ph was approximately 7 . the oily suspension was extracted with etoac ( 400 ml ) , washed with sat . nacl ( 300 ml ) , and the organic layer was dried over anhydrous sodium sulfate . the solution was concentrated to yield a solid that was purified by flash column chromatography ( sio2 ) , eluting with a gradient of hexanes to 40% etoac in hexanes to yield the product as an orange crystalline solid ( 1.79 g , 64% from 42 ) . the h nmr chemical shifts for this compound are identical to those previously reported by inglis et al.h nmr ( 500 mhz ; cdcl3 ) : 8.01 ( d , j = 8.6 hz , 1 h ) , 7.91 ( d , j = 9.2 hz , 1 h ) , 7.577.55 ( m , 2 h ) , 7.34 ( d , j = 8.6 hz , 1 h ) , 2.53 ( s , 3 h ) . chloride 44 ( 0.300 g , 1.69 mmol ) was diluted with molten anhydrous acetamide ( 8 g , 135 mmol ) , and k2co3 ( 1.17 g , 8.45 mmol ) was added . the mixture was heated in a sand bath , at reflux ( 230 c ) for 16 h. the mixture was cooled , poured into h2o ( 120 ml ) , and extracted with etoac ( 4 30 ml ) . the organic layers were washed with h2o ( 3 100 ml ) and sat . purification of the residue by flash column chromatography ( sio2 ) , eluting with a gradient of 10% etoac in ch2cl2 to 30% etoac in ch2cl2 , afforded the desired compound as a white solid ( 0.250 g , 74% ) . h nmr chemical shifts for this compound are consistent with those reported by inglis et al.h nmr ( 500 mhz ; cdcl3 ) : 8.36 ( br d , j = 8.6 hz , 1 h ) , 8.27 ( br s , 1 h ) , 8.09 ( d , j = 8.9 hz , 1 h ) , 7.70 ( d , j = 8.6 hz , 1 h ) , 7.55 ( s , 1 h ) , 7.50 ( dd , j = 8.6 , 1.9 hz , 1 h ) , 2.51 ( s , 3 h ) , 2.24 ( s , 3 h ) . compound 45 ( 0.300 g , 1.50 mmol ) was diluted in anhydrous benzene ( 10 ml ) . n - bromosuccinimide ( 0.280 g , 1.57 mmol ) and a catalytic amount ( 0.020 g ) of benzoyl peroxide were added , and the mixture was heated to reflux under nitrogen until an orange tint was no longer visible in the solution refluxing in the condenser ( around 2 h 40 min ) . the mixture was cooled , concentrated , and purified by flash column chromatography ( sio2 ) , eluting with a gradient of 10% to 12% etoac in ch2cl2 to yield the product ( 0.262 g , 63% ) as a flocculent yellow solid . the h nmr chemical shifts for this compound are identical to those previously reported by inglis et al.h nmr ( 500 mhz ; cdcl3 ) : 8.44 ( br d , j = 8.6 hz , 1 h ) , 8.30 ( br s , 1 h ) , 8.17 ( d , j = 9.0 hz , 1 h ) , 7.82 ( m , j = 9.1 hz , 2 h ) , 7.72 ( dd , j = 8.6 , 2.1 hz , 1 h ) , 4.67 ( s , 2 h ) , 2.29 ( s , 3 h ) . compound 46 ( 0.254 g , 0.91 mmol ) was diluted with anhydrous dmf ( 10 ml ) , and nacn ( 0.230 g , 4.55 mmol ) was added . the orange mixture was stirred at room temperature for 17 h. the mixture was concentrated and partitioned between etoac and h2o ( 50 ml each ) , and the layers were separated . the aqueous phase was extracted with etoac ( 2 50 ml ) , and the combined organic layers were washed with h2o ( 2 80 ml ) and sat . the residue was purified by flash column chromatography ( sio2 ) , eluting with a gradient of 15% etoac in ch2cl2 to 25% etoac in ch2cl2 to yield the title compound as a white solid ( 0.170 g , 83% ) : mp 154155 c . h nmr ( 500 mhz ; cdcl3 ) : 8.48 ( d , j = 8.6 hz , 1 h ) , 8.22 ( br s , 1 h ) , 8.20 ( d , j = 9.0 hz , 1 h ) , 7.85 ( d , j = 8.7 hz , 1 h ) , 7.81 ( d , j = 1.0 hz , 1 h ) , 7.61 ( dd , j = 8.7 , 2.1 hz , 1 h ) , 3.96 ( s , 2 h ) , 2.30 ( s , 3 h ) . c nmr ( 126 mhz ; cdcl3 ) : 169.1 ( 1 c ) , 151.3 ( 1 c ) , 145.8 ( 1 c ) , 138.6 ( 1 c ) , 129.8 ( 1 c ) , 128.3 ( 1 c ) , 126.74 ( 1 c ) , 126.64 ( 1 c ) , 126.2 ( 1 c ) , 117.6 ( 1 c ) , 114.9 ( 1 c ) , 25.0 ( 1 c ) , 23.7 ( 1 c ) . esims m / z ( rel . compound 47 ( 0.060 g , 0.266 mmol ) was diluted in absolute etoh ( 7 ml ) , and methanolic ammonia ( 7 n , 7 ml ) was added . raney nickel ( 1.5 g , washed with h2o and meoh ) was added , and the mixture was degassed and hydrogenated with a balloon at room temperature for 30 min while stirring rapidly . the clear solution was decanted away from the nickel and was filtered through a pall 0.2 m syringe filter to remove fine particulate matter . the solution was concentrated and dried in vacuo to yield a colorless gum that became a white semisolid upon standing ( 0.050 g , 82% ) . conversion to this amine was confirmed by h nmr spectrometry , tlc , ms , and ninhydrin staining , and it was used crude without any further characterization or purification . rat and human nnos , murine macrophage inos , and bovine enos were recombinant enzymes , expressed in e. coli and purified as previously reported . to test for enzyme inhibition , the assay was performed at 37 c in hepes buffer ( 100 mm , with 10% glycerol , ph 7.4 ) in the presence of 10 m l - arginine . also included were 100 m nadph , 0.83 mm cacl2 , approximately 320 units / ml of calmodulin , 10 m tetrahydrobiopterin , and human oxyhemoglobin ( 3 m ) . for inos , cacl2 and calmodulin were omitted and replaced with hepes buffer ( as neither are required for activation of inos ) . this assay was performed in 96-well plates using a synergy 4 biotek hybrid reader , and the dispensing of nos enzyme and hemoglobin were automated ; after 30 s ( maximum delay ) , no production was read by monitoring the absorbance at 401 nm ( resulting from the conversion of oxyhemoglobin to methemoglobin ) . each compound was assayed at least in duplicate , and nine concentrations ( 500 m50 nm or 100 m10 nm for enos and inos ; 50 m to 5 nm for nnos ) were used to construct dose ic50 values were calculated by nonlinear regression using graphpad prism software ( standard error values reported are from the logic50 calculations ) , and ki values were obtained using the cheng prusoff equation [ ki = ic50/(1 + [ s]/km ) ] using the following km values : 1.3 ( rat nnos ) , 1.6 ( human nnos ) , 8.2 ( murine macrophage inos ) , and 1.7 m ( bovine enos ) . the nnos or enos heme domain proteins used for crystallographic studies were produced by limited trypsin digest from the corresponding full length enzymes and further purified through a superdex 200 gel filtration column ( ge healthcare ) as described previously . the nnos heme domain ( at 9 mg / ml containing 20 mm histidine ) or the enos heme domain ( at 12 mg / ml containing 2 mm imidazole ) was used for the sitting drop vapor diffusion crystallization setup under conditions previously reported . fresh crystals ( 12 days old ) were first passed stepwise through cryoprotectant solutions and then soaked with a 10 mm inhibitor for 46 h at 4 c before being flash cooled with liquid nitrogen . the cryogenic ( 100 k ) x - ray diffraction data were collected remotely at the stanford synchrotron radiation lightsource ( ssrl ) or advanced light source ( als ) through the data collection control software blu - ice and a crystal mounting robot . when a q315r ccd detector was used , 90100 of data were typically collected with 0.5 per frame . if a pilatus pixel array detector was used , 120130 of fine - sliced data were collected with 0.2 per frame . raw ccd data frames were indexed , integrated , and scaled using hkl2000 , but the pixel array data were processed with xds and scaled with scala . the binding of inhibitors was detected by the initial difference fourier maps calculated with refmac . disordering in portions of inhibitors bound in the nos active sites was often observed , sometimes resulting in poor density quality . however , partial structural features usually could still be visible if the contour level of the sigmaa weighted 2m|fo| d|fc| map dropped to 0.5 , which afforded the building of reasonable models into the disordered regions . the tls protocol was implemented in the final stage of refinements with each subunit as one tls group . fc density maps were calculated by repeating the last round of tls refinement with the inhibitor coordinate removed from the input pdb file to generate the map coefficients delfwt and phdelwt . the refined structures were validated in coot before deposition in the rcsb protein data bank . the crystallographic data collection and structure refinement statistics are summarized in table s1 of the supporting information , with the pdb accession codes included . ( watertown , ma ) using the following standard procedure : caco-2 cells , grown in tissue culture flasks , were trypsinized , resuspended , and grown and differentiated in 96-well plates for three weeks ; monolayer formation was determined by measuring transport of lucifer yellow , an impermeable dye . all assays were performed at a concentration of 10 m for 2 h. for apical to basolateral ( ab ) permeability , compounds were added on the apical side ( a ) , with permeation determined at the receiving ( basolateral , b ) side , where the receiving buffer was removed for analysis by lc / ms / ms using an agilent 6410 mass spectrometer ( esi , mrm mode ) coupled with an agilent 1200 hplc . buffers used were 100 m lucifer yellow in transport buffer ( 1.98 g / l glucose in 10 mm hepes , 1 hank s balanced salt solution , ph 6.5 ) ( apical side ) and tranport buffer , ph 7.4 ( basolateral side ) . apparent permeability ( papp ) is expressed using the following equation : papp = ( dq / dt)/c0a , where the numerator is the rate of permeation , c0 is initial concentration , and a is the monolayer area . for bidirectional permeability , the efflux ratio was defined as papp ( ba)/papp ( ab ) ; high efflux ratio values ( > 3 ) indicate that a compound may be a substrate for p - gp or other active transport systems .

since high levels of nitric oxide ( no ) are implicated in neurodegenerative disorders , inhibition of the neuronal isoform of nitric oxide synthase ( nnos ) and reduction of no levels are therapeutically desirable . nonetheless , many nnos inhibitors mimic l - arginine and are poorly bioavailable . 2-aminoquinoline - based scaffolds were designed with the hope that they could ( a ) mimic aminopyridines as potent , isoform - selective arginine isosteres and ( b ) possess chemical properties more conducive to oral bioavailability and cns penetration . a series of these compounds was synthesized and assayed against purified nnos enzymes , endothelial nos ( enos ) , and inducible nos ( inos ) . several compounds built on a 7-substituted 2-aminoquinoline core are potent and isoform - selective ; x - ray crystallography indicates that aminoquinolines exert inhibitory effects by mimicking substrate interactions with the conserved active site glutamate residue . the most potent and selective compounds , 7 and 15 , were tested in a caco-2 assay and showed good permeability and low efflux , suggesting high potential for oral bioavailability .

Introduction Chemistry Results and Discussion Conclusions Experimental Section

nitric oxide ( no ) is an important second messenger in the human body , and dysregulation of its production is implicated in many pathologies . no is produced by the nitric oxide synthase enzymes , of which there are three isoforms : endothelial nitric oxide synthase ( enos ) , which regulates blood pressure and flow , inducible nitric oxide synthase ( inos ) , involved in immune system activation , and nnos , which is required for normal neuronal signaling . nonetheless , overexpression of nnos in neural tissue and increased levels of no can result in protein nitration and oxidative damage to neurons , especially if peroxynitrite is formed from excess no . each monomer consists of both a reductase domain with fad , fmn , and nadph binding sites , and a heme - containing oxygenase domain , where the substrate ( l - arginine ) and cofactor ( 6r)-5,6,7,8-tetrahydrobiopterin ( h4b ) bind . these compounds are highly potent and selective nnos inhibitors , and compound 1 reverses a hypoxic - ischemic brain damage phenotype in newborn rabbit kits when administered intravenously to the dam . many attempts to improve the bioavailability of these compounds have been made , including alkylation , fluorination , introduction of lipophilic tails , and replacement of the amines ; most of these strategies either diminished potency or selectivity or were synthetically challenging . nonetheless , these simplified molecules are not without fault ; their isoform selectivities are lower , 3 suffers from poor caco-2 permeability , and 4 is much less potent in cell - based assays than against isolated enzymes , both likely the result , in part , of the amidine moiety , which will be charged at physiological ph . additionally , a recent report indicates that 2-aminoquinoline - based bace-1 inhibitors have high cellular activity and good blood brain barrier permeability in a rat model . preliminary docking studies and crystallography indicated that elongation of the chain between the aminoquinoline system and the distal fluorophenyl ring of 5 , moving the position of the secondary amine , or a combination of both , might provide the right length and orientation to reach this hydrophobic pocket , and a series of analogues investigating chain length ( 69 ) and nitrogen position was , therefore , prepared ( figure 2 ) . all compounds were assayed against purified rat nnos , and select compounds were assayed against enos , inos , and human nnos , and for cellular permeability in a caco-2 model . to prepare phenethyl analogues 7 , 14 , 15 , and 16 ( see scheme 4 ) , requisite phenylacetaldehydes 3538 were prepared by dess - martin oxidation of commercially available phenethyl alcohols 3134 , respectively . compounds 516 were assayed against purified rat nnos , bovine enos , and murine macrophage inos using the hemoglobin capture assay , as previously described . the lead 7-substituted 2-aminoquinoline , compound 5 , has potent nnos inhibitory activity ( 74 nm ) and high n / i selectivity ( 124-fold ) , yet it is only weakly selective for nnos over enos ( around 6-fold ) . in both cases , the aminoquinoline acts as an arginine mimic and interacts with the active site glutamate as arginine does ( glu592 in nnos ( 1om4 ) ; glu363 in enos ( 2nse ) ) , while the secondary amine sits between the heme propionates , making h - bonds to both . active site structures of lead 5 bound to rat nnos ( a ) and bovine enos ( b ) . while residues in chain a of nnos ( a ) and enos ( b ) are colored green and cyan , respectively , the residue from chain b ( second monomer in the homodimeric structure ) is distinguished by a different color . however , as the 4-atom linker ( of 5 and 6 ) is not long enough to bring the fluorophenyl ring in contact with the aforementioned hydrophobic pocket ( as in figure 3 ) , the majority of the stabilization results from the hydrogen bonds from the aminoquinoline and the linker amine . ( b ) overlay of 5 ( cyan ) and 6 ( yellow ) in nnos . nnos inhibitory activity is very similar between 7 and 9 ( table 1 ) with the nitrogen placement not drastically affecting potency ; it appears that the influence of the amine position is weakened in these inhibitors with longer linker lengths . the omit electron density map reveals that 7 ( figure 5a ) , which lacks a strong secondary amine heme propionate interaction , is more flexible / disordered in the fluorophenyl tail region relative to the structure of 9 ( which does show the amine nonetheless , the similar potencies of 7 and 9 indicate that the nitrogen position is not crucial for these compounds with longer linkers , likely because additional stabilization results from contact with the hydrophobic pocket . active site structure of 7 ( a ) or 9 ( b ) bound to nnos . active site structure of 8 ( a ) or 15 ( b ) bound to nnos . nonetheless , even the short - chain inhibitors ( 5 and 6 ) still possess good n / i selectivity , despite not reaching this distal pocket , indicating that interactions with residues at this end of the binding site are not the full determinant of this poor inos inhibition . however , the length of the linker in 7 enables the fluorophenyl ring to make good hydrophobic contacts with the residues met336 , leu337 , tyr706 , and trp306 ( from the other monomer of the nnos homodimer ) . encouraged by the high potency and selectivity of 7 and 15 , we assayed these compounds ( and lead 5 ) against purified human nnos ( table 2 ) . the human isoform has an active site that is nearly identical to that in the rat enzyme , with the exception of the hydrophobic pocket , where leu337 is replaced by a histidine ( his341 ) . previously , aminopyridine - based inhibitors showed lower potency against the human enzyme when compared to the rat enzyme , and the same trend is observed for the aminoquinolines , although 5 , 7 , and 15 still display good human nnos inhibition . because of the very similar selectivities ( ki - human / ki - rat ) among these three compounds , it can be concluded that the modifications that are well tolerated by the rat isoform ( chain elongation and replacement of fluorine with chlorine ) are likewise tolerated similarly by human nnos , including the introduction of the bulkier chlorine . finally , compounds 7 and 15 were assayed in a caco-2 monolayer permeability assay ( table 3 ) . compound 15 even shows improved membrane permeation relative to compound 4 , and both 7 and 15 display relatively low efflux ratios , diminishing the possibility that p - gp or other active transport mechanisms are significantly acting on these compounds ( especially on 15 ) . in summary , we have prepared a series of novel simplified 2-aminoquinolines based on the rationale that they might bind to and inhibit nnos in a manner similar to that of aminopyridines , while being less polar , less basic , more lipophilic , and , therefore , more bioavailable . crystal structures indicate that these compounds act as competitive arginine mimics , where the aminoquinoline moiety makes hydrogen bonds with the active - site glutamate residue , and that the noncoordinating aryl rings are stabilized in a hydrophobic pocket on the far end of the substrate access channel . most promisingly , two of these highly effective compounds , 7 and 15 , show good permeability in a caco-2 assay . these results indicate that these compounds have high potential for oral bioavailability and brain penetration and that the 7-substituted 2-aminoquinoline cores offer very promising leads for further nnos inhibitor development . compound 29 ( 0.064 g , 0.280 mmol ) was diluted in 7:1 chcl3/meoh ( 7 ml ) , and aldehyde 41 ( 0.049 g , 0.322 mmol ) was added , followed by glacial acetic acid ( 6 l ) and anhydrous mgso4 ( 0.5 g ) . the residue was diluted with etoac ( 10 ml ) , and the solution was washed with h2o / sat . the residue was diluted with ch2cl2 ( 5 ml ) , filtered to remove particulate matter , and reconcentrated . c nmr ( 126 mhz ; dmso - d6 ) : ( 163.2 + 161.3 , 1 c ) , 154.6 ( 1 c ) , ( 143.67 + 143.61 , 1 c ) , 142.6 ( 1 c ) , 136.6 ( 1 c ) , ( 130.32 + 130.25 , 1 c ) , 129.0 ( 1 c ) , 126.4 ( 1 c ) , ( 124.47 + 124.45 , 1 c ) , 120.9 ( 1 c ) , 118.7 ( 1 c ) , ( 115.08 + 114.91 , 1 c ) , 114.5 ( 1 c ) , ( 112.95 + 112.78 , 1 c ) , 49.3 ( 1 c ) , 46.0 ( 1 c ) , 31.5 ( 1 c ) , 26.7 ( 1 c ) ; one of the aminoquinoline carbons is not visible due to baseline broadening ; esims m / z ( rel . this syrup was dissolved in meoh ( 5 ml ) , and k2co3 ( 0.026 g , 0.148 mmol ) was added . this compound was immediately diluted in meoh ( 5 ml ) , and k2co3 ( 0.021 g , 0.154 mmol ) was added . nacl ( 4 ml ) , dried over anhydrous sodium sulfate , and concentrated to yield a sticky residue that was diluted with ch2cl2 ( 3 ml ) and filtered to remove particulate matter . this substance was immediately dissolved in meoh ( 3 ml ) , k2co3 ( 0.030 g , 0.217 mmol ) was added , and the mixture was heated to vigorous reflux for 2 h. the mixture was cooled and concentrated , and the residue was partitioned between etoac ( 6 ml ) and 1:1 h2o / sat . nacl ( 3 ml ) , dried over anhydrous sodium sulfate , and concentrated to yield a sticky residue that was diluted with ch2cl2 ( 3 ml ) and filtered to remove particulate matter . compound 29 ( 0.076 g , 0.333 mmol ) was diluted in 7:1 chcl3/meoh ( 7 ml ) , and aldehyde 36 ( 0.045 g , 0.327 mmol ) was added as a solution in 1 ml of chcl3 , followed by glacial acetic acid ( 7 l ) and anhydrous mgso4 ( 0.5 g ) . nahco3 ( 10 ml ) , and the aqueous layer was extracted with chcl3 ( 2 5 ml ) . nacl ( 20 ml ) , dried over anhydrous sodium sulfate , and concentrated . the aqueous layer was extracted with etoac ( 3 6 ml ) , and the combined organic layers were washed with sat . compound 29 ( 0.076 g , 0.333 mmol ) was diluted in 8:1 chcl3/meoh ( 7 ml ) , and aldehyde 37 ( 0.051 g , 0.330 mmol ) was added as a solution in 1 ml of chcl3 , followed by glacial acetic acid ( 7 l ) and anhydrous mgso4 ( 0.5 g ) . compound 29 ( 0.076 g , 0.333 mmol ) was diluted in 7:1 chcl3/meoh ( 7 ml ) , and aldehyde 38 ( 0.051 g , 0.330 mmol ) was added as a solution in 1 ml of chcl3 , followed by glacial acetic acid ( 7 l ) and anhydrous mgso4 ( 0.5 g ) . this substance was immediately diluted with anhydrous meoh ( 7 ml ) , and k2co3 ( 0.024 g , 0.174 mmol ) was added . evaporation and retreatment of the residue with methanolic hcl ( 1 ml ) and ether ( 1 ml ) afforded the pure compound as a white flocculent solid ( 0.0094 g , 7.4% from 29 ) : mp 288290 c ( dec ) . a mixture of 18a and 18b ( 2.53 g , 15.9 mmol ) was diluted in pocl3 ( 35 ml ) , and the mixture was heated at reflux for 70 min , before the clear orange solution was cooled to room temperature and poured into ice the beaker was immersed in ice and cooled to 0 c with stirring , and solid naoh was added until the ph of the mixture was approximately 7 . the resultant cloudy suspension was extracted with etoac ( 300 ml ) , and the organic layers were washed with h2o ( 100 ml ) and sat . chloride 19a ( 0.300 g , 1.69 mmol ) was diluted with molten anhydrous acetamide ( 8 g , 135 mmol ) , and k2co3 ( 1.17 g , 8.45 mmol ) was added . the mixture was heated in a sand bath , at reflux ( 230 c ) for 17 h. the mixture was cooled , poured into h2o ( 120 ml ) , and extracted with etoac ( 4 30 ml ) . for the 7-isomer.h nmr ( 500 mhz ; cdcl3 ) : 8.438.41 ( m , 2 h ) , 8.16 ( d , j = 8.9 hz , 1 h ) , 7.797.77 ( m , 2 h ) , 7.49 ( dd , j = 8.4 , 1.7 hz , 1 h ) , 4.65 ( s , 2 h ) , 2.27 ( s , 3 h ) . compound 24 ( 0.180 g , 1.21 mmol ) was diluted in etoh ( 3 ml ) , and methanolic ammonia ( 7 m , 6 ml ) and raney nickel ( 1 g ) were added . the aqueous phase was extracted with etoac ( 2 50 ml ) , and the combined organic layers were washed with h2o ( 2 80 ml ) and sat . compound 28 ( 0.060 g , 0.266 mmol ) was diluted in absolute etoh ( 7 ml ) , and methanolic ammonia ( 7 n , 7 ml ) was added . the aqueous layer was extracted with etoac ( 2 5 ml ) , and the combined organic layers were washed with h2o ( 15 ml ) and sat . anhydrous ch2cl2 ( 10 ml ) was cooled to 78 c , and anhydrous dmso ( 0.546 g , 7.00 mmol ) was added , followed , dropwise , by oxalyl chloride ( 0.380 g , 3.00 mmol ) . the mixture was heated to 90 c under nitrogen for 2 h , upon which the mixture became black , was subsequently cooled , and poured into ice the resulting suspension was extracted with etoac ( 700 ml ) , and the organic layer was washed with h2o ( 300 ml ) and dried over anhydrous sodium sulfate . this was not characterized but was instead diluted in pocl3 ( 30 ml ) and heated at reflux for 70 min before cooling and pouring into ice the beaker was immersed in a cooler of ice , with stirring , and solid naoh was added until the ph was approximately 7 . the mixture was heated in a sand bath , at reflux ( 230 c ) for 16 h. the mixture was cooled , poured into h2o ( 120 ml ) , and extracted with etoac ( 4 30 ml ) . the aqueous phase was extracted with etoac ( 2 50 ml ) , and the combined organic layers were washed with h2o ( 2 80 ml ) and sat . compound 47 ( 0.060 g , 0.266 mmol ) was diluted in absolute etoh ( 7 ml ) , and methanolic ammonia ( 7 n , 7 ml ) was added . each compound was assayed at least in duplicate , and nine concentrations ( 500 m50 nm or 100 m10 nm for enos and inos ; 50 m to 5 nm for nnos ) were used to construct dose ic50 values were calculated by nonlinear regression using graphpad prism software ( standard error values reported are from the logic50 calculations ) , and ki values were obtained using the cheng prusoff equation [ ki = ic50/(1 + [ s]/km ) ] using the following km values : 1.3 ( rat nnos ) , 1.6 ( human nnos ) , 8.2 ( murine macrophage inos ) , and 1.7 m ( bovine enos ) . all assays were performed at a concentration of 10 m for 2 h. for apical to basolateral ( ab ) permeability , compounds were added on the apical side ( a ) , with permeation determined at the receiving ( basolateral , b ) side , where the receiving buffer was removed for analysis by lc / ms / ms using an agilent 6410 mass spectrometer ( esi , mrm mode ) coupled with an agilent 1200 hplc .

cell membranes contain dynamic , nanoscale sterol- and/or sphingolipid - enriched ordered assemblies containing specific proteins , sometimes called lipid rafts . simplified ternary mixtures of lipids phase - separate in vitro into liquid - disordered ld and ordered lo regions , but these conditions are far from those in cells . more convincingly , recent work shows that vesicles derived from the plasma membrane of living cells do spontaneously phase separate upon cooling below physiological temperatures . critical fluctuations of this underlying miscibility transition could provide thermodynamically easy modes for domain assembly at physiological temperatures , under the additional influence of the cytoskeleton and membrane proteins . the fact that rafts in vivo do not coarsen to larger size has sparked a variety of theoretical explanations , including the critical fluctuation hypothesis , elastic repulsion , hybrid lipids , or a microemulsion - like state . we do not adjudicate on these explanations here , though we will closely study the consequences of such limited raft size for transbilayer organization . interleaflet interactions are a key aspect of domain formation and are potentially crucial in cell membrane biology . communication between domains in apposed leaflets is implicated in protein clustering and signaling , while work on model lipid systems shows that the phase behavior of leaflets is coupled . for example , interleaflet dimerization of cholesterol can cause cholesterol precipitation in bilayers , whereas similarly prepared monolayers remain uniform . hydrophobic lipid tail length mismatch implicitly couples the leaflets as well as affecting lateral phase separation . traditional phase diagrams of model ternary bilayers ( typically saturated and unsaturated lipids plus cholesterol ) employ a gibbs triangle to map coexisting phases in ternary composition space , while ignoring the distinct leaflets . to address this , phase separation can instead be described in space with a free - energy landscape f( , ) ( see figure 1b ) . the top ( bottom ) leaflet is described by a composition variable for the area fraction of , e.g. , saturated lipid this amounts to a pseudobinary mapping of the ternary mixture within each leaflet . bilayers may separate into registered r or antiregistered ar phases , in which the two leaflets are locally approximately symmetric , or strongly asymmetric , respectively . ( a ) a semimicroscopic theoretical model includes competing direct ( b ) and indirect ( j , hydrophobic mismatch ) interleaflet couplings , which respectively favor transbilayer symmetry and asymmetry . ( b ) a schematic leaflet leaflet free - energy landscape f( , ) with an axis for each leaflet s composition , which determines phase coexistences through the common tangent plane construction . tie - lines ( dashed ) for r r and ar ar coexistences are sketched beneath the landscape . for the overall leaflet compositions simulated here r ( c ) schematic map of linear instability growth rates for r versus ar demixing modes , obtained from f( , ) plus gradient terms for thickness or composition boundaries between domains . for a longer saturated lipid ( 5:0 pc ) we expect the ar mode to grow fastest , leading to initial demixing into an ar ar state . ( d ) the overall composition is a mixture of 3:5:2 molar ratios dlipc : dnpc : cholesterol . ( e ) a snapshot after 10 s of simulation showing how the lipids have separated into registered ordered and disordered phases ( water is not shown ) . ( f ) altering the number of beads in the tail of the saturated lipid relative to dppc tunes the tail length mismatch with the unsaturated lipid , dlipc . two- or three - phase coexistences are then derived by drawing common tangent planes touching the surface f( , ) . for instance , the conventional coexistence of two symmetric phases in a bilayer of symmetric overall compositions corresponds to an r r tie - line linking the two registered ( r ) minima of f( , ) in figure 1b . ar ar tie - lines allow coexistence of two antiregistered ( ar ) phases . ( 18 ) for asymmetric overall leaflet compositions was explained as a triangle of r r ar coexistence in which one phase is strongly asymmetric and two are approximately symmetric . in this paper , we use molecular simulations to explore phase separation upon quenching ternary lipid bilayers whose overall leaflet compositions are symmetric . we link our simulation observations to a recent theory in which the free energy f( , ) and associated phase - transition kinetics were derived from a semimicroscopic model of coupled leaflets , in order to infer the underlying intra- and interleaflet interactions which , in turn , could have biological implications . martini and its predecessors can accurately model the insertion of proteins into membranes , the bending rigidity of simple bilayers , the diffusion of membrane proteins , and multicomponent asymmetric representations of the plasma membrane . our results verify a prediction that large hydrophobic tail length mismatch between the lipids induces a two - step or nonmonotonic kinetics . upon a quench , metastable antiregistered phases can initially grow fastest , before the equilibrium registered phases take over and dominate . by systematically varying simulation size we find evidence of competing area- and line - dependent energies ; we obtain a quantitative estimate of a length scale below which antiregistration can be thermodynamically preferable due to restricted domain size , 20 nm for the strongest hydrophobic mismatch studied . this implies a length scale dependence characterizing competing contributions to transbilayer communication in cell membranes . in addition , we measure the bulk phase compositions within the leaflets , and show how they exhibit the influence of interleaflet coupling and hydrophobic mismatch . there is evidence for a direct coupling acting over the area of the bilayer to favor domain registration . this has been incorporated into phenomenological theories , wherein separate effective free - energies for each leaflet are augmented with a term proportional to ( ) , thereby coupling one leaflet composition to the other . such an area - dependent coupling may in general comprise both enthalpic and entropic contributions and has been attributed to , e.g. , the properties of lipid tails , cholesterol flip - flop or chain interdigitation . recent molecular simulations suggest that chloroform ( a small hydrophobic molecule ) can induce an entropic contribution to direct coupling by rapidly flipping between leaflets . other proposed sources of direct coupling include undulations or curvature of the leaflets . in contrast , hydrophobic thickness mismatch ( e.g. , thick lo phases versus thinner ld ) may encourage antiregistration , in order to maintain uniform bilayer thickness ( figure 1a ) . this is an indirect coupling in the sense that lipids in apposed leaflets interact via the bilayer hydrophobic thickness of their surroundings . in some regimes , hydrophobic mismatch the theoretical model employs a two - layer lattice to represent the two leaflets . the lattice contains s and u species , where s ( saturated ) is taken to have a larger preferred hydrophobic length than the u ( unsaturated ) lipid . ( 1 ) microscopically , each lipid apposes a lipid of the same or different species in the other leaflet . ( 2 ) a direct coupling b encourages like species to appose ( analogously to the phenomenological coupling term used previously ) . ( 3 ) a multibody indirect coupling j encourages unlike species to appose to minimize hydrophobic mismatch with their surroundings . explanation of the model s hamiltonian appears in the supporting information , while detailed calculations and discussion appear in ref . a similar model has successfully captured experimental measurements of complementarity , a preference for short lipids in one leaflet to appose long ones in the other , at the single - lipid level . the coupled lattice model yields a free - energy landscape f( , ) ( figure 1b ) playing the same role as the simpler phenomenological free energies used in previous approaches . once this landscape is found , common tangent planes ( analogous to the common tangent line construction ) are used to determine two- or three - phase coexistences . consider , as in the molecular simulations presented below , roughly equal overall leaflet compositions and area fractions of lo and ld - forming lipids in each leaflet , i.e. , the center ( 0.5 , 0.5 ) of the free - energy landscape in figure 1b . the equilibrium ( lowest bulk free energy ) tangent plane for this composition corresponds to a tie - line of r r coexistence , but ( 0.5 , 0.5 ) can also access an ar ar tie - line describing a metastable state of two coexisting antiregistered phases . for general overall compositions a variety of metastable and equilibrium states exist ( e.g. , ar - ar - r and r - r - ar ) , in which the degree of local transbilayer asymmetry is either greater or lesser than if the leaflets were completely uncorrelated . upon a quench , a bilayer may experience instabilities to the two competing demixing modes indicated by curved arrows on figure 1b . this competition can be studied by linear stability analysis as a function of the semimicroscopic model s parameters ( figure 1c ) . the analysis compares the free energy gain of demixing ( downward curvature of the bulk free energy in figure 1b ) with penalties for creating interfaces between domains of differing composition and/or thickness . for large enough hydrophobic mismatch , the asymmetric ar mode can grow faster than the symmetric r mode ( figure 1c ) . a key prediction of the theoretical model is thus the possibility of two - step kinetics in which , after a quench , a bilayer first responds by preferentially forming ar phases before later reaching its equilibrium r this picture of competing area- and line - dependent energies further implies that domains restricted to sufficiently small size may thermodynamically prefer the ar ar state , such that compositional perturbations in one leaflet colocalize with asymmetric perturbations in the apposed leaflet . this nucleation - like scenario was proposed theoretically and invoked to explain molecular simulations showing antiregistered phases . however , neither the existence of this crossover behavior nor the relevant length scale have yet been determined explicitly . ( 36 ) estimated 2 nm for a single parameter set , while in ref . ( 48 ) the predicted critical size depended sensitively on the direct and indirect coupling strengths . we now present the results of our martini molecular simulations ( see methods ) . we first study a ternary mixture of an unsaturated lipid ( dlipc ) , a saturated lipid ( dppc ) , and cholesterol in the ratio 3:5:2 ( figure 1d ) . this has a moderate degree of hydrophobic mismatch and has been previously shown to rapidly phase - separate into registered ordered and disordered regions ( figure 1e ) . martini replaces every four heavy atoms in the lipid tail by a single coarse - grained bead . therefore , the 16 carbon atoms in each tail of dppc are represented by four beads in the coarse - grained simulations ( figure 1f ) , so that this lipid can also be described as 4:0 pc . removing one coarse - grained bead from each tail of dppc gives dlpc ( or 3:0 pc ) , which has minimal hydrophobic mismatch with the unsaturated lipid dlipc . conversely , adding one bead to each tail yields dapc ( or 5:0 pc ) , which has the largest amount of hydrophobic mismatch . hereafter we shall use the notations 4:0 pc and dppc ( etc . ) interchangeably . three bilayers of 6000 lipids were simulated for each of the three lipid mixtures ( table s1 ) and analyzed as described in the methods . in analogy with the semimicroscopic model , we define four categories of transbilayer phospholipid arrangement : saturated lipids aligned with saturated lipids in the apposed leaflet ( ss ) , unsaturated lipids apposing unsaturated ( uu ) ; or saturated apposing unsaturated ( su or us ) . thus , at the local scale lipids are considered pairwise registered ( ss , uu ) or pairwise antiregistered ( su , us ) , cf . we use this fine - grained description to simply and directly inspect the degree of local symmetry , i.e. , the proportion of bilayer area where lipids appose one of the same species . one should not describe , e.g. , an isolated saturated lipid as an lo phase . rather , we will define s - enriched and u - enriched regions as lo and ld phases only at the scale of at least a few lipids , which becomes necessary when measuring the bulk phase compositions . plane views of all three different mixtures ( figure 2 ) reveal separation into two contiguous registered domains after 10 s , which can be identified as the bulk , registered lo and ld phases . for most physical parameter regimes in the semimicroscopic model , and as in figure 1b , this r r coexistence is predicted to be the equilibrium state in bulk . for the smallest hydrophobic tail length mismatch ( figure 2a ) , the equilibrium r r state exhibits significant compositional impurities within the bulk phases , which become less apparent as hydrophobic mismatch increases , indicating stronger segregation . we address this quantitatively later . in terms of kinetics , we observe that significant antiregistered domains form in the first few s of the simulation with the largest hydrophobic mismatch ( figure 2c ) . increasing hydrophobic mismatch leads to two - step kinetics , but a registered equilibrium state is always reached . ( a ) images mapping evolution of the local lipid compositions in both leaflets for the dlpc ( 3:0 pc ) mixture . there are four categories of transbilayer arrangements : both leaflets saturated ( ss , red ) , both unsaturated ( uu , blue ) and asymmetric arrangements ( su , pink or us , light blue ) . the hydrophobic mismatch is small and the bilayer separates directly into registered ordered and disordered phases . the images on the far right show how the area of each local arrangement varies with time . also shown are the corresponding distributions of the thickness of the bilayer , showing a final thickness difference of 0.4 nm between the registered ordered and disordered phases . ( b ) increasing the tail length of the saturated lipid by one bead ( dppc ) leads to a larger hydrophobic mismatch between the disordered and ordered phases . there is a small initial increase in the area of antiregistration ( su and us ) until t 0.05 s . ( c ) increasing the number of beads in each tail of the saturated lipid to five ( dapc ) further increases the degree of hydrophobic mismatch , leading to a final thickness difference of 1.7 nm between the registered ordered and disordered phases . initial demixing is dominated by antiregistered domains causing an increase in the area of su and us up to t 1.1 s , after which registration takes over to complete the two - step kinetics . as expected , the bilayer thickness of the antiregistered phases is intermediate between the registered ordered and disordered phases . to examine the kinetics in more detail we plot the time dependence of the area fraction of each transbilayer arrangement ( ss , uu , su , or us ) , along with snapshots of the distribution of bilayer thickness measured by the distance between the phosphate beads of the phospholipids ( figure 2 ) . because the lipids are initially randomly mixed and the area fractions in each leaflet are roughly equal , there are initially approximately equal areas of ss , uu , su , us . for the dlpc ( 3:0 pc ) mixture , the amount of antiregistration ( su and us ) decreases monotonically throughout the simulation . this signifies dominance of the r demixing mode leading to a continuous evolution toward the equilibrium r the overall thickness distribution does not change significantly during the simulation , and the saturated ss and unsaturated uu registered regions have only a small ( 0.4 nm ) mismatch in thickness . the lo phase of all three simulations of this mixture converted to the gel phase after the registered phases became established ; a simulation at a higher temperature ( 323 k ) did not become gel - like but otherwise showed similar kinetics ( figure s4 ) . increasing the tail length of the saturated lipid to 4:0 ( dppc ) results in a larger thickness difference 0.9 nm between saturated ss and unsaturated uu registered regions ( figure 2b ) , indicating significant hydrophobic mismatch . this system again exhibits a decrease in antiregistration over the simulation time , albeit with slightly different kinetics . there is , however , a very brief initial increase in the proportion of antiregistration in the first tens of nanoseconds which was absent in the 3:0 pc case . finally , dapc ( 5:0 pc ) induces a large hydrophobic mismatch ( 1.7 nm ) between the registered saturated ss and unsaturated uu regions ( figure 2c ) . the kinetics is now markedly nonmonotonic , or two - step . ar phases initially dominate demixing , leading to a clear increase in the proportion of antiregistered ( su and us ) regions . the schematic dashed line superimposed on the instability analysis ( figure 1c ) illustrates our explanation for the emergence of two - step kinetics . a greater tail length mismatch should predominantly affect the indirect coupling j , and for the 5:0 pc system this is sufficient to render the ar demixing mode fastest . these results therefore verify quantitative estimates that appropriate model parameters for typical phospholipids straddle the line between dominant r or ar demixing modes , so that small changes to molecular properties can tip the balance one way or the other . in the final equilibrium states ( especially for 5:0 pc ) there are significant regions of su and us antiregistered lipids confined to the interfaces between the large ss and uu - dominated ( registered lo and ld ) domains . these are not true phases but slip regions that spread the cost of hydrophobic mismatch . hence , we conclude that at late times the expected thermodynamic equilibrium of two bulk registered phases ( r r ) has been reached , even with the largest hydrophobic mismatch studied . we have verified that the two - step kinetics of the 5:0 pc mixture is also exhibited when compositions with smaller or larger fractions of cholesterol ( figure s7b , c ) are used . with less cholesterol ( figure s7b ) cholesterol has been suggested as a contributor to direct coupling so a smaller fraction could decrease the direct coupling and lead to a weaker and slower transition to registration . however , extensive further studies would be needed to confirm whether the difference suggested in figure s7b is statistically valid and to explain it . the role of cholesterol is highly complex , including effects on the position in the phase diagram and hence on the properties of the lo and ld phases . ar background is expected to involve a line - dependent hydrophobic mismatch cost and an area - dependent energy gain for registration . this implies restricting the domains to below a critical size could favor the ar ar state . to test this we ran a series of smaller simulations of the mixture with the largest degree of hydrophobic mismatch ( dapc , dlipc , cholesterol ) , with between 100 and 6000 lipids ( figure s8 ) . each simulation was run for 10 s , which had been long enough to form equilibrium registered states in all the main 6000-lipid simulations ( figure 2c , s6 ) . with an equal area fraction lo ld mixture in each leaflet , the expected area fraction of ar lipids if the leaflets were completely uncorrelated ( the high - temperature limit ) is 50% . values lower than this benchmark indicate that interleaflet correlations associated with phase separation favor registration , while higher values indicate a preference for antiregistration . in marked contrast to the large main simulations , bilayers with 800 or fewer lipids strongly exhibited > 50% ar area after 10 s ( figure 3 ) . for the 1600-lipid simulation , neither registered nor antiregistered phases dominated , perhaps indicating disruption of nucleation energetics by the small system size to create a complex free - energy landscape . the ratio of registered to antiregistered area was the same for times between t = 8 s and t = 10 s ( figure 3 , s9 ) . decreasing the size of the simulation unit cell favors antiregistered phases . a series of simulations with the 5:0 saturated lipid ( 3:5:2 dlipc : dapc : cholesterol ) of decreasing size , from 6000 to 100 lipids / bilayer . restricting the domains to smaller length scales in this manner should increase the importance of line energies . after 10 s , simulations with 800 lipids ( size 14.9 nm ) show a preference for antiregistration indicated by > 50% ar area . in contrast the simulation within 6000 lipids ( size = 39.6 nm ) became fully registered . for the simulation with 1600 lipids significant registered regions have appeared but not dominated . supporting information figures s10 , s11 show corresponding results for the 4:0 pc and 3:0 pc systems . unlike the clear behavior in figure 3 , 4:0 pc required significantly smaller system sizes to show an effect , as would be expected for smaller hydrophobic mismatch . the attainment of > 50% ar area was in fact barely resolvable with the available statistics . the 3:0 pc system tended toward 50% ar area but showed no sign of exceeding 50% . this may indicate that increased compositional impurity due to weak phase segregation for 3:0 pc ( cf . figure 2a ) tends to bring the measurement toward its high - temperature limit of 50% , as opposed to a preference for ar that should push the measurement above 50% . that a restricted domain size induces a weak ar preference ( 4:0 pc ) or none at all ( 3:0 pc ) is compatible with the kinetics discussed in the previous section , where 4:0 pc showed weak initial growth of ar and 3:0 pc showed none ( figure 2 ) . analysis of bulk phase leaflet compositions in the dapc ( 5:0 pc ) system at intermediate ( a , b , c ) and late ( d , e , f ) time . at intermediate time the leaflet compositions in the bulk antiregistered phases are less pure than in the registered . at late time as described in the methods , the main interfaces are identified ( black ) and ignored in the analysis , while each contiguous region they enclose is assigned to a bulk phase : registered lo , ld , or antiregistered , either lo that apposes ld , denoted lo(ar ) , or vice versa ld(ar ) . ( b ) compositions averaged over t = 4.55 s in one representative simulation . ( c ) information from ( b ) plotted as crosses on a gibbs triangle , with a separate data point for each of the three simulations of this mixture . ( d , e , f ) at late time the contiguous antiregistered regions are confined to slip regions at the interfaces between the equilibrium registered phases . figure 3 supports the idea that competing area- and line - dependent interleaflet couplings lead to a crossover length scale , below which the thermodynamic preference can be for one leaflet to organize asymmetrically to the pattern of local composition within the other . intriguingly , this is approximately the size of some previous martini simulations with dapc that , instead of evolving toward registration , found stable antiregistration . further , it falls within the putative size range of lipid rafts in vivo . the biophysical ramifications of a length scale set by competing interleaflet couplings are explored further in the discussion . to measure the leaflet compositions within the different phases , we first identified the bulk phases as described in the methods and illustrated in figure 4a . each contiguous region enclosed by the main interfaces , including its small fluctuating impurities , was assigned to a bulk phase . we begin with the dapc ( 5:0 pc ) system and consider intermediate time ( figure 4a c ) at which there are significant contiguous bulk regions of both r and ar phases . figure 4b shows that the registered ld phase predominantly comprises the unsaturated lipid dlipc , with a small proportion of dapc and a tiny amount of cholesterol . the registered lo phase contains dapc enriched in cholesterol with negligible unsaturated lipid . the leaflet compositions within the bulk antiregistered phases , either lo that apposes ld ( denoted lo(ar ) ) , or vice versa ( ld(ar ) ) , are qualitatively similar to their respective registered compositions but are quantitatively less pure . these leaflet phase compositions can be plotted on a standard gibbs triangle ( figure 4c ) . the measured tie - lines are roughly consistent with a previous martini study also using the doubly unsaturated lipid dlipc . from our comparative measurements of bulk r and ar compositions , we infer the influence of an area - dependent direct interleaflet coupling , which would tend to make the ar minima of figure 1b less well - separated than the r minima , and therefore make the ar phases less pure . ar coexistence was found to have less pure leaflet compositions than the r phases , which was explained by phenomenological theories similarly invoking a direct interleaflet composition coupling term . at late times ( figure 4d f ) contiguous ar regions are only present at interfaces , and again exhibit reduced purity relative to the r phases . this implies that , as well as forming slip regions , the equilibrium r r interfaces further reduce their energy via interfacial mixing . we next compare the systems with shorter saturated lipids to determine the effect of hydrophobic mismatch on composition , focusing on the bulk registered phase compositions at late times ( figure 5 ) . the composition of the lo phase changes slightly , while the proportion of saturated lipid in the ld phase increases strongly from 4.8% to 14.7% as the number of martini beads in the tail is reduced from 5 to 3 . this points to a role for hydrophobic mismatch in driving phase separation , which we consider in the discussion along with related experiments . equilibrium lo and ld phases become purer with increasing hydrophobic tail length mismatch ( 3:0 pc , 4:0 pc , and 5:0 pc saturated lipids ) . the bulk compositions are measured as for figure 4 , outlined in the methods . ( a ) representative snapshots at t = 9.75 s for each mixture ( compare those in figure 2 ) . ( b ) the compositions of the phases are identified as in figure 4 and averaged over the final 0.5 s , and points for the three separate simulations of each mixture plotted on gibbs triangles . the nature of interleaflet coupling , and the role of hydrophobic tail length mismatch are key to the fundamental interactions and thermodynamics of mixed bilayers , which biological membranes may exploit or resist in a given physiological context . we have investigated these via a molecular dynamics study of the kinetics and compositions of registered ( r , approximately symmetric ) and antiregistered ( ar , strongly asymmetric ) phases , using model ternary mixtures in which the overall composition of each leaflet is the same , comprising roughly equal area fractions of lo and ld -forming lipids . we used the coarse - grained martini force field , which has been repeatedly shown to reproduce both physical chemical properties of bilayers and biological phenomena . we studied ternary mixtures comprising of dlipc , cholesterol , and one of three saturated lipids exhibiting increasing hydrophobic mismatch with dlipc : 3:0 pc , 4:0 pc and 5:0 pc ( martini representations of dlpc , dppc and dapc ) . we have the following principal findings:1.novel two - step phase - transition kinetics occurs for bilayers with significant hydrophobic mismatch , observable clearly in the 5:0 pc mixture and slightly in the 4:0 pc mixture . the bilayer first becomes locally more asymmetric , developing antiregistration , before the registered equilibrium state takes over . this behavior is predicted for estimated physical parameters in a semimicroscopic theory , and exhibited in simple lattice simulations . the early stages of kinetics , i.e. , a bilayer s initial response to a change in external conditions , may be important for cell membrane domains which form transiently and are small.2.smaller simulations for the 5:0 pc system ( figure 3 ) show that restricting domains below a certain length scale ca a recent theory claimed that registration can be explained by line tensions alone . however , that study did not properly calculate the total line energy , and when this is accounted for one finds that a bulk ( i.e. , area - dependent ) free - energy difference between r and ar phases is needed to explain registration in general.the estimated crossover length scale is 20 nm for the largest hydrophobic mismatch studied , consistent with theoretical predictions , though expected to be dependent on parameters such as tail length or structure mismatch . it is comparable to the simulation size of previous martini studies , perhaps contributing to their observing stable antiregistration with dapc rather than the registration that was reached in all large simulations here . ( 41 ) even a control dapc simulation with increased size apparently failed to reach registration , which may indicate that the lower temperature ( 295 k versus 310 k ) of ref . . it would be interesting to systematically study the role of parameters such as temperature or pressure on the interplay of interleaflet couplings.in complex biological membranes , domain size is not restricted by a simulation box but by a variety of proposed mechanisms , so that true stability of antiregistration in practice depends on the independent mechanism(s ) restricting the domain size . we have not addressed these , instead focusing on the consequences of restricted domain size for the associated transbilayer organization . more generally , the complexity of biomembranes and inherently nonequilibrium features like turnover preclude the wholesale transfer of findings from equilibrium model systems . nevertheless , the stable antiregistration simulated here and in refs . ( 25,41 ) manifests a thermodynamic preference for local asymmetry given domains of a certain size , and the fundamental interactions responsible have clear biological implications.for a cell membrane , the direct interleaflet coupling alone would imply that local composition perturbations in one leaflet ( e.g. , a cluster of longer - than - average lipids and proteins ) can colocalize similar perturbations in the other ( perhaps similar tail structure and/or cholesterol content ) . conversely , our study reveals a lateral length scale below which asymmetric organization of the leaflets perturbations can be thermodynamically preferable ( e.g. , a complementary domain of shorter - than - average bilayer constituents ) . that is , the equilibrium thermodynamic contribution to transbilayer organization in cells is predicted to be length scale dependent.3.measurements of bulk phase composition reveal direct ( area - dependent ) interleaflet coupling . at intermediate times in the 5:0 pc system ( figure 4a c ) bulk regions of ar phases are present , but with leaflet compositions of reduced purity ( figure 4c ) relative to those in the r phases . this can be attributed to a direct compositional coupling between the leaflets , which penalises the difference between apposed leaflets compositions within an ar phase , causing shorter ar ar tie - lines and hence less pure leaflet compositions . this agrees with previous experiments and simulations , which were explained by a direct interleaflet coupling.the eventual formation of equilibrium registered phases in all of the large simulations here may be contrasted to a particular calculation in ref . the authors predicted that , without an area - dependent coupling , antiregistration would be equilibrium in silico ( though not , they argued , in a real system but see ref . significant ar phases form but give way to registration instead of persisting , as would have been expected if ar were equilibrium in silico . this suggests that area - dependent coupling should be accounted for in the calculation as well . which enthalpic or entropic effects contribute most to such a direct coupling remains to be determined.4.increasing hydrophobic tail length mismatch increased the purity of the phases , seen qualitatively in figure 2 and measured in the tie - lines of figure 5 . this agrees with previous simulation measurements showing more intense phase separation as hydrophobic mismatch was increased . we propose that hydrophobic mismatch increases the immiscibility of the lipids at the microscopic scale , and therefore the separation of the minima in the free - energy landscape figure 1b , resulting in purer phases . ( one would expect an analogous effect on immiscibility by increasing the unsaturation in the unsaturated lipids , which was similarly found to lengthen the tie - lines between separated phases . ) such a role for hydrophobic mismatch arises naturally from our semimicroscopic theoretical model ( see supporting information and ref . ( 21 ) ) , while in phenomenological approaches it would be incorporated into an effective ( flory - like ) parameter for the intraleaflet lipid immiscibility . the simultaneous influence of hydrophobic mismatch on both lateral phase separation and transbilayer organization underlines the intimate link between intra- and interleaflet interactions.thus , hydrophobic tail length mismatch appears to drive phase separation , as argued for on the basis of experiments that showed increased mixing temperatures for greater hydrophobic mismatch . in theories of phase separation , higher mixing temperature at given overall composition ( as in ref . ( 53 ) ) and greater phase purity at a given temperature / composition ( as found here ) typically share common dependence on an effective immiscibility parameter . though this relationship need not always hold in real systems , our results can thus be considered in accord with ref . first , the mixing temperature depends on the entire phase boundary for the given system and on the chosen overall composition , so there can not be a unique relationship between mixing temperature and hydrophobic mismatch . second , using a variety of unsaturated and saturated lipids ( including noncanonical cases where the pure saturated lipids had shorter carbon chains than the unsaturated ones ) , ref . ( 54 ) found no general trend between the highest mixing temperature of a given mixture and any single parameter such as hydrophobic mismatch or number of carbons . nonetheless , in the case most comparable to ours , a monotonic trend was found ; increasing the saturated chain length for fixed other components led to an increase in the highest mixing temperature . novel two - step phase - transition kinetics occurs for bilayers with significant hydrophobic mismatch , observable clearly in the 5:0 pc mixture and slightly in the 4:0 pc mixture . the bilayer first becomes locally more asymmetric , developing antiregistration , before the registered equilibrium state takes over . this behavior is predicted for estimated physical parameters in a semimicroscopic theory , and exhibited in simple lattice simulations . the early stages of kinetics , i.e. , a bilayer s initial response to a change in external conditions , may be important for cell membrane domains which form transiently and are small . smaller simulations for the 5:0 pc system ( figure 3 ) show that restricting domains below a certain length scale ca . along with the two - step kinetics , this behavior further evidences the competing line- and area - dependent interleaflet couplings . a recent theory claimed that registration can be explained by line tensions alone . however , that study did not properly calculate the total line energy , and when this is accounted for one finds that a bulk ( i.e. , area - dependent ) free - energy difference between r and ar phases is needed to explain registration in general . the estimated crossover length scale is 20 nm for the largest hydrophobic mismatch studied , consistent with theoretical predictions , though expected to be dependent on parameters such as tail length or structure mismatch . it is comparable to the simulation size of previous martini studies , perhaps contributing to their observing stable antiregistration with dapc rather than the registration that was reached in all large simulations here . ( 41 ) even a control dapc simulation with increased size apparently failed to reach registration , which may indicate that the lower temperature ( 295 k versus 310 k ) of ref . . it would be interesting to systematically study the role of parameters such as temperature or pressure on the interplay of interleaflet couplings . in complex biological membranes , domain size is not restricted by a simulation box but by a variety of proposed mechanisms , so that true stability of antiregistration in practice depends on the independent mechanism(s ) restricting the domain size . we have not addressed these , instead focusing on the consequences of restricted domain size for the associated transbilayer organization . more generally , the complexity of biomembranes and inherently nonequilibrium features like turnover preclude the wholesale transfer of findings from equilibrium model systems . nevertheless , the stable antiregistration simulated here and in refs . ( 25,41 ) manifests a thermodynamic preference for local asymmetry given domains of a certain size , and the fundamental interactions responsible have clear biological implications . for a cell membrane , the direct interleaflet coupling alone would imply that local composition perturbations in one leaflet ( e.g. , a cluster of longer - than - average lipids and proteins ) can colocalize similar perturbations in the other ( perhaps similar tail structure and/or cholesterol content ) . conversely , our study reveals a lateral length scale below which asymmetric organization of the leaflets perturbations can be thermodynamically preferable ( e.g. , a complementary domain of shorter - than - average bilayer constituents ) . that is , the equilibrium thermodynamic contribution to transbilayer organization in cells is predicted to be length scale dependent . measurements of bulk phase composition reveal direct ( area - dependent ) interleaflet coupling . at intermediate times in the 5:0 pc system ( figure 4a c ) bulk regions of ar phases are present , but with leaflet compositions of reduced purity ( figure 4c ) relative to those in the r phases . this can be attributed to a direct compositional coupling between the leaflets , which penalises the difference between apposed leaflets compositions within an ar phase , causing shorter ar ar tie - lines and hence less pure leaflet compositions . this agrees with previous experiments and simulations , which were explained by a direct interleaflet coupling . the eventual formation of equilibrium registered phases in all of the large simulations here may be contrasted to a particular calculation in ref . the authors predicted that , without an area - dependent coupling , antiregistration would be equilibrium in silico ( though not , they argued , in a real system but see ref . significant ar phases form but give way to registration instead of persisting , as would have been expected if ar were equilibrium in silico . this suggests that area - dependent coupling should be accounted for in the calculation as well . which enthalpic or entropic effects contribute most to such a direct coupling remains to be determined . increasing hydrophobic tail length mismatch increased the purity of the phases , seen qualitatively in figure 2 and measured in the tie - lines of figure 5 . this agrees with previous simulation measurements showing more intense phase separation as hydrophobic mismatch was increased . we propose that hydrophobic mismatch increases the immiscibility of the lipids at the microscopic scale , and therefore the separation of the minima in the free - energy landscape figure 1b , resulting in purer phases . ( one would expect an analogous effect on immiscibility by increasing the unsaturation in the unsaturated lipids , which was similarly found to lengthen the tie - lines between separated phases . ) such a role for hydrophobic mismatch arises naturally from our semimicroscopic theoretical model ( see supporting information and ref . ( 21 ) ) , while in phenomenological approaches it would be incorporated into an effective ( flory - like ) parameter for the intraleaflet lipid immiscibility . the simultaneous influence of hydrophobic mismatch on both lateral phase separation and transbilayer organization underlines the intimate link between intra- and interleaflet interactions . thus , hydrophobic tail length mismatch appears to drive phase separation , as argued for on the basis of experiments that showed increased mixing temperatures for greater hydrophobic mismatch . in theories of phase separation , ( 53 ) ) and greater phase purity at a given temperature / composition ( as found here ) typically share common dependence on an effective immiscibility parameter . though this relationship need not always hold in real systems , our results can thus be considered in accord with ref . first , the mixing temperature depends on the entire phase boundary for the given system and on the chosen overall composition , so there can not be a unique relationship between mixing temperature and hydrophobic mismatch . second , using a variety of unsaturated and saturated lipids ( including noncanonical cases where the pure saturated lipids had shorter carbon chains than the unsaturated ones ) , ref . ( 54 ) found no general trend between the highest mixing temperature of a given mixture and any single parameter such as hydrophobic mismatch or number of carbons . nonetheless , in the case most comparable to ours , a monotonic trend was found ; increasing the saturated chain length for fixed other components led to an increase in the highest mixing temperature . our study reveals generic interactions and thermodynamics that can be expected to contribute to the lateral and transbilayer organization in complex biological membranes . membrane proteins will certainly perturb the behaviour relative to lipid - only model systems , leading to a variety of possible additional effects . for example , an integral membrane protein matching the hydrophobic thickness at the interface between registered phases could behave as a linactant to reduce the interface energy between registered phases . alternatively , other membrane proteins could promote antiregistered regions , leading to changes in both the organisation and types of domains in the membrane and potentially also the sorting of proteins . indeed experiments have shown that protein - membrane hydrophobic mismatch induces protein clustering , and simulations suggest that both symmetric and asymmetric modes of aggregation are possible for non - transmembrane proteins , determined by their hydrophobic length . whilst the coarse - grained forcefield martini is well - established for the study of the lipid bilayers , it requires the doubly - unsaturated lipid dlipc in order to display phase separation on simulation timescales . this degree of unsaturation is not unusual in biology , but does lead to the relatively long tie - lines measured here , and in the martini simulations of ackerman and feigenson . these authors found that reducing the average degree of unsaturation by adding a fraction of pupc lipids progressively shortened the simulation tie - lines into a range more typical of experimental model systems that employ singly - unsaturated lipids . identical patches of 6000 coarse - grained lipids comprising an unsaturated lipid dilinoleyl - phosphatidylcholine ( dlipc ) , cholesterol , and one of three saturated lipids dilauroyl - phosphatidylcholine ( dlpc ) , dipalmitoyl - phosphatidylcholine ( dppc ) or diarachidoyl - phosphatidylcholine ( dapc ) ( figure 1f)were prepared by mutating lipids as described elsewhere . three separate patches of 6000 lipids were prepared for each of the three mixtures and solvated using coarse - grained water and sodium and chloride ions as described in table s1 . the total number of beads ranged from 137 000 to 167 000 . in addition , two small patches of 100 and 200 lipids , with the same composition as above , were prepared . these were tessellated to yield a series of patches where the number of lipids doubled with each step : 100 , 200 , 400 , 800 , 1600 and 3200 lipids . finally , lipids were deleted from the 3200 lipid 3:5:2 dlipc : dapc : cholesterol patch to create two further patches with altered compositions . first half the cholesterol was deleted , yielding a patch of 2880 lipids with a 3:5:1 composition . then some of the saturated and unsaturated lipids were deleted , producing a patch of 2560 lipids with a 2:4:2 composition . the energy of each patch was minimized for 5000 steps using the steepest descent algorithm of gromacs 4.5.5 before 10 s of dynamics was simulated using an integration time step of 20 fs . the exception was the three 6000 lipid simulations containing dppc for which corresponding 5 s simulations from a previous paper were simply extended . electrostatic forces were calculated using a reaction - field scheme in conjunction with periodic boundary conditions , a switching distance of 1.2 nm , and a dielectric constant of 15 . van der waals interactions were cut off at 1.2 nm and switched from 0.9 nm . a berendsen thermostat with a relaxation time of 1 ps applied separately to the lipids and the solute was used to maintain the temperature at 310 k. the pressure was kept at 1 atm by a berendsen barostat that was applied semi - isotropically with a relaxation time of 2 ps and a compressibility of 3 10 bar . coordinates were written to disc every 2 ns . to measure bilayer symmetry and determine the bulk phases and interfaces we treated each bilayer as an image ( figure s1 ) and applied image - processing techniques . each leaflet was described by three separate arrays of pixels , one for each lipid species . the pixels are 0.1 0.1 nm square and represent the ( x , y ) plane of the bilayer . a pixel was set if it coincides with the ( x , y ) cartesian coordinates of the center of the phosphate bead ( or the hydroxyl bead for cholesterol ) of the relevant lipid species . these arrays were then convolved with a gaussian of width 0.8 nm and the density of the species at each pixel was determined by cubic interpolation ( figure s1a ) . the width of the gaussian was chosen to mimic the average area per lipid . subtracting the saturated density from the unsaturated density yields an array of the difference in densities for each leaflet . if this is greater than zero then locally there are more unsaturated lipids ( and vice versa ) . applying a threshold at zero defined regions either with more saturated lipids ( drawn throughout in red ) or with more unsaturated lipids ( drawn in blue ) : we call this the region_mask . cholesterol is ignored when defining these regions . using the scikit - image python module we applied the canny edge detection algorithm with a smoothing gaussian of 0.2 nm to this image to identify the interfaces between the regions . these were also convolved with a gaussian of width 0.8 nm . a routine from the same module that identifies contiguous regions was then used and only the main interfaces that were larger than 1% of the total area were kept . in addition , the z - values of the phosphate beads were cubically interpolated onto a grid to create an array of the height of each leaflet ( figure s1b ) . these arrays from each leaflet can be then combined to analyze the entire bilayer ( figure s2 ) . adding 2 the regions [ upper ] to the regions [ lower ] arrays ( figure s2a ) yields the regions [ overlap ] array which distinguishes saturated lipids apposing saturated lipids ( ss regions ) along with the other three combinations ( uu , su , us ) . simple masks can be extracted from these to calculate properties of each region type . summing the number of pixels of each mask array gives , for example , the proportion of the total area taken up by unsaturated lipids apposing unsaturated lipids ( uu regions ) . to define the compositions of bulk phases , small fluctuations of the species within a contiguous bulk region ( e.g. , a small droplet of su within a surrounding uu domain ) should be included as part of the bulk phase in question . such impurities arise from the equilibrium solubility of , e.g. , saturated lipids in the ld phase . ( strictly , such droplets can be defined as fluctuations within a parent phase if their size is of order or less than the correlation length for composition fluctuations . ) to achieve this we multiplied the interface_masks of each leaflet together ( figure s2b ) and identified contiguous regions , as before . each region was defined according to the dominant lipid arrangement , which yielded a array in which the fine interfaces are masked out , and small regions of impurity have been reassigned as members of the surrounding contiguous phase in which they are solvated . thereafter , masks can be extracted from this array which can then be multiplied by , for example , the density of a specific lipid to calculate the lipid fractions present in that bulk phase . lastly , the difference between the two surface arrays simply yields the variation in thickness across the patch of bilayer .

characterizing the nanoscale dynamic organization within lipid bilayer membranes is central to our understanding of cell membranes at a molecular level . we investigate phase separation and communication across leaflets in ternary lipid bilayers , including saturated lipids with between 12 and 20 carbons per tail . coarse - grained molecular dynamics simulations reveal a novel two - step kinetics due to hydrophobic mismatch , in which the initial response of the apposed leaflets upon quenching is to increase local asymmetry ( antiregistration ) , followed by dominance of symmetry ( registration ) as the bilayer equilibrates . antiregistration can become thermodynamically preferred if domain size is restricted below 20 nm , with implications for the symmetry of rafts and nanoclusters in cell membranes , which have similar reported sizes . we relate our findings to theory derived from a semimicroscopic model in which the leaflets experience a direct area - dependent coupling , and an indirect coupling that arises from hydrophobic mismatch and is most important at domain boundaries . registered phases differ in composition from antiregistered phases , consistent with a direct coupling between the leaflets . increased hydrophobic mismatch purifies the phases , suggesting that it contributes to the molecule - level lipid immiscibility . our results demonstrate an interplay of competing interleaflet couplings that affect phase compositions and kinetics , and lead to a length scale that can influence lateral and transverse bilayer organization within cells .

Introduction Theory Results Discussion Methods

bilayers may separate into registered r or antiregistered ar phases , in which the two leaflets are locally approximately symmetric , or strongly asymmetric , respectively . ( a ) a semimicroscopic theoretical model includes competing direct ( b ) and indirect ( j , hydrophobic mismatch ) interleaflet couplings , which respectively favor transbilayer symmetry and asymmetry . in this paper , we use molecular simulations to explore phase separation upon quenching ternary lipid bilayers whose overall leaflet compositions are symmetric . we link our simulation observations to a recent theory in which the free energy f( , ) and associated phase - transition kinetics were derived from a semimicroscopic model of coupled leaflets , in order to infer the underlying intra- and interleaflet interactions which , in turn , could have biological implications . martini and its predecessors can accurately model the insertion of proteins into membranes , the bending rigidity of simple bilayers , the diffusion of membrane proteins , and multicomponent asymmetric representations of the plasma membrane . our results verify a prediction that large hydrophobic tail length mismatch between the lipids induces a two - step or nonmonotonic kinetics . by systematically varying simulation size we find evidence of competing area- and line - dependent energies ; we obtain a quantitative estimate of a length scale below which antiregistration can be thermodynamically preferable due to restricted domain size , 20 nm for the strongest hydrophobic mismatch studied . this implies a length scale dependence characterizing competing contributions to transbilayer communication in cell membranes . in addition , we measure the bulk phase compositions within the leaflets , and show how they exhibit the influence of interleaflet coupling and hydrophobic mismatch . there is evidence for a direct coupling acting over the area of the bilayer to favor domain registration . this has been incorporated into phenomenological theories , wherein separate effective free - energies for each leaflet are augmented with a term proportional to ( ) , thereby coupling one leaflet composition to the other . such an area - dependent coupling may in general comprise both enthalpic and entropic contributions and has been attributed to , e.g. other proposed sources of direct coupling include undulations or curvature of the leaflets . this is an indirect coupling in the sense that lipids in apposed leaflets interact via the bilayer hydrophobic thickness of their surroundings . ( 2 ) a direct coupling b encourages like species to appose ( analogously to the phenomenological coupling term used previously ) . , ar - ar - r and r - r - ar ) , in which the degree of local transbilayer asymmetry is either greater or lesser than if the leaflets were completely uncorrelated . a key prediction of the theoretical model is thus the possibility of two - step kinetics in which , after a quench , a bilayer first responds by preferentially forming ar phases before later reaching its equilibrium r this picture of competing area- and line - dependent energies further implies that domains restricted to sufficiently small size may thermodynamically prefer the ar ar state , such that compositional perturbations in one leaflet colocalize with asymmetric perturbations in the apposed leaflet . therefore , the 16 carbon atoms in each tail of dppc are represented by four beads in the coarse - grained simulations ( figure 1f ) , so that this lipid can also be described as 4:0 pc . removing one coarse - grained bead from each tail of dppc gives dlpc ( or 3:0 pc ) , which has minimal hydrophobic mismatch with the unsaturated lipid dlipc . conversely , adding one bead to each tail yields dapc ( or 5:0 pc ) , which has the largest amount of hydrophobic mismatch . in analogy with the semimicroscopic model , we define four categories of transbilayer phospholipid arrangement : saturated lipids aligned with saturated lipids in the apposed leaflet ( ss ) , unsaturated lipids apposing unsaturated ( uu ) ; or saturated apposing unsaturated ( su or us ) . for the smallest hydrophobic tail length mismatch ( figure 2a ) , the equilibrium r r state exhibits significant compositional impurities within the bulk phases , which become less apparent as hydrophobic mismatch increases , indicating stronger segregation . in terms of kinetics , we observe that significant antiregistered domains form in the first few s of the simulation with the largest hydrophobic mismatch ( figure 2c ) . increasing hydrophobic mismatch leads to two - step kinetics , but a registered equilibrium state is always reached . also shown are the corresponding distributions of the thickness of the bilayer , showing a final thickness difference of 0.4 nm between the registered ordered and disordered phases . ( b ) increasing the tail length of the saturated lipid by one bead ( dppc ) leads to a larger hydrophobic mismatch between the disordered and ordered phases . ( c ) increasing the number of beads in each tail of the saturated lipid to five ( dapc ) further increases the degree of hydrophobic mismatch , leading to a final thickness difference of 1.7 nm between the registered ordered and disordered phases . initial demixing is dominated by antiregistered domains causing an increase in the area of su and us up to t 1.1 s , after which registration takes over to complete the two - step kinetics . as expected , the bilayer thickness of the antiregistered phases is intermediate between the registered ordered and disordered phases . to examine the kinetics in more detail we plot the time dependence of the area fraction of each transbilayer arrangement ( ss , uu , su , or us ) , along with snapshots of the distribution of bilayer thickness measured by the distance between the phosphate beads of the phospholipids ( figure 2 ) . this signifies dominance of the r demixing mode leading to a continuous evolution toward the equilibrium r the overall thickness distribution does not change significantly during the simulation , and the saturated ss and unsaturated uu registered regions have only a small ( 0.4 nm ) mismatch in thickness . the lo phase of all three simulations of this mixture converted to the gel phase after the registered phases became established ; a simulation at a higher temperature ( 323 k ) did not become gel - like but otherwise showed similar kinetics ( figure s4 ) . increasing the tail length of the saturated lipid to 4:0 ( dppc ) results in a larger thickness difference 0.9 nm between saturated ss and unsaturated uu registered regions ( figure 2b ) , indicating significant hydrophobic mismatch . the schematic dashed line superimposed on the instability analysis ( figure 1c ) illustrates our explanation for the emergence of two - step kinetics . a greater tail length mismatch should predominantly affect the indirect coupling j , and for the 5:0 pc system this is sufficient to render the ar demixing mode fastest . we have verified that the two - step kinetics of the 5:0 pc mixture is also exhibited when compositions with smaller or larger fractions of cholesterol ( figure s7b , c ) are used . with less cholesterol ( figure s7b ) cholesterol has been suggested as a contributor to direct coupling so a smaller fraction could decrease the direct coupling and lead to a weaker and slower transition to registration . ar background is expected to involve a line - dependent hydrophobic mismatch cost and an area - dependent energy gain for registration . to test this we ran a series of smaller simulations of the mixture with the largest degree of hydrophobic mismatch ( dapc , dlipc , cholesterol ) , with between 100 and 6000 lipids ( figure s8 ) . ( c ) information from ( b ) plotted as crosses on a gibbs triangle , with a separate data point for each of the three simulations of this mixture . figure 3 supports the idea that competing area- and line - dependent interleaflet couplings lead to a crossover length scale , below which the thermodynamic preference can be for one leaflet to organize asymmetrically to the pattern of local composition within the other . the biophysical ramifications of a length scale set by competing interleaflet couplings are explored further in the discussion . the leaflet compositions within the bulk antiregistered phases , either lo that apposes ld ( denoted lo(ar ) ) , or vice versa ( ld(ar ) ) , are qualitatively similar to their respective registered compositions but are quantitatively less pure . from our comparative measurements of bulk r and ar compositions , we infer the influence of an area - dependent direct interleaflet coupling , which would tend to make the ar minima of figure 1b less well - separated than the r minima , and therefore make the ar phases less pure . ar coexistence was found to have less pure leaflet compositions than the r phases , which was explained by phenomenological theories similarly invoking a direct interleaflet composition coupling term . we next compare the systems with shorter saturated lipids to determine the effect of hydrophobic mismatch on composition , focusing on the bulk registered phase compositions at late times ( figure 5 ) . this points to a role for hydrophobic mismatch in driving phase separation , which we consider in the discussion along with related experiments . ( b ) the compositions of the phases are identified as in figure 4 and averaged over the final 0.5 s , and points for the three separate simulations of each mixture plotted on gibbs triangles . the nature of interleaflet coupling , and the role of hydrophobic tail length mismatch are key to the fundamental interactions and thermodynamics of mixed bilayers , which biological membranes may exploit or resist in a given physiological context . we have investigated these via a molecular dynamics study of the kinetics and compositions of registered ( r , approximately symmetric ) and antiregistered ( ar , strongly asymmetric ) phases , using model ternary mixtures in which the overall composition of each leaflet is the same , comprising roughly equal area fractions of lo and ld -forming lipids . we studied ternary mixtures comprising of dlipc , cholesterol , and one of three saturated lipids exhibiting increasing hydrophobic mismatch with dlipc : 3:0 pc , 4:0 pc and 5:0 pc ( martini representations of dlpc , dppc and dapc ) . we have the following principal findings:1.novel two - step phase - transition kinetics occurs for bilayers with significant hydrophobic mismatch , observable clearly in the 5:0 pc mixture and slightly in the 4:0 pc mixture . , a bilayer s initial response to a change in external conditions , may be important for cell membrane domains which form transiently and are small.2.smaller simulations for the 5:0 pc system ( figure 3 ) show that restricting domains below a certain length scale ca a recent theory claimed that registration can be explained by line tensions alone . , area - dependent ) free - energy difference between r and ar phases is needed to explain registration in general.the estimated crossover length scale is 20 nm for the largest hydrophobic mismatch studied , consistent with theoretical predictions , though expected to be dependent on parameters such as tail length or structure mismatch . it would be interesting to systematically study the role of parameters such as temperature or pressure on the interplay of interleaflet couplings.in complex biological membranes , domain size is not restricted by a simulation box but by a variety of proposed mechanisms , so that true stability of antiregistration in practice depends on the independent mechanism(s ) restricting the domain size . ( 25,41 ) manifests a thermodynamic preference for local asymmetry given domains of a certain size , and the fundamental interactions responsible have clear biological implications.for a cell membrane , the direct interleaflet coupling alone would imply that local composition perturbations in one leaflet ( e.g. conversely , our study reveals a lateral length scale below which asymmetric organization of the leaflets perturbations can be thermodynamically preferable ( e.g. that is , the equilibrium thermodynamic contribution to transbilayer organization in cells is predicted to be length scale dependent.3.measurements of bulk phase composition reveal direct ( area - dependent ) interleaflet coupling . this can be attributed to a direct compositional coupling between the leaflets , which penalises the difference between apposed leaflets compositions within an ar phase , causing shorter ar ar tie - lines and hence less pure leaflet compositions . this agrees with previous experiments and simulations , which were explained by a direct interleaflet coupling.the eventual formation of equilibrium registered phases in all of the large simulations here may be contrasted to a particular calculation in ref . the authors predicted that , without an area - dependent coupling , antiregistration would be equilibrium in silico ( though not , they argued , in a real system but see ref . this suggests that area - dependent coupling should be accounted for in the calculation as well . which enthalpic or entropic effects contribute most to such a direct coupling remains to be determined.4.increasing hydrophobic tail length mismatch increased the purity of the phases , seen qualitatively in figure 2 and measured in the tie - lines of figure 5 . we propose that hydrophobic mismatch increases the immiscibility of the lipids at the microscopic scale , and therefore the separation of the minima in the free - energy landscape figure 1b , resulting in purer phases . ( 21 ) ) , while in phenomenological approaches it would be incorporated into an effective ( flory - like ) parameter for the intraleaflet lipid immiscibility . the simultaneous influence of hydrophobic mismatch on both lateral phase separation and transbilayer organization underlines the intimate link between intra- and interleaflet interactions.thus , hydrophobic tail length mismatch appears to drive phase separation , as argued for on the basis of experiments that showed increased mixing temperatures for greater hydrophobic mismatch . novel two - step phase - transition kinetics occurs for bilayers with significant hydrophobic mismatch , observable clearly in the 5:0 pc mixture and slightly in the 4:0 pc mixture . along with the two - step kinetics , this behavior further evidences the competing line- and area - dependent interleaflet couplings . the estimated crossover length scale is 20 nm for the largest hydrophobic mismatch studied , consistent with theoretical predictions , though expected to be dependent on parameters such as tail length or structure mismatch . in complex biological membranes , domain size is not restricted by a simulation box but by a variety of proposed mechanisms , so that true stability of antiregistration in practice depends on the independent mechanism(s ) restricting the domain size . conversely , our study reveals a lateral length scale below which asymmetric organization of the leaflets perturbations can be thermodynamically preferable ( e.g. this can be attributed to a direct compositional coupling between the leaflets , which penalises the difference between apposed leaflets compositions within an ar phase , causing shorter ar ar tie - lines and hence less pure leaflet compositions . the eventual formation of equilibrium registered phases in all of the large simulations here may be contrasted to a particular calculation in ref . the authors predicted that , without an area - dependent coupling , antiregistration would be equilibrium in silico ( though not , they argued , in a real system but see ref . this suggests that area - dependent coupling should be accounted for in the calculation as well . increasing hydrophobic tail length mismatch increased the purity of the phases , seen qualitatively in figure 2 and measured in the tie - lines of figure 5 . we propose that hydrophobic mismatch increases the immiscibility of the lipids at the microscopic scale , and therefore the separation of the minima in the free - energy landscape figure 1b , resulting in purer phases . ( 21 ) ) , while in phenomenological approaches it would be incorporated into an effective ( flory - like ) parameter for the intraleaflet lipid immiscibility . the simultaneous influence of hydrophobic mismatch on both lateral phase separation and transbilayer organization underlines the intimate link between intra- and interleaflet interactions . our study reveals generic interactions and thermodynamics that can be expected to contribute to the lateral and transbilayer organization in complex biological membranes . whilst the coarse - grained forcefield martini is well - established for the study of the lipid bilayers , it requires the doubly - unsaturated lipid dlipc in order to display phase separation on simulation timescales . this degree of unsaturation is not unusual in biology , but does lead to the relatively long tie - lines measured here , and in the martini simulations of ackerman and feigenson . identical patches of 6000 coarse - grained lipids comprising an unsaturated lipid dilinoleyl - phosphatidylcholine ( dlipc ) , cholesterol , and one of three saturated lipids dilauroyl - phosphatidylcholine ( dlpc ) , dipalmitoyl - phosphatidylcholine ( dppc ) or diarachidoyl - phosphatidylcholine ( dapc ) ( figure 1f)were prepared by mutating lipids as described elsewhere . three separate patches of 6000 lipids were prepared for each of the three mixtures and solvated using coarse - grained water and sodium and chloride ions as described in table s1 . then some of the saturated and unsaturated lipids were deleted , producing a patch of 2560 lipids with a 2:4:2 composition . each region was defined according to the dominant lipid arrangement , which yielded a array in which the fine interfaces are masked out , and small regions of impurity have been reassigned as members of the surrounding contiguous phase in which they are solvated .

atrial fibrillation ( af ) is the most common cardiac arrhythmia , affecting 1%2% of the general population,1 although this may be an underestimate . studies assessing silent af using modern implantable rhythm monitoring devices suggest that up to 50%60% of patients older than 65 years may have unsuspected episodes of af.2,3 the relevance of these data is best understood in view of the increased mortality and morbidity associated with af : nonvalvular af confers a fivefold risk of stroke,1 which increases to 17-fold in patients with mitral stenosis.4 at the present time , vitamin k antagonists ( vkas ) remain the mainstay for stroke prevention in af . in a meta - analysis by hart et al,5 a 64% reduction in overall stroke risk was observed in patients using adjusted - dose vkas ( the reduction increased to 68% when only ischemic strokes were considered ) . despite such strong evidence , warfarin is still underused because of its associated bleeding risk , need for regular monitoring , narrow therapeutic range , and common interactions with other medications as well as food.4,6 in certain subgroups of patients , or when bleeding risk is elevated , acetylsalicylic acid ( asa ) , alone or in combination with clopidogrel , may be considered as an alternative to vkas.7 however , antiplatelet agents are inferior to vkas for stroke prevention and may be associated with similar bleeding risk , particularly in elderly patients.5,710 as an alternative to vkas , novel oral anticoagulant agents ( noacs ) have been increasingly used . the re - ly ( randomized evaluation of long - term anticoagulant therapy ) trial11 ( dabigatran 110 or 150 mg twice daily ) , the rocket - af ( rivaroxaban once - daily , oral , direct factor xa inhibition compared with vitamin k antagonism for prevention of stroke and embolism trial in atrial fibrillation)12 trial ( rivaroxaban 20 mg once daily ) , the aristotle ( apixaban for the prevention of stroke in subjects with atrial fibrillation)13 trial ( apixaban 5 mg twice daily ) , and the engage af timi 48 ( effective anticoagulation with factor xa next generation in atrial fibrillation thrombolysis in myocardial infarction 48)14 trial ( edoxaban 60 or 30 mg once daily ) , using dose - adjusted warfarin as standard - of - care control , showed noninferiority or superiority for the efficacy outcome of combined ischemic stroke and systemic embolism in patients with nonvalvular af . however , the composite outcome of major bleeding does not differ substantially from warfarin.1518 the exact mechanism of thrombus formation in af - related strokes , although not yet fully understood , involves the presence of endocardial injury , hypercoagulability , and blood stasis in the atria.3 the latter seems to play a pivotal role ; in fact , the failure of the left atrium to effectively contract results in atrial stretch and dilation , favoring thrombus formation.3 approximately 75% of embolic events associated with af result from atrial thrombosis.19 a review of autopsy , echocardiographic , and operative reports demonstrate that atrial thrombi can be identified in the left atrial appendage ( laa ) in 91% of nonvalvular af cases and 57% of rheumatic af cases.20 af is associated with a systemic prothrombotic state characterized by endothelial dysfunction and platelet activation.21 there are several anatomical features and physiological characteristics that explain the pivotal role played by the laa as a major site of thrombus formation and source of thromboembolic stroke . first , the wall of the laa has numerous trabeculae ( pectinate muscles ) , forming crypts that can harbor blood clots . these features differentiate the laa from the left atrium that has a smooth wall . a different embryologic origin explains this different characteristic . douglas et al22 described that the laa originates by the incorporation of the left pulmonary vein in the body of the left atrium . second , the macroscopic anatomy of the laa is complex and highly variable , with a long , tubular , and often multilobed body extending over the atrioventricular groove and left ventricular surface . recently , a correlation between the morphology of the laa and embolic risk has been described.23 the laa ostium is usually oval - shaped and located anterior and inferior to the left superior pulmonary vein . third , the laa is also more distensible than the left atrium itself and can receive a large volume of blood when the pressure in the left atrium is high . fourth , a reduction in stroke volume and cardiac output has been described after removal of the laa.24,25 fifth , anatomically , the laa lies superior and anterior to the left ventricle in close contact with the left ventricular wall . as a consequence , changes in the left ventricular volume and pressure can be transmitted to the laa wall , which affects blood flow through the laa . finally , the laa plays an important physiologic function in regulating intravascular volume through the release of atrial natriuretic factor . of note , surgical laa exclusion has been performed to reduce stroke risk in cardiac valve surgery in patients with af,26 but this procedure is too invasive to be used as a stand - alone operation in patients who do not have any other concurrent indication to undergo cardiac surgery . percutaneous laa closure is a relatively new procedure that obviates the need for sternotomy and cardiopulmonary bypass and is an attractive alternative to chronic anticoagulation with vkas or noacs in reducing stroke risk in nonvalvular af . exclusion of the laa cavity from the atrium can be achieved with devices that are positioned at the neck of the laa by either an endovascular or epicardial approach ( table 1 ) . this review describes the current technologies used in percutaneous laa closure ( table 2 ) , as well as a summary of the published trials concerning their efficacy in reducing stroke risk in af . the plaato ( percutaneous left atrial appendage transcatheter occlusion ) device ( appriva medical , inc . , sunnyvale , ca , usa ) was the first laa occlusion device designed ; however , as of 2007 , it is no longer in production.27 it consists of a self - expanding nitinol cage covered by an impermeable polymeric membrane . the diameters of the plaato device range between 15 and 35 mm , and it is 20%40% larger than the laa ostium . it is deployed through a catheter introduced via the femoral vein and is advanced from the right to the left atrium by transseptal puncture . during the months after the procedure , the device endothelializes with proliferation of a new layer of neointima , starting at the edges of the device . the neointimal growth is continued with the atrial wall and is usually tighter at the border of the implant surface and looser at the center.28 the time span for a complete sealing of the device in the atrial wall is usually around 3 months . however , the amount of time an antithrombotic agent should be administered to avoid thrombotic complications is highly debated in the literature . after plaato implantation , lifelong asa administration is , in general , recommended . in 2002 , sievert et al29 published a pilot study evaluating the feasibility and safety of the plaato device . fifteen patients underwent a successful procedure , although one patient required a second attempt after sustaining hemopericardium . subsequent studies on the plaato device focused on the effectiveness of percutaneous laa occlusion in preventing strokes . the international multi - center feasibility trial30 enrolled 111 patients with contraindication to anticoagulation therapy . the success rate of implantation was 97.3% ; reasons for unsuccessful implantation were thrombus in the laa in one patient , perforation of the femoral artery during femoral puncture in another patient , and cardiac tamponade in the third patient . pericardial effusion or hemopericardium occurred in 4.5% of patients . during a 9.8 month follow - up , a 65% reduction in stroke rate was observed compared with the expected stroke risk , as calculated using the chads2 score ( representing congestive heart failure , hypertension , age , diabetes mellitus , and stroke , the chads2 score is a clinical prediction rule widely used for estimating the risk of stroke in patients with nonvalvular af ) . it is worth emphasizing that the authors excluded three transient ischemic attacks ( tias ) from the number of strokes and justify this on the basis that the chads2 score was elaborated to calculate the risk for strokes and not for tias.31 residual peridevice leaks were observed not uncommonly by fluoroscopy or echocardiogram , and laa occlusion was defined as absent , trace , or a mild leak on the basis of these imaging modalities . this is a point of interest that has been the focus of critique not only for the plaato device but also for the newer devices as well and will be discussed later on in this review . the laa closure procedure was successful in 162 ( 90% ) but failed in 18 ( 10% ) patients because of anatomic characteristics of the laa ( 14 cases ) , impossibility of accessing the laa ( 2 cases ) , complications during transseptal puncture ( 1 case ) , and device instability ( 1 case ) . one plaato device embolized requiring retrieval by snaring catheter , and a larger device was successfully implanted in the same procedure . one case of device thrombus documented at 1 month eventually resolved with low - molecular - weight heparin . the observed stroke rate was 2.3% , correlating to a 65% reduction compared with a chads2-based estimate . a composite of the international multi - center feasibility trial and the european - only plaato registry study,27 including almost 300 patients , demonstrated a relative reduction of stroke rate of 54% with plaato compared with the stroke rate in a historic control group , using the chads2.33 in this registry , thrombus formation was reported in 1% of cases within the first 6 months and was not associated with any neurological events . long - term follow - up of the plaato device is reported in three trials . the observed annual stroke rate was 3.8% , which correlated with a 42% reduction compared with the chads2-expected risk . the success rate of laa occlusion was 97.3% , and procedural complications included one death resulting from device embolization , one pericardial effusion , and one periprocedural stroke . during follow - up , no strokes were observed , diverging from an expected incidence of seven events . finally , ussia et al36 successfully implanted the plaato device for laa occlusion in 18 of 20 patients . nevertheless , financial problems of the manufacturer and a significant rate of adverse events , as mentioned earlier , led to discontinuation of the device in 2006.37 the watchman laa system ( figure 1 ) became available in 2002 . similar to the plaato device , it is a nitinol - cage device with a row of fixation barbs around the surface , but it differs from the plaato device in that its polymeric membrane is permeable . the watchman laa closure device is available in five different sizes , ranging from 21 to 33 mm , and is usually selected 10%20% larger than the laa ostium . the protect af ( watchman left atrial appendage system for embolic protection in patients with atrial fibrillation ) trial,38 published in 2009 , was a randomized controlled trial that involved the randomization of 707 af patients with at least one stroke risk factor to the watchman device versus warfarin in a 2:1 fashion . the device was found to be noninferior to warfarin for the combined end - point of stroke , systemic embolism , and cardiovascular death . the success rate for implantation was 91% , with procedural complications including pericardial effusions requiring intervention in 4.8% and device embolization in 0.6% . thrombus formation on the device was observed in 3.7% of cases at 6 weeks postprocedure . those receiving the device were treated with warfarin for approximately 45 days , followed by clopidogrel for 4.5 months , and then lifelong asa . because only 30% of patients had been followed for more than 2 years in the original publication of the protect af trial , the success of the device could have been attributed in part to the ongoing anticoagulation that was received . however , a recently published 2.3 year ( 1.1 years ) follow - up study of protect af confirmed the noninferiority of the watchman device.39 there were no statistically significant differences detected in individual comparisons between the two groups ; however , the confidence interval was wide ( 95% confidence interval , 0.361.76).38 the device group exhibited more ischemic strokes , but less cardiovascular death and less hemorrhagic stroke . of note , all patients in the control group received warfarin ( time in therapeutic international normalized ratio range was 66% ) , with 34% of control patients having had interrupted anticoagulation at some point for various reasons . the primary safety endpoint , which included both procedure - related events ( including pericardial effusion requiring intervention of hospitalization , procedure - related stroke , or device embolization ) and major bleeding ( intracranial bleeding or gastrointestinal bleeding requiring transfusion ) was higher in the device group ( 5.5% per year ) than the control group ( 3.6% per year ) , for a risk ratio of 1.53 ( 95% confidence interval , 0.952.70 ) . importantly , patients with a previous history of stroke or tia appeared to receive sustained benefit from the device that was at least as good as warfarin therapy.39 peridevice leaks with the watchman device detected by transesophageal echocardiography were common ( up to 30% at 12 months),40 although a recent retrospective analysis of the protect af trial showed no effect on the effectiveness of the device.41 laa closure with the watchman device also resulted in improved quality of life.4 gangireddy et al43 estimated the net clinical benefit ( ncb ) of percutaneous laa closure in a post hoc analysis of outcomes among 707 af patients in the protect af trial and 566 in the continued access registry who were undergoing laa closure with the watchman device compared with anticoagulation with a vka . outcomes were ischemic stroke , intracranial hemorrhage , major bleeding , pericardial effusion , and death . the ncb was calculated as the sum of annualized rates of these outcomes after intervention minus rates when receiving warfarin . the ncb initially favored anticoagulation , but 69 months after the procedure , the ncb then favored the device - based intervention , driven mainly by reductions in intracranial hemorrhage and death in patients undergoing laa closure . importantly , operator experience significantly affected the safety and efficacy end - points of the intervention : the incidence of procedure - related events was considerably lower in the nonrandomized continued access registry that followed the protect af trial , with the ncb clearly favoring intervention in the registry . as expected , the highest net clinical benefit was observed for patients with high chads2 scores . sick et al20 performed 75 watchman device implantations , of which 66 ( 88% ) were successful . thrombus formation in four patients , in addition to two device embolizations , was reported . originally , vka therapy was required for 45 days after laa closure with the watchman device , followed by dual - antiplatelet therapy for up to 6 months , followed by asa alone . the asap study ( asa plavix feasibility study with watchman left atrial appendage closure technology ) , presented in 2012 , was a feasibility study designed to evaluate whether the watchman device could be used for patients who were unable to take warfarin . in this nonrandomized study enrolling 150 patients with an average chads2 score of 2.8 who had a contraindication to anticoagulation , patients were treated with both asa and clopidogrel for 6 months after implantation instead of the standard warfarin . during follow - up , three ischemic strokes were observed , corresponding to 1.7 events per 100 patient - years ( a 77% reduction in the expected stroke rate based on the chads2 score estimate ) . in fact , despite not using warfarin , the event rate was lower than in the protect af study ( 2.2 strokes per 100 patient - years).44 these new data permitted the use of the watchman device in patients with nonvalvular af and contraindications to vka therapy . an editorial published by whitlock et al21 showed that patients undergoing laa closure still had a considerable incidence of stroke in the randomized and nonrandomized control trials at follow - up . specifically alluding to the protect af trial , incidence of strokes at follow up were 50% more in the device group compared with in the warfarin group , at 3.0% versus 2.0%.45 one can argue the need for anticoagulation or antiplatelet therapy added to laa closure indefinitely . last , the prevail ( watchman laa closure device in patients with atrial fibrillation versus long term warfarin therapy ) study46 was a randomized controlled trial including 269 patients in the watchman laa closure group compared with 138 patients in the warfarin group . although the study group ( watchman ) did not achieve the prespecified criteria for noninferiority at 18 months follow - up compared with the control group ( warfarin ) , the overall periprocedural early events were lower than in the protect af trial , at 2.2% versus 4.9% , respectively . overall , adverse events in the prevail trial were significantly lower than in the protect af trial , at 4.2% versus 8.7% ( p=0.004 ) . the amplatzer cardiac plug ( acp ; figures 2 and 3 ) is a nitinol device . unlike the plaato and watchman devices , the acp consists of two parts , the lobe and the disc , which are connected by a central waist . twelve stabilizing wires are equally displaced around the main disc and contribute to device retention and stabilization inside the laa . the sizes of the lobe range from 16 to 30 mm , and it is designed to hug the inner wall of the laa . the lobe enters into the laa neck with a depth of 10 mm or more , anchoring the device , whereas the disc covers the laa orifice , thereby occluding it . after the procedure , 1 month of double antiplatelet therapy the initial european experience by park et al47 is a retrospective analysis of 143 patients who received the acp device . six patients were excluded because of difficult laa anatomy or thrombus , and 137 procedures were attempted , of which 133 were successful ( 96% ) . ten patients ( 7% ) experienced complications : three periprocedural strokes , two device embolizations , five cardiac tamponades requiring pericardiocentesis , and four pericardial effusions with spontaneous resolution . the study showed the feasibility of laa closure with the acp device , as well as its relatively low rate of complications . lam et al48 reported 20 cases , of whom 19 ( 95% ) underwent successful implantation of the acp device . they also reported one episode of transient myocardial ischemia and one esophageal lesion resulting from transesophageal echocardiography . no strokes were observed during a 12.7 month follow - up period compared with an estimated risk of 5.3% . lpez - mnguez et al49 recently published a trial of 35 patients treated with the acp device . no cardiac complications were observed , one femoral arteriovenous fistula was reported , and one severe gastrointestinal bleeding was observed , at which point clopidogrel was stopped and asa alone was continued . thrombus formation on the acp surface was documented in five cases ( 14.28% ) , and in four of them , enoxaparin was administered , with eventual thrombus resolution . during a 21-month follow up period , only one tia was observed in the patient who exhibited thrombus formation and did not receive enoxaparin . a transesophageal echocardiography performed in this patient showed three minor leaks at the border of the device . urena et al50 published a trial on 52 patients treated with the acp device who demonstrated a contraindication to warfarin . one patient experienced a tia 24 hours after the procedure despite no visualized intracardiac thrombus and evidence of complete laa sealing by transesophageal echocardiography . during a mean 20 months of follow - up , a lacunar stroke and second tia were reported . nevertheless , the number of strokes , thromboembolic events , and major bleeding events were significantly lower than expected based on cha2ds2-vasc ( congestive heart failure , hypertension , age 75 years or older , diabetes mellitus , prior stroke or tia or thromboembolism , vascular disease , age 65 to 74 years , sex ) and has - bled ( hypertension , abnormal renal / liver function , stroke , bleeding history or predisposition , labile international normalized ratio , elderly , drugs / alcohol concomitantly ) score estimates . peridevice residual leaks as assessed using transesophageal echo were observed in 16.2% of cases at 6 months ; however , none of these patients experienced a stroke or tia . leaks were significantly associated with low ejection fractions , suggesting modification in laa dimensions through remodeling can cause incomplete sealing . the lower rate of leaks using the acp device as compared with that observed with the watchman device is likely a result of the peculiar form of this device , which allows the disc to cover the laa ostium , whereas the closure in the watchman device is performed by positioning the device inside the laa neck . a recent systematic review51 evaluated the success and adverse event rates of percutaneous laa device occlusion by combining 14 trials ( 1,737 patients ) . no distinction was made among individual devices despite combining trials , which evaluated the plaato , watchman , or acp devices . overall , the authors concluded that laa closure was successfully performed in 93% of cases with a 1.1% periprocedural mortality . systemic embolism occurred in 1.6% , cardiac perforation in 0.9% , pericardial effusion and cardiac tamponade in 4% , and device embolization in 0.7% . major bleeding occurred in 0.7% of patients . when the observed stroke incidence of eight trials was compared with the estimated stroke risk , as predicted from the chads2 score , the newest amplatzer amulet ( acp-2 ) is the evolution of the original acp device , a dedicated device for percutaneous laa occlusion ( figure 4 ) . the main design of the first generation acp-1 is maintained , although several features were modified to improve feasibility of implantation procedure and orifice sealing : the stabilizing wires are stiffer and the number is increased from six to ten pairs , larger sizes are available ( 31 and 34 mm ) , the length of the distal lobe is increased by 23 mm , and the diameter of the proximal disc was increased by 5.58 mm . freixa et al52 reported successful implantation in 24 of 25 patients with no periprocedural complications and no strokes at 3 months , suggesting feasibility and safety . although most of the experience with laa occlusion devices has been with endocardial devices , isolation of the laa using an epicardial approach has more recently emerged as an attempt to simulate laa closure with suture ligation during cardiac surgery . laa occlusion with a percutaneous suture ligation method has been shown to be feasible for acute closure of the laa and has been shown to result in laa necrosis and atrophy.53 endocardial suture ligation during cardiac surgery has been demonstrated to be incomplete in 10%30% of patients , thus predisposing the patient to thromboembolic events.54,55 potential reasons for incomplete closure include that the procedure is performed when the heart is in a flaccid state during cardiopulmonary bypass , that the access for suturing may be difficult , and that the success of laa closure can not be determined until the patient resumes cardiopulmonary bypass . these reasons may be mitigated by using a percutaneous epicardial approach without the need for cardiopulmonary bypass . further , laa isolation has been reported as a successful therapy for laa tachycardias56 through electrical isolation and could allow elimination of focal laa triggers that potentiate atrial tachycardias and af . in addition , they offer further benefit because epicardial devices negate the need to implant a permanent endovascular device , as with the endocardial approach . the disadvantage with the epicardial approach is the need for pericardial access ; patients who have had previous cardiac surgery are therefore not candidates for an epicardial device . there is also a percentage of the population that is not eligible for an epicardial occlusion device because of the unsuitable anatomy of the laa . at the present time , the aegis system is a percutaneous device that is positioned around the laa to perform ligation . the most interesting aspect of this procedure is represented by an electrocardiogram - guided research of the laa by the two electrodes mounted at the extremity of the grabber . once the appendage is identified , a transesophageal echocardiogram or intracardiac ultrasound can be used to confirm the correct capture of the appendage . the second step of the procedure consists of ligating the appendage through a wire loop advanced over the grabber . in cases in which the appendage has multiple lobes , results of this new approach are encouraging.57,58 the lariat device is an laa exclusion device that consists of a pretied suture enclosed in on a closed snare and that employs a hybrid endocardial - epicardial approach . through a transseptal puncture another magnetic probe is introduced in the pericardial space via a percutaneous epicardial approach . over this endocardial - epicardial magnetic wire bridge , a lariat suture is inserted over the wire in the pericardial space to find the laa and snare it . bartus et al59 first performed successful laa exclusion using the lariat device in 13 patients : eleven with a closed - chest procedure and two during open - chest valvular cardiac surgery . a subsequent nonrandomized , single - center study60 evaluated a total of 119 patients for laa ligation with the lariat device . because of the thrombi within the laa or unsuitable anatomy , only 89 patients ( 75% ) underwent laa ligation . there were three procedure - related complications ( two of which were related to pericardial access and one to transseptal access ) . two deaths occurred more than 6 months after the procedure that were not related to the lariat device . another small study published by stone et al61 showed a success rate of 92.6% via a percutaneous laa ligation approach with the sentreheart lariat snare device . periprocedural complications included one patient with laa perforation , three patients developing pericarditis , and one patient having a stroke . on follow - up , han et al62 published a study on the effect of laa ligation with the lariat device on laa electrical activity . ninety - four percent of the patients had a reduction in the laa voltage after the closure of the snare , with 33% of the patients having complete elimination of laa voltage with the initial tightening of the suture . these preliminary results are interesting in the context in which laa occlusion with this particular device may help decrease recurrence of atrial fibrillation in this patient population . considering all these studies , one can state that the laa occlusion procedure can be performed with the lariat device with high success rates and acceptably low access complications and adverse events , except pericarditis , which is reported in all studies using that device . because this technique requires an epicardial approach , further research on device / suture material or adjuvant anti - inflammatory therapy that may reduce risk for pericarditis is required to improve procedural safety . risks from the catheter - based access of laa occlusion also have their limitations . as mentioned in table 1 , many procedural complications have been noted with every device , including the plaato , watchman , acp , and lariat devices . it is encouraging to see , for example , in the prevail study , that as this technique evolves , the rate of complications decreases . laa closure is an intervention of increasing interest , in particular for patients with either contraindications to anticoagulation therapy , embolic events while receiving anticoagulation therapy , or intolerance to anticoagulants . laa device closure has been recently been introduced into the 2012 focused update of the european society of cardiology as well as the 2014 american college of cardiology / american heart association / heart rhythm society guidelines for the management of af in patients at high risk for embolic stroke and contraindication to long - term therapy with an oral anticoagulant , for which it has been given a class iib recommendation for both.63,64 at this time , no evidence supports the systematic closure of laa in patients with no contraindications to oral anticoagulants . moreover , the advent of the noac will have a deep effect on the management of patients with nonvalvular af and may further limit the indications for percutaneous laa closure . notably , no trials have been designed to compare noacs with laa closure or to evaluate the use of noacs as adjunctive therapy in patients undergoing laa closure . furthermore , laa occlusion with current devices does not obviate the need for long - term antithrombotic therapy with aspirin . percutaneous laa closure can be associated with several procedural complications such as major bleeding , pericardial effusion , pericarditis , cardiac perforation , and air embolism . moreover , the differences in laa anatomy can increase the complexity of the procedure , posing challenges to accessing the laa and sealing the laa orifice . several device - related aspects still limit this approach , such as the high rate of incomplete laa closure reported at long - term follow - up with transesophageal echocardiography and the occurrence of device embolization and migration . the coherex wave - crest is a new device for laa closure furnished with anchoring points and the possibility of assessing device stability through a distal injection port during the procedure . preliminary animal studies report durable laa occlusion and an improved biocompatibility.65,66 future refinements of laa occlusion technology are expected to improve the feasibility of the procedure . the device design and coating should aim to overcome the limitations of incomplete laa orifice sealing and device dislodgement . in contrast , acute procedural complications have been shown to be influenced by a learning curve , and operator experience deeply affects outcomes of the procedure.67 the evolution and application of laa closure in clinical practice will be related to an extensive knowledge of physiopathology of laa and its effect on af . its has been shown that laa is an alternative site for af trigger,68 and as stated earlier , laa closure has been linked to a reduction in recurrence of paroxysmal af episodes.62 moreover , hemodynamic consequences of laa exclusion such as reduction in stroke volume and cardiac output have been amply documented.69 the occurrence of laa thrombus increases in patients with af and severe heart disease.29 the reduced ejection fraction and increased heart rate are a major determinant of the blood flow through the laa and blood clot formation . indications for laa closure will depend on further studies to define subgroups of patients achieving maximal benefit from laa exclusion .

atrial fibrillation ( af ) , the most common cardiac arrhythmia , confers a 5-fold risk of stroke that increases to 17-fold when associated with mitral stenosis . at this time , the most effective long - term solution to protect patients from stroke and thromboembolism is oral anticoagulation , either with vitamin k antagonists ( vkas ) or a novel oral anticoagulant ( noac ) . despite the significant benefits they confer , both vkas and noacs are underused because of their increased potential for bleeding , and vkas are underused because of their narrow therapeutic range , need for regular international normalized ratio checks , and interactions with food or medications . in patients with nonvalvular af , approximately 90% of strokes originate from the left atrial appendage ( laa ) ; in patients with rheumatic mitral valve disease , many patients ( 60% ) have strokes that originate from the left atrium itself . surgical laa amputation or closure , although widely used to reduce stroke risk in association with cardiac surgery , is not currently performed as a stand - alone operation for stroke risk reduction because of its invasiveness . percutaneous laa closure , as an alternative to anticoagulation , has been increasingly used during the last decade in an effort to reduce stroke risk in nonvalvular af . several devices have been introduced during this time , of which one has demonstrated noninferiority compared with warfarin in a randomized controlled trial . this review describes the available technologies for percutaneous laa closure , as well as a summary of the published trials concerning their safety and efficacy in reducing stroke risk in af .

Introduction Percutaneous LAA closure: endovascular approach Percutaneous LAA closure: epicardial approach Current state of percutaneous LAA closure and future directions

atrial fibrillation ( af ) is the most common cardiac arrhythmia , affecting 1%2% of the general population,1 although this may be an underestimate . studies assessing silent af using modern implantable rhythm monitoring devices suggest that up to 50%60% of patients older than 65 years may have unsuspected episodes of af.2,3 the relevance of these data is best understood in view of the increased mortality and morbidity associated with af : nonvalvular af confers a fivefold risk of stroke,1 which increases to 17-fold in patients with mitral stenosis.4 at the present time , vitamin k antagonists ( vkas ) remain the mainstay for stroke prevention in af . in a meta - analysis by hart et al,5 a 64% reduction in overall stroke risk was observed in patients using adjusted - dose vkas ( the reduction increased to 68% when only ischemic strokes were considered ) . despite such strong evidence , warfarin is still underused because of its associated bleeding risk , need for regular monitoring , narrow therapeutic range , and common interactions with other medications as well as food.4,6 in certain subgroups of patients , or when bleeding risk is elevated , acetylsalicylic acid ( asa ) , alone or in combination with clopidogrel , may be considered as an alternative to vkas.7 however , antiplatelet agents are inferior to vkas for stroke prevention and may be associated with similar bleeding risk , particularly in elderly patients.5,710 as an alternative to vkas , novel oral anticoagulant agents ( noacs ) have been increasingly used . the re - ly ( randomized evaluation of long - term anticoagulant therapy ) trial11 ( dabigatran 110 or 150 mg twice daily ) , the rocket - af ( rivaroxaban once - daily , oral , direct factor xa inhibition compared with vitamin k antagonism for prevention of stroke and embolism trial in atrial fibrillation)12 trial ( rivaroxaban 20 mg once daily ) , the aristotle ( apixaban for the prevention of stroke in subjects with atrial fibrillation)13 trial ( apixaban 5 mg twice daily ) , and the engage af timi 48 ( effective anticoagulation with factor xa next generation in atrial fibrillation thrombolysis in myocardial infarction 48)14 trial ( edoxaban 60 or 30 mg once daily ) , using dose - adjusted warfarin as standard - of - care control , showed noninferiority or superiority for the efficacy outcome of combined ischemic stroke and systemic embolism in patients with nonvalvular af . however , the composite outcome of major bleeding does not differ substantially from warfarin.1518 the exact mechanism of thrombus formation in af - related strokes , although not yet fully understood , involves the presence of endocardial injury , hypercoagulability , and blood stasis in the atria.3 the latter seems to play a pivotal role ; in fact , the failure of the left atrium to effectively contract results in atrial stretch and dilation , favoring thrombus formation.3 approximately 75% of embolic events associated with af result from atrial thrombosis.19 a review of autopsy , echocardiographic , and operative reports demonstrate that atrial thrombi can be identified in the left atrial appendage ( laa ) in 91% of nonvalvular af cases and 57% of rheumatic af cases.20 af is associated with a systemic prothrombotic state characterized by endothelial dysfunction and platelet activation.21 there are several anatomical features and physiological characteristics that explain the pivotal role played by the laa as a major site of thrombus formation and source of thromboembolic stroke . first , the wall of the laa has numerous trabeculae ( pectinate muscles ) , forming crypts that can harbor blood clots . these features differentiate the laa from the left atrium that has a smooth wall . douglas et al22 described that the laa originates by the incorporation of the left pulmonary vein in the body of the left atrium . second , the macroscopic anatomy of the laa is complex and highly variable , with a long , tubular , and often multilobed body extending over the atrioventricular groove and left ventricular surface . recently , a correlation between the morphology of the laa and embolic risk has been described.23 the laa ostium is usually oval - shaped and located anterior and inferior to the left superior pulmonary vein . third , the laa is also more distensible than the left atrium itself and can receive a large volume of blood when the pressure in the left atrium is high . fourth , a reduction in stroke volume and cardiac output has been described after removal of the laa.24,25 fifth , anatomically , the laa lies superior and anterior to the left ventricle in close contact with the left ventricular wall . as a consequence , changes in the left ventricular volume and pressure can be transmitted to the laa wall , which affects blood flow through the laa . of note , surgical laa exclusion has been performed to reduce stroke risk in cardiac valve surgery in patients with af,26 but this procedure is too invasive to be used as a stand - alone operation in patients who do not have any other concurrent indication to undergo cardiac surgery . percutaneous laa closure is a relatively new procedure that obviates the need for sternotomy and cardiopulmonary bypass and is an attractive alternative to chronic anticoagulation with vkas or noacs in reducing stroke risk in nonvalvular af . this review describes the current technologies used in percutaneous laa closure ( table 2 ) , as well as a summary of the published trials concerning their efficacy in reducing stroke risk in af . it is deployed through a catheter introduced via the femoral vein and is advanced from the right to the left atrium by transseptal puncture . during the months after the procedure , the device endothelializes with proliferation of a new layer of neointima , starting at the edges of the device . during a 9.8 month follow - up , a 65% reduction in stroke rate was observed compared with the expected stroke risk , as calculated using the chads2 score ( representing congestive heart failure , hypertension , age , diabetes mellitus , and stroke , the chads2 score is a clinical prediction rule widely used for estimating the risk of stroke in patients with nonvalvular af ) . it is worth emphasizing that the authors excluded three transient ischemic attacks ( tias ) from the number of strokes and justify this on the basis that the chads2 score was elaborated to calculate the risk for strokes and not for tias.31 residual peridevice leaks were observed not uncommonly by fluoroscopy or echocardiogram , and laa occlusion was defined as absent , trace , or a mild leak on the basis of these imaging modalities . this is a point of interest that has been the focus of critique not only for the plaato device but also for the newer devices as well and will be discussed later on in this review . the laa closure procedure was successful in 162 ( 90% ) but failed in 18 ( 10% ) patients because of anatomic characteristics of the laa ( 14 cases ) , impossibility of accessing the laa ( 2 cases ) , complications during transseptal puncture ( 1 case ) , and device instability ( 1 case ) . a composite of the international multi - center feasibility trial and the european - only plaato registry study,27 including almost 300 patients , demonstrated a relative reduction of stroke rate of 54% with plaato compared with the stroke rate in a historic control group , using the chads2.33 in this registry , thrombus formation was reported in 1% of cases within the first 6 months and was not associated with any neurological events . long - term follow - up of the plaato device is reported in three trials . nevertheless , financial problems of the manufacturer and a significant rate of adverse events , as mentioned earlier , led to discontinuation of the device in 2006.37 the watchman laa system ( figure 1 ) became available in 2002 . the watchman laa closure device is available in five different sizes , ranging from 21 to 33 mm , and is usually selected 10%20% larger than the laa ostium . the protect af ( watchman left atrial appendage system for embolic protection in patients with atrial fibrillation ) trial,38 published in 2009 , was a randomized controlled trial that involved the randomization of 707 af patients with at least one stroke risk factor to the watchman device versus warfarin in a 2:1 fashion . the device was found to be noninferior to warfarin for the combined end - point of stroke , systemic embolism , and cardiovascular death . those receiving the device were treated with warfarin for approximately 45 days , followed by clopidogrel for 4.5 months , and then lifelong asa . because only 30% of patients had been followed for more than 2 years in the original publication of the protect af trial , the success of the device could have been attributed in part to the ongoing anticoagulation that was received . however , a recently published 2.3 year ( 1.1 years ) follow - up study of protect af confirmed the noninferiority of the watchman device.39 there were no statistically significant differences detected in individual comparisons between the two groups ; however , the confidence interval was wide ( 95% confidence interval , 0.361.76).38 the device group exhibited more ischemic strokes , but less cardiovascular death and less hemorrhagic stroke . of note , all patients in the control group received warfarin ( time in therapeutic international normalized ratio range was 66% ) , with 34% of control patients having had interrupted anticoagulation at some point for various reasons . importantly , patients with a previous history of stroke or tia appeared to receive sustained benefit from the device that was at least as good as warfarin therapy.39 peridevice leaks with the watchman device detected by transesophageal echocardiography were common ( up to 30% at 12 months),40 although a recent retrospective analysis of the protect af trial showed no effect on the effectiveness of the device.41 laa closure with the watchman device also resulted in improved quality of life.4 gangireddy et al43 estimated the net clinical benefit ( ncb ) of percutaneous laa closure in a post hoc analysis of outcomes among 707 af patients in the protect af trial and 566 in the continued access registry who were undergoing laa closure with the watchman device compared with anticoagulation with a vka . outcomes were ischemic stroke , intracranial hemorrhage , major bleeding , pericardial effusion , and death . the ncb initially favored anticoagulation , but 69 months after the procedure , the ncb then favored the device - based intervention , driven mainly by reductions in intracranial hemorrhage and death in patients undergoing laa closure . importantly , operator experience significantly affected the safety and efficacy end - points of the intervention : the incidence of procedure - related events was considerably lower in the nonrandomized continued access registry that followed the protect af trial , with the ncb clearly favoring intervention in the registry . as expected , the highest net clinical benefit was observed for patients with high chads2 scores . the asap study ( asa plavix feasibility study with watchman left atrial appendage closure technology ) , presented in 2012 , was a feasibility study designed to evaluate whether the watchman device could be used for patients who were unable to take warfarin . in this nonrandomized study enrolling 150 patients with an average chads2 score of 2.8 who had a contraindication to anticoagulation , patients were treated with both asa and clopidogrel for 6 months after implantation instead of the standard warfarin . in fact , despite not using warfarin , the event rate was lower than in the protect af study ( 2.2 strokes per 100 patient - years).44 these new data permitted the use of the watchman device in patients with nonvalvular af and contraindications to vka therapy . specifically alluding to the protect af trial , incidence of strokes at follow up were 50% more in the device group compared with in the warfarin group , at 3.0% versus 2.0%.45 one can argue the need for anticoagulation or antiplatelet therapy added to laa closure indefinitely . last , the prevail ( watchman laa closure device in patients with atrial fibrillation versus long term warfarin therapy ) study46 was a randomized controlled trial including 269 patients in the watchman laa closure group compared with 138 patients in the warfarin group . although the study group ( watchman ) did not achieve the prespecified criteria for noninferiority at 18 months follow - up compared with the control group ( warfarin ) , the overall periprocedural early events were lower than in the protect af trial , at 2.2% versus 4.9% , respectively . six patients were excluded because of difficult laa anatomy or thrombus , and 137 procedures were attempted , of which 133 were successful ( 96% ) . ten patients ( 7% ) experienced complications : three periprocedural strokes , two device embolizations , five cardiac tamponades requiring pericardiocentesis , and four pericardial effusions with spontaneous resolution . the study showed the feasibility of laa closure with the acp device , as well as its relatively low rate of complications . thrombus formation on the acp surface was documented in five cases ( 14.28% ) , and in four of them , enoxaparin was administered , with eventual thrombus resolution . nevertheless , the number of strokes , thromboembolic events , and major bleeding events were significantly lower than expected based on cha2ds2-vasc ( congestive heart failure , hypertension , age 75 years or older , diabetes mellitus , prior stroke or tia or thromboembolism , vascular disease , age 65 to 74 years , sex ) and has - bled ( hypertension , abnormal renal / liver function , stroke , bleeding history or predisposition , labile international normalized ratio , elderly , drugs / alcohol concomitantly ) score estimates . when the observed stroke incidence of eight trials was compared with the estimated stroke risk , as predicted from the chads2 score , the newest amplatzer amulet ( acp-2 ) is the evolution of the original acp device , a dedicated device for percutaneous laa occlusion ( figure 4 ) . the main design of the first generation acp-1 is maintained , although several features were modified to improve feasibility of implantation procedure and orifice sealing : the stabilizing wires are stiffer and the number is increased from six to ten pairs , larger sizes are available ( 31 and 34 mm ) , the length of the distal lobe is increased by 23 mm , and the diameter of the proximal disc was increased by 5.58 mm . although most of the experience with laa occlusion devices has been with endocardial devices , isolation of the laa using an epicardial approach has more recently emerged as an attempt to simulate laa closure with suture ligation during cardiac surgery . laa occlusion with a percutaneous suture ligation method has been shown to be feasible for acute closure of the laa and has been shown to result in laa necrosis and atrophy.53 endocardial suture ligation during cardiac surgery has been demonstrated to be incomplete in 10%30% of patients , thus predisposing the patient to thromboembolic events.54,55 potential reasons for incomplete closure include that the procedure is performed when the heart is in a flaccid state during cardiopulmonary bypass , that the access for suturing may be difficult , and that the success of laa closure can not be determined until the patient resumes cardiopulmonary bypass . further , laa isolation has been reported as a successful therapy for laa tachycardias56 through electrical isolation and could allow elimination of focal laa triggers that potentiate atrial tachycardias and af . there is also a percentage of the population that is not eligible for an epicardial occlusion device because of the unsuitable anatomy of the laa . the most interesting aspect of this procedure is represented by an electrocardiogram - guided research of the laa by the two electrodes mounted at the extremity of the grabber . because of the thrombi within the laa or unsuitable anatomy , only 89 patients ( 75% ) underwent laa ligation . as mentioned in table 1 , many procedural complications have been noted with every device , including the plaato , watchman , acp , and lariat devices . laa closure is an intervention of increasing interest , in particular for patients with either contraindications to anticoagulation therapy , embolic events while receiving anticoagulation therapy , or intolerance to anticoagulants . laa device closure has been recently been introduced into the 2012 focused update of the european society of cardiology as well as the 2014 american college of cardiology / american heart association / heart rhythm society guidelines for the management of af in patients at high risk for embolic stroke and contraindication to long - term therapy with an oral anticoagulant , for which it has been given a class iib recommendation for both.63,64 at this time , no evidence supports the systematic closure of laa in patients with no contraindications to oral anticoagulants . moreover , the advent of the noac will have a deep effect on the management of patients with nonvalvular af and may further limit the indications for percutaneous laa closure . notably , no trials have been designed to compare noacs with laa closure or to evaluate the use of noacs as adjunctive therapy in patients undergoing laa closure . furthermore , laa occlusion with current devices does not obviate the need for long - term antithrombotic therapy with aspirin . percutaneous laa closure can be associated with several procedural complications such as major bleeding , pericardial effusion , pericarditis , cardiac perforation , and air embolism . several device - related aspects still limit this approach , such as the high rate of incomplete laa closure reported at long - term follow - up with transesophageal echocardiography and the occurrence of device embolization and migration . in contrast , acute procedural complications have been shown to be influenced by a learning curve , and operator experience deeply affects outcomes of the procedure.67 the evolution and application of laa closure in clinical practice will be related to an extensive knowledge of physiopathology of laa and its effect on af . its has been shown that laa is an alternative site for af trigger,68 and as stated earlier , laa closure has been linked to a reduction in recurrence of paroxysmal af episodes.62 moreover , hemodynamic consequences of laa exclusion such as reduction in stroke volume and cardiac output have been amply documented.69 the occurrence of laa thrombus increases in patients with af and severe heart disease.29 the reduced ejection fraction and increased heart rate are a major determinant of the blood flow through the laa and blood clot formation .

aneurysmal subarachnoid hemorrhage ( asah ) is a relatively rare cause of stroke , occurring in approximately 30,000 persons in the united states each year . however , its impact equals that of cerebral ischemia , the most common cause of stroke , due to its higher morbidity , higher mortality , and occurrence in younger individuals . approximately 20 to 30 percent of patients with asah suffer from cerebral ischemia and infarction due to cerebral vasospasm , which is the number one cause of treatable death and disability following asah . vasospasm occurs most frequently at 7 to 8 days after asah and can last for a prolonged period . clinical vasospasm is defined as a decline in neurologic status due to vasospasm and can result in severe morbidity and mortality . however , clinical vasospasm has also been observed after other invasive and traumatic processes such as craniotomy and traumatic brain injury . the pathogenesis and etiology of this vasospasm are very poorly understood , and treatments to prevent post - sah vasospasm are widely varied and have greatly different magnitudes of effectiveness . new methods to predict , diagnosis , and manage vasospasm are an active field of research , and there is great controversy regarding the effectiveness of these various methods to affect the course of vasospasm . further research is needed to settle some of these controversies and to establish evidence to justify the use of some of the more common interventions already used in clinical practice . the aim of this paper is to address controversies in the prediction , diagnosis , and management of cerebral vasospasm based on a comprehensive review of the recent literature regarding these topics . previous studies on asah have studied a variety of different outcomes , making the direct comparison of the results difficult . while delayed cerebral ischemia ( dci ) has generally been utilized as the outcome in many studies , its definition has not been applied consistently . while it is defined most accurately as the occurrence of focal neurological impairment or a decrease of at least 2 points on the glasgow coma scale which lasts for at least one hour , it is not apparent immediately after aneurysm occlusion , and can not be due to other causes by means of clinical assessment , ct , mri , or appropriate laboratory studies . a recent literature review by vergouwen et al . found that a variety of different terms had been used to reflect dci , including delayed ischemic neurologic deficit ( dind ) , secondary cerebral ischemia , symptomatic ischemia , vasospasm , clinical vasospasm , symptomatic vasospasm , and cerebral infarction . while dci is most purely a clinical definition , many previous studies had combined its definition with angiographic or anatomic evidence of vasospasm , making it impossible to compare many studies . vasospasm , although often associated with dci , is defined as an anatomic narrowing of the intracranial arteries from various causes such as constriction , swelling , endothelial remodeling , and fibrosis . according to poiseuille 's law it is hypothesized that this increase in resistance in vasospasm decreases blood flow to dependent regions of the brain , resulting in dci and eventually cerebral infarction . vasospasm can be visualized directly with imaging such as angiography or inferred indirectly from increased blood flow velocities as would be expected according to bernoulli 's law in an artery with a decreased luminal cross - sectional area . while it is generally true that vasospasm is strongly associated with dci and cerebral infarction [ 24 ] , patients with asah can have poor clinical and functional outcomes without vasospasm . at best , vasospasm serves as a surrogate marker for dci , but it has incorrectly been used as a synonym for dci in many studies . recently , the international multidisciplinary consensus conference on the critical care management of subarachnoid hemorrhage published guidelines for a uniform definition of dci for use in clinical trials and observational studies . it recommended that separately defined outcomes for angiographic vasospasm , dci , cerebral infarction , and functional outcome be utilized in future studies . there was consensus that cerebral infarction on neuroimaging would be a better outcome measure than dci or vasospasm because it is more objective ( with a higher interobserver reliability ) , has a higher sensitivity in sedated / comatose patients , and has been shown to be a superior predictor of 3-month functional outcome . it is important to interpret the results of the studies described in this paper in this context . there is disagreement in the literature , however , as to the relationship between age and incidence of vasospasm after ruptured asah . other studies have indicated that older patients are at greater risk of vasospasm or that there is no relationship between age and vasospasm [ 1417 ] . the definition of vasospasm used in these studies differed along with the method of diagnosing vasospasm , which makes the interpretation of these studies difficult . two of the studies performed only bivariate analyses of age as a predictor of vasospasm and were not controlled for other common confounders [ 7 , 12 ] . three studies suggesting an inverse relationship between increasing age and increased risk of vasospasm utilized threshold cerebral blood flow velocity ( cbfv ) values from transcranial doppler ( tcd ) to define vasospasm . the sensitivity of a high cbfv for detecting symptomatic vasospasm in elderly sah patients has been shown to be particularly low when using the same criteria as in younger patients , because older patients have significantly lower maximum mean cbfvs . in elderly patients , the differences in the definition of vasospasm , measurement modality , and age categories used in these studies make the interpretation of their results difficult . smoking has been suggested for decades as one of the most important risk factors for subarachnoid hemorrhage and recent evidence firmly supports this relationship [ 1921 ] . cigarette smoking also appears to increase the risk of angiographic vasospasm and dci in patients with ruptured asah . two prospective studies found that cigarette smoking was an independent predictor of dci after asah [ 15 , 22 ] . this is plausible , as cigarette smoking has previously been identified as the most important risk factor in the development of coronary arterial vasospasm [ 2325 ] . however , it is unclear which components of cigarette smoke are associated with the increased risk of symptomatic vasospasm . a retrospective study involving 258 active smokers found that there was a decreased risk of symptomatic vasospasm amongst those given nicotine replacement therapy . yet another recent retrospective study of a similar size showed no association between nicotine replacement therapy and vasospasm or dci . however , there is consensus in the recent literature suggesting that smoking is associated with an increased risk of dci after asah . cocaine use can also cause coronary artery vasospasm and is one established cause of prinzmetal angina . additionally , it has been associated with neurological complications such as intraparenchymal hemorrhage , intraventricular hemorrhage , and cerebral infarction . however , there have been fewer studies on the angiographic effects of cocaine on human cerebral arteries . there is controversy in the literature on the effect of cocaine use in the aftermath of asah . in 2001 , conway and tamargo determined that of 440 patients who experienced asah over a 7-year period , 27 patients had used cocaine within 72 hours of asah rupture . cocaine use was found to be an independent predictor of dci with an odds ratio of 6.41 ( 95% ci , 2.1419.24 , p = 0.0009 ) . more recently , boco and macdonald reported no significant differences in mean internal carotid , middle cerebral , anterior cerebral , and basilar artery angiographic diameters between a group of 13 cocaine users and 26 control asah patients . a study in 2010 casts further doubt on the association between cocaine use and vasospasm . showed that of 600 patients with asah included in the study , 31 ( 5% ) had a history of recent cocaine use . cocaine users were younger ( 45.1 years versus 54.1 , p < 0.0003 ) and were more likely to also use tobacco and alcohol . however , no association was found between cocaine use and hunt - hess score , fisher grade , short - term outcome ( modified rankin scale > 3 ) , symptomatic vasospasm , radiologic vasospasm , stroke , or mortality . the largest study to date of 142 patients with cocaine use - associated asah by chang et al . in 2013 reported that cocaine users tended to be younger ( 49 years versus 53 years , p < 0.001 ) and had no difference in hunt - hess score , intraventricular hemorrhage , and hydrocephalus , which was consistent with previous studies . most importantly , however , cocaine use was not an independent predictor of dci when controlled for with other known independent predictors such as age , hunt - hess , wfns , and admission glasgow coma scale ( gcs ) score . this result conflicts with the study by conway et al . but had a much larger sample size ( n = 142 versus n = 27 cocaine users ) . while cocaine users were more likely to have hospital mortality with an odds ratio of 2.9 ( 95% ci , 1.764.63 , p < 0.001 ) , there was no difference in 3-month functional outcome . overall , there seem to be few studies on the effect of cocaine use on vasospasm after asah and there is no definite consensus between them . there is a need for well - powered studies with well - defined outcomes comparing angiographic vasospasm , clinical outcome , cerebral infarction , and mortality in cocaine - associated asah . the relationship between the amount and density of blood detected on an initial computed tomography ( ct ) scan of patients admitted for a diagnosis of ruptured asah and angiographic vasospasm was first observed in the late 1970s . in 1980 , mizukami et al . found that amongst 26 patients with high density hemorrhage on initial ct scans , 84.6% developed cerebral vasospasm while none of 8 patients did not develop cerebral vasospasm . additionally , earlier ct scans have greater predictive power for symptomatic vasospasm . in 1980 , davis et al . demonstrated a direct correlation between the extent of blood and the severity of vasospasm that developed only in ct scans taken within 4 days of sah . more recently , dupont et al . found that there was a relationship between the extent of subarachnoid blood on admission ct imaging and the subsequent development of vasospasm only if the ct was performed within 24 hours of aneurysmal rupture . these data suggest that the earlier the ct scan after ruptured asah , the greater the prognostic value in predicting cerebral vasospasm . one of the best predictors of cerebral vasospasm to date is the characteristic of asah on admission ct shortly after aneurysmal rupture of the aneurysm [ 33 , 34 ] . the most commonly used and well - known grading scale for classification of admission ct is the fisher grading scale [ 35 , 36 ] . in the well - known analysis between the amount of subarachnoid blood and development of cerebral vasospasm , fisher et al . found that there was a high correlation between symptomatic vasospasm and grade iii sah , classified by the presence of subarachnoid clots greater than 5 3 mm in diameter in cisterns and fissures in the horizontal plane , or clots greater than 1 mm thick in the vertical plane [ 35 , 36 ] . in 2001 , classen et al . suggested an asah grading system based on an analysis of 276 patients who had a ct scan within 72 hours of rupture , which found that the best predictors of dci were blood in both lateral ventricles ( odds ratio ( or ) 4.1 , 95% ci 1.79.8 ) and a thick clot completely filling any cistern or fissure ( odds ratio ( or ) 2.3 , 95% ci 1.59.5 ) these two characteristics were found to be independently predictive of cerebral vasospasm , and the claassen scale incorporated separate categories for cisternal blood with and without bilateral intraventricular hemorrhage . the role of cisternal blood in predicting vasospasm has been firmly established in the literature . in 1978 , takemae et al . reported that there was a significant relationship between cerebral vasospasm and a high density of blood in basal subarachnoid cisterns on admission ct scans . in a report of the cooperative aneurysm study , adams et al . discovered that focal , thick collections of blood in the cisternae were highly predictive of dci . grosset et al . studied the admission ct scans of 121 patients presenting after asah . there have been published studies indicating no relationship between ivh on admission ct scan and symptomatic vasospasm [ 31 , 37 , 38 ] . the majority of studies , however , found that ivh was predictive of symptomatic vasospasm [ 31 , 34 , 3942 ] . a modified fisher ct grading scale taking into account thick cisternal and ventricular blood outperformed the original fisher scale in predicting symptomatic vasospasm in a study of 1355 asah patients in the placebo arm of a randomized controlled trial ( rct ) of tirilazad . in 2011 , ko et al . used imaging software to calculate cisternal plus intraventricular hemorrhage volume ( chiv ) from ct scans taken within 24 hours of aneurysmal rupture patients in the highest quartile of chiv ( 31.0 ml ) were significantly more likely to develop dci ( odds ratio ( or ) 4.6 , 95% ci 1.3427.83 ) than those in the lowest quartile ( < 9.6 ml ) . cisternal blood volumes have uniformly been found to be a predictor of symptomatic vasospasm , and recent research utilizing large datasets indicates that intraventricular hemorrhage is also predictive , although this has been controversial . however , other authors have voiced concerns over several limitations in the implementation and effectiveness of the scheme . the highest score ( grade iv sah ) does not have the highest risk of vasospasm , which can be confusing , and there is no category for patients who have both thick cisternal blood and intraventricular hemorrhage , even though these may be independent and additive predictors of vasospasm . in a randomized controlled trial of nimodipine in patients with sah , . found that in modern practice , the fisher grade only predicted symptomatic vasospasm in half of the patients . van norden et al . also noted that fisher grade iii and grade iv did not predict dci . this may indicate that in the era of modern practice , where nimodipine , hypertensive therapy , and endovascular therapy have become established , the fisher grade may be outdated . in addition , many studies have raised concern that the fisher scale is a qualitative , categorical scale and may have considerable interobserver variability that may adversely affect its usefulness for clinical practice and research . an earlier study by ogilvy and carter collapsed the fisher scale into grades 0 to ii versus grades iii and iv and noted that there was almost perfect agreement ( kappa 0.90 ) . ibrahim et al . observed that the fisher scale had only fair - to - moderate agreement ( kappa 0.41 , 95% ci 0.330.49 ) compared to a semiquantitative scale , the hijdra scale ( icc 0.56 , 95% 0.490.62 ) . van norden et al . also found the hijdra scale ( kappa ranging from 0.67 to 0.75 ) to have greater interobserver reliability than the fisher scale ( kappa ranging from 0.37 to 0.55 ) . svensson et al . found slightly greater agreement for the fisher scale ( kappa 0.500.63 , moderate to substantial agreement ) . kramer et al . published consistent results observing that the claassen scale had substantial agreement ( kappa 0.64 ) , while the modified fisher scale and fisher scale had moderate agreement ( kappa 0.59 and 0.45 , resp . ) . the modified fisher scale , however , was the only grading scale in which each unit increase was associated with an incremental risk for all 3 outcomes and was thus recommended by the authors . the hijdra scale was also found to be superior to the original fisher scale in terms of interobserver reliability and ability to predict symptomatic vasospasm [ 47 , 49 , 52 ] . it has been criticized by other authors , however , for being difficult to use in clinical practice . while there are a variety of grading schemes for interpretation of ct scans , each possesses limitations to its validity , both internal and external , and/or ease of implementation in the clinical practice setting . future research is needed to produce a valid , reliable grading scheme that provides strong prognostic information for development of symptomatic vasospasm . various methods are currently used to detect vasospasm including tcd , ct angiography , digital subtraction angiography ( dsa ) , and ct perfusion imaging . of these , tcd is the most widely used but the standard of reference for the anatomic demonstration of cerebral vasospasm is dsa . however , dsa is costly and has a small but nonnegligible risk of neurologic complications , making the consideration of the other modalities of diagnosis attractive . however , there is controversy over which method is the best at detecting vasospasm . while tcd is inexpensive , available at the bedside , noninvasive , and without known adverse side effects , it is operator dependent , requires a good acoustic window , and has been known to have a high false negative rate for vasospasm when compared to dsa [ 56 , 57 ] . it has been shown to be specific , but not sensitive for vasospasm and poorly predictive of developing secondary cerebral infarction compared to angiography [ 58 , 59 ] . ct perfusion imaging has shown good specificity and accuracy in detecting vasospasm with a specificity of 100% and an accuracy of 92.3% . specifically , the cerebral blood flow reduction on perfusion ct was found to significantly predict the clinical outcome of patients . a meta - analysis comparing the accuracy of ct angiography and ct perfusion for detecting vasospasm found that they had similar specificity and sensitivity , with an overall sensitivity and specificity of 79.6% and 93.1% , respectively , for ct angiography and 74.1% and 93.0% , respectively , for ct perfusion . unfortunately , both of these modalities can be limited by beam - hardening artifact from clips and coils and are of limited utility in evaluating posterior fossa territories . because of the different detection characteristics for these modalities , there is controversy over the modern incidence of vasospasm and that with increased specificity of more recent techniques , the actual incidence of vasospasm may be lower than noted in previous studies . a prospective study systemically comparing the ability of these techniques to predict dci would provide valuable evidence to guide monitoring strategies for vasospasm . a mainstay in the prophylaxis and treatment of cerebral vasospasm in the past was hyperdynamic therapy , also known as triple - h therapy , which utilized the three approaches of hypervolemia , induced hypertension , and slight hemodilution with the aim of improving cerebral blood flow ( cbf ) . in contemporary practice , there is much disagreement on the use of hyperdynamic therapy in the prophylaxis and treatment of vasospasm after asah . a survey of 626 physicians ( anesthesiologists , internists , neurologists , and neurosurgeons ) in europe and the united states found that 39% and 52% of respondents would use hyperdynamic therapy for the prophylaxis and treatment of vasospasm after asah , respectively , . this combined treatment approach has not yet been assessed for efficacy in any large rcts , and as such , evidence for efficacy has been based mostly on case series of patients with dci attributed to vasospasm . although originally patients were treated with a combination of all three of these therapies ( hypervolemia , hypertension , and hemodilution ) , recently the efficacy of each component of triple - h therapy has been questioned . in addition , studies have been conducted recently to specifically address the role of these therapies to prophylactically prevent vasospasm . in the early 2000s , two rcts compared the effect of prophylactic hypervolemia to normovolemia . in 82 patients assigned to prophylactic normovolemia or hypervolemia after aneurysm clipping , lennihan et al . demonstrated that hypervolemic therapy resulted in increased cardiac filling pressures and fluid intake but did not increase cbf or blood volume compared with normovolemic therapy , concluding that prophylactic hypervolemic therapy is unlikely to confer benefit . in 2001 , egge et al . published results from an rct of 32 patients who were randomized to prophylactic normovolemia or in addition to having no benefit , prophylactic hyperdynamic therapy was found to have greater costs and more frequent complications such as excess bleeding , congestive heart failure , and infections . a recent , comprehensive review of 11 studies in the literature on prophylactic hyperdynamic therapy concluded that the available evidence failed to show a benefit for the use of prophylactic hyperdynamic therapy and suggested harm in using overly aggressive hydration . there seems to be evidence that suggests that the use of hemodynamic therapy after the occurrence of vasospasm may be beneficial and could reverse symptomatic vasospasm and neurological deficits . in 1982 , kassell et al . induced arterial hypertension in 58 patients with intravascular volume expansion , blockade of the vagal depressor response , and the administration of antidiuretics and vasopressor agents , resulting in a permanent reversal of neurological deterioration in 43 patients . in 1995 , mori et al . found that hypervolemic hemodilution therapy after symptomatic vasospasm could reverse neurological deterioration due to cerebral vasospasm . in 2003 , a systematic review of 4 prospective studies of triple - h therapy found that it reduced symptomatic vasospasm ( relative risk ( rr ) 0.45 , 95% ci 0.530.87 ) and death ( rr 0.68 , 95% ci 0.530.87 ) but had no effect on dind ( rr 0.54 , 95% ci 0.21.49 ) . however , the individual roles of the separate components of triple - h therapy are still controversial . a study of the recent literature indicates that hypertension may be the most important component of hyperdynamic therapy . a retrospective study of 45 patients conducted in 2005 by raabe et al . found that hypervolemia could be associated with increased risks but that moderate hypertension ( cerebral perfusion pressure 80120 mmhg ) in a normovolemic , hemodiluted patient helped to improve cerebral oxygenation and had a lower complication rate compared to hypervolemic therapy . more recently in 2010 , frontera et al . found that 43% of patients having volume expansion after symptomatic vasospasm had a clinical improvement , while 68% of those with hypertensive therapy responded , suggesting that induced hypertension was more effective in treating vasospasm . in 1990 , otsubo published a study reporting that induced hypertension in the presence of normovolemia reduced the signs and symptoms of vasospasm in 54% of patients . a possible drawback to the use of hypertensive therapy is the possibility of rebleeding , rerupture , or rupture of other , unsecured aneurysms . however , recent evidence seems to suggest that hypertensive therapy is generally safe for secured , ruptured aneurysms and unruptured aneurysms . in 2002 , hoh et al . observed that amongst 40 patients requiring blood pressure augmentation up to 200 mmhg with unsecured unruptured aneurysms , no bleeding events occurred . in a study of 25 consecutive patients with asah undergoing induced hypertension after coiling up to a peak systolic blood pressure from 195 mmhg to 205 mmhg , there were no episodes of aneurysm rebleeding . in 2011 , platz et al . conducted a retrospective study of 71 unsecured , unruptured aneurysms with an average size of 4.0 mm during induced hypertension and found that no aneurysm ruptured during therapy . these studies suggest that induced hypertension is efficacious independent of hypervolemia and that it should not be omitted based on the presence of unsecured aneurysms . with evidence cautiously suggesting the efficacy of hypertensive therapy in preventing dci with a low risk of aneurysmal rupture further research is needed in the form of a large rct , however , to elucidate the role of induced hypertension and to determine its risk profile in patients with symptomatic vasospasm . in patients undergoing surgery for acute asah , there is usually a decrease in circulating blood volume for at least 3 days after surgery . therefore , there is at least theoretical basis in the reasoning that the maintenance of normovolemia via relative hypervolemia within 3 days after surgery for asah may improve outcomes . however , hypervolemia has been associated with a higher rate of complications such as pulmonary edema and congestive heart failure . in 2002 , ekelund et al . published a study comparing the effects of iso- and hypervolemic hemodilution on regional cerebral blood flow and oxygen delivery in patients with tcd - defined vasospasm . no difference was found between normovolemic and hypervolemic hemodilution , and rather hemodilution was found to result in a pronounced reduction in oxygen delivery capacity . reported that the administration of a saline bolus of 15 ml / kg/1 hr in patients with normovolemia at baseline with new symptoms of vasospasm produced an increase in cbf . in 2007 , tseng et al . reported that in patients with a poor - grade sah , administration of 2 ml / kg of 23.5% hypertonic saline solution decreased intracranial pressure and increased cerebral perfusion pressure , transcranial doppler velocities , and cbf [ 76 , 77 ] . the literature regarding hypervolemic therapy is therefore still inconclusive regarding the efficacy of hypervolemia . despite this , there is some evidence that hypovolemia is associated with increased risk of dci , and therefore , normovolemia should be attained via initial fluid resuscitation in patients with hypovolemia . hemodilution , particularly the lowering of hematocrit , attempts to decrease the viscosity of blood and thereby improves blood flow to ischemic regions of the brain via the poiseuille equation , which states that the resistance to flow is inversely proportional to viscosity . an optimal hematocrit aims to increase oxygen delivery to ischemic tissues by balancing these two opposing factors . recommended maintaining a hematocrit of 3032% utilizing hemodilution with colloids to maximize oxygen delivery to ischemic brain in the setting of vasospasm . in humans , the role of hemodilution in treatment of cerebral vasospasm has also been scrutinized , but with no good consensus in the literature . in 2006 , a study by naidech et al . found that asah patients with higher initial and mean hemoglobin values had improved outcomes . a larger study in 2007 of 611 consecutive patients by the same authors supported their earlier conclusions . higher hemoglobin levels were associated with improved outcomes at 14 days and 3 months , but the authors did not recommend an optimal goal hemoglobin nor routine packed red blood cell transfusions . retrospectively reviewed the management of 441 patients and found that postoperative packed rbc transfusion actually had a positive correlation with angiographic vasospasm with an odds ratio of 1.68 ( 95% ci , 1.022.75 ) additionally , intraoperative rbc transfusion was found to be correlated with worse outcome . the authors concluded that patients should be assessed for anemia to determine whether such transfusion would be necessary . a recent 2010 study by levine et al . the authors cautiously declared that the data did not infer causation and that further study was necessary to better define the indications for transfusion after sah . published in 2010 found no significant difference in cerebral infarction between a hemoglobin goal concentration of at least 10 or 11.5 g / ld . but a trend favoring higher goal hemoglobin ( p > 0.1 ) . this suggested that perhaps higher goal hemoglobin in asah patients may be feasible and that a larger rct to determine optimal goal hemoglobin may be warranted . contrary results were obtained in two separate computational studies modeling the fluid dynamics of blood flow in the middle cerebral artery which showed that hemodilution did not improve oxygen transport , but to the contrary , ischemia may be worsened [ 85 , 86 ] . these same models also showed that the middle cerebral artery required a large increase in intracranial artery inlet pressure for a beneficial effect on cbf , with a minimal effect of decreased hematocrit . in 2012 , neither routine hemodilution nor transfusion was recommended for routine treatment of dci or vasospasm in the guidelines for the management of aneurysmal subarachnoid hemorrhage by the american stroke association and based on the current literature ; this seems reasonable . there were conflicting results in the literature regarding the usage of albumin for intravascular volume expansion . in 1987 , yamakami et al . administered 5% albumin ( 500 ml in 30 minutes ) to assess the effect of volume expansion and found a reduction in cbf following albumin administration in patients with vasospasm during the first 2 weeks after asah with no net change in the 3rd week . more recently , in 2004 , suarez et al . found that there was a beneficial effect of albumin when used as a fluid for volume expansion in patients with sah . patients in the albumin group were more likely to have better outcomes at 3 months . more recent studies are needed to readdress this topic and no conclusion on albumin therapy can be drawn at this point . the first randomized controlled trial of nimodipine in patients with subarachnoid hemorrhage was published by allen et al . in 1983 in the new england journal of medicine . the placebo group had neurologic deficit that was severe or resulted in death by the end of the 21-day treatment period in 8 of 60 patients , while the nimodipine treatment group had an occurrence of 1 of 56 ( p = 0.03 ) . , the authors presumed that the efficacy of nimodipine resulted from its effects on the prevention of cerebral arterial spasm . in 1998 , however , a systematic review by feigin et al . found that nimodipine reduced the frequency of ischemic deficit by 33% ( 95% ci , 25%41% ) but it has no significant effect on angiographically detected cerebral vasospasm . thus , it is now thought that the beneficial effect of nimodipine may be through neuroprotective factors rather than the prevention of vasospasm . the use of nimodipine in the prevention of symptomatic vasospasm is no longer a controversial topic in cerebral vasospasm management . there is consensus in the published literature supporting the safety of nimodipine and its efficacy to improve clinical outcomes in patients with asah . at least 5 double - blind placebo - controlled trials have consistently demonstrated that nimodipine is efficacious in improving morbidity and mortality in patient with asah and is safe for routine use with mild hypertension occurring as a relatively infrequent adverse event [ 46 , 9194 ] . an extensive and exhaustive systematic review by the cochrane collaboration of rct of oral nimodipine showed that prophylactic oral nimodipine reduced the risk of poor outcome defined as death or dependence ( risk ratio ( rr ) 0.67 , 95% ci 0.550.81 , sample size 853 ) and secondary ischemia ( risk ratio ( rr ) 0.64 , 95% ci 0.490.83 , sample size 390 ) . nimodipine is now accepted as efficacious , safe , and cost - effective and is recommended as standard of care [ 87 , 96100 ] . statins are widely utilized for their cholesterol lowering effect , but they also exhibit many pleiotropic actions . they have been known to improve endothelial function , possibly via direct upregulation of endothelial nitrogen oxide synthase [ 102 , 103 ] . in 2002 , mcgirt et al . recently , there has been controversy over the prophylactic use of statins to prevent the occurrence of cerebral vasospasm . there have been at least 4 rcts [ 105108 ] and 6 observational studies [ 104 , 109114 ] published on the effects of statins of symptomatic vasospasm . a meta - analysis in 2006 by sillberg et al . based on 3 rcts and 158 patients found that the incidence of vasospasm ( rr = 0.73 , 95% ci 0.540.99 ) , delayed ischemic deficits ( rr = 0.38 , 95% ci 0.170.83 ) , and mortality ( rr = 0.22 , 95% ci 0.060.82 ) were reduced in the group receiving statin versus placebo . more mixed results were found by kramer and fletcher who in 2009 performed a meta - analysis of the same 3 rcts and an additional rct published in 2009 by vergouwen et al . , and 2 pseudo - rcts which were published as abstracts in the literature . they found that amongst a total of 309 patients , statins significantly reduced the occurrence of dinds ( or 0.38 , 95% ci 0.230.65 ) but did not reduce the occurrence of mortality ( or 0.51 , 95% ci 0.251.02 ) or poor neurological recovery ( or 0.81 , 95% ci 0.491.32 ) . in the same year , vergouwen et al . performed another meta - analysis on just the 4 truly randomized rcts and found that statin use did not significantly reduce radiographic vasospasm , dci , poor outcome , or mortality . these inconclusive results highlight the need for future , well - designed rcts to determine the effectiveness of statins in preventing symptomatic vasospasm . currently , there are four such controlled rcts ongoing and results from these studies are expected in the near future . magnesium is a widely used , a cost - effective therapy that is well - established in the fields of obstetrics and cardiology . its effectiveness in the prevention of cerebral vasospasm after asah , however , is much more controversial . although the exact neuroprotective mechanism of magnesium remains speculative [ 119 , 120 ] , there exists a sound theoretical framework to suggest that magnesium sulfate administration may be beneficial after asah . magnesium is known to dilate cerebrovascular arteries , block glutamate release and neurotoxicity , block the glutamate nmda receptor , voltage - dependent calcium channels , and buffer against the depletion of intracellular atp . promisingly , in numerous earlier small pilot clinical trials , magnesium sulfate showed a trend towards improving clinical outcomes in patients with asah [ 123130 ] . however , the results of three recent phase iii , randomized , placebo - controlled , double - blind clinical trials of magnesium sulfate have shown that intravenous magnesium sulfate may not be as effective as previously hoped . a study in 2010 by wong et al . found that in 327 patients randomized to intravenous magnesium sulfate or saline placebo for 10 to 14 days , 64% of the magnesium sulfate group and 63% of the placebo group had a favorable outcome at 6 months using the extended glasgow outcome scale . secondary outcome analyses such as the modified rankin scale , barthel index , sf-36 , and clinical vasospasm also showed no significant differences . consistent with these finding , no differences in radiographic vasospasm defined by changes in tcd mean mca velocities were found . in a study published in 2010 , westermaier et al . reported that amongst 110 patients randomized to 10 days of intravenous magnesium sulfate to titrate serum magnesium to 2.02.5 mmol / l and control , the incidence of delayed ischemic infarction was significantly lower in the magnesium group ( or 0.28 , 95% ci 0.120.64 ) than in the control group . however , this did not translate into a difference in the number reaching good outcome ( 63% in magnesium versus 51% in control , p = 0.209 ) or in dind ( or 0.51 , 95% ci 0.201.29 ) . interestingly , there was a difference in tcd and dsa defined radiographic vasospasm ( 67% in magnesium versus 85% in control , p = 0.028 ) . the results of the latest phase iii clinical trial published in 2012 by dorhout mees et al . showed that in 1204 patients randomized to magnesium and placebo , there was no difference in the incidence of poor outcome ( rr 1.03 , 95% ci 0.851.25 ) . the authors also performed an updated meta - analysis with 2047 patients , the largest study to date , and concluded that magnesium is not superior to placebo for reduction of poor outcome after asah and that routine administration of magnesium after asah could not be recommended . these results were consistent with another recent meta - analysis by wong et al . which included a total of 875 patients and found no difference in dci ( rr 0.87 , 95% ci 0.362.09 ) , delayed cerebral infarction ( rr 0.61 , 95% ci 0.351.22 ) , favorable outcome at 3 months ( rr 1.14 , 95% ci 0.991.31 ) , or favorable outcome at 6 months ( rr 1.08 , 95% ci 0.941.24 ) . these recent reviews incorporating the latest phase iii clinical trial data suggest that magnesium sulfate may not be as beneficial in patient outcomes after asah as previously thought . based on this new evidence , the routine administration of magnesium sulfate after asah can not be recommended . endothelin is a 21-residue vasoconstrictor peptide which was discovered as one of the most potent vasoconstrictors known in 1998 by yanagisawa et al . . a specific subtype of endothelin , endothelin-1 , drew much attention as a target for prevention of cerebral vasospasm because it was shown to produce dose - dependent and very long - lasting constrictive effects on cerebral vasculature both in vivo and in vitro [ 133136 ] . the eta receptor is located in vascular endothelial cells and is known to mediate the endothelium - dependent vasodilative actions of endothelins . recently , a specific antagonist to the eta receptor , clazosentan ( actelion pharmaceuticals , allschwil , switzerland ) , has been the focus of research . an initial phase iia study , clazosentan to overcome neurological ischemia and infarction occurring after subarachnoid hemorrhage-1 ( conscious-1 ) , was performed . in this randomized , placebo - controlled , double - blind study by macdonald et al . , the highest dose of clazosentan , 15 mg / hr , demonstrated a relative risk reduction in angiographic vasospasm of 65% ( 95% ci 4778% ; p < 0.0001 ) . in a phase iii trial of aneurysmal clipping patients by the same investigators , conscious-2 , with 1157 patients , clazosentan had no significant effect on the primary endpoint , which was defined as mortality or cerebral vasospasm related morbidity 6 weeks after sah . there was also no significant effect on poor functional outcome at week 12 , which was defined as a gos - e score of at least 4 . a similar phase iii study , conscious-3 , was conducted in patients with asah secured by endovascular coiling . patients received up to 14 days of intravenous clazosentan ( 5 or 15 mg / h ) or placebo . consistent with the results of conscious-2 , there was no effect of the lower dose of clazosentan , 5 mg / h , on the primary outcome , defined as all - cause mortality , cerebral vasospasm - related new cerebral infarcts , dind , or rescue therapy . there was also no effect of this lower dose on the secondary outcome , defined as a gos - e of at least 4 at week 12 . however , the larger dose of clazosentan , 15 mg / h , did significantly reduce cerebral vasospasm - related morbidity and mortality within 6 weeks asah . the authors theorized that the complications of clazosentan , including pulmonary complications , anemia , and hypotension , may have biased the effect of clazosentan on cerebral vasospasm towards the null . these recent phase iii trials showed a promising statistically and clinically significant effect of endothelin-1 receptor antagonist on angiographic vasospasm . further research is needed to determine whether this failure of clazosentan to affect long - term outcomes is due to its associated side effects or due to the lack of a relationship between angiographic vasospasm and outcomes . this review has focused on some risk factors for vasospasm and dci after asah , the use of ct imaging schemes to grade asah . additionally , it presents recent data on methods to prevent and treat vasospasm after asah . experimental strategies that may be useful in the near and distant future are also discussed . hemodilution and hypervolemia , components of triple - h therapy which remains a mainstay of vasospasm treatment in the modern practice of neurosurgery , have been controversial . other therapies , such as magnesium sulfates , statins , and endothelin-1a receptor antagonists showed initial promise but have shown disappointing results in the most recent literature and are thus unproven . interestingly , roughly one - third of patients develop dci without angiographic vasospasm , which suggests that vasospasm may be only part of the picture in dci development after asah . recent research efforts have been vigorously undertaken to elucidate the role of other factors such as delayed effects of global cerebral ischemia , thromboembolism , microcirculatory dysfunction , and cortical spreading depression , which likely also play a role in the outcome after asah . in this literature review , we have chosen to focus on the factors specifically related to vasospasm . in the future , hypotheses based on the known pathophysiology of vasospasm are expected to lead to well - powered rcts that will help to elucidate the efficacy of these unproven management strategies for the prevention and treatment of vasospasm after asah .

aneurysmal subarachnoid hemorrhage occurs in approximately 30,000 persons in the united states each year . around 30 percent of patients with aneurysmal subarachnoid hemorrhage suffer from cerebral ischemia and infarction due to cerebral vasospasm , a leading cause of treatable death and disability following aneurysmal subarachnoid hemorrhage . methods used to predict , diagnose , and manage vasospasm are the topic of recent active research . this paper utilizes a comprehensive review of the recent literature to address controversies surrounding these topics . evidence regarding the effect of age , smoking , and cocaine use on the incidence and outcome of vasospasm is reviewed . the abilities of different computed tomography grading schemes to predict vasospasm in the aftermath of subarachnoid hemorrhage are presented . additionally , the utility of different diagnostic methods for the detection and visualization of vasospasm , including transcranial doppler ultrasonography , ct angiography , digital subtraction angiography , and ct perfusion imaging is discussed . finally , the recent literature regarding interventions for the prophylaxis and treatment of vasospasm , including hyperdynamic therapy , albumin , calcium channel agonists , statins , magnesium sulfate , and endothelin antagonists is summarized . recent studies regarding each topic were reviewed for consensus recommendations from the literature , which were then presented .

1. Introduction 2. Outcomes Used in Vasospasm Research 3. Conclusion

aneurysmal subarachnoid hemorrhage ( asah ) is a relatively rare cause of stroke , occurring in approximately 30,000 persons in the united states each year . however , its impact equals that of cerebral ischemia , the most common cause of stroke , due to its higher morbidity , higher mortality , and occurrence in younger individuals . approximately 20 to 30 percent of patients with asah suffer from cerebral ischemia and infarction due to cerebral vasospasm , which is the number one cause of treatable death and disability following asah . the pathogenesis and etiology of this vasospasm are very poorly understood , and treatments to prevent post - sah vasospasm are widely varied and have greatly different magnitudes of effectiveness . new methods to predict , diagnosis , and manage vasospasm are an active field of research , and there is great controversy regarding the effectiveness of these various methods to affect the course of vasospasm . the aim of this paper is to address controversies in the prediction , diagnosis , and management of cerebral vasospasm based on a comprehensive review of the recent literature regarding these topics . previous studies on asah have studied a variety of different outcomes , making the direct comparison of the results difficult . while it is defined most accurately as the occurrence of focal neurological impairment or a decrease of at least 2 points on the glasgow coma scale which lasts for at least one hour , it is not apparent immediately after aneurysm occlusion , and can not be due to other causes by means of clinical assessment , ct , mri , or appropriate laboratory studies . found that a variety of different terms had been used to reflect dci , including delayed ischemic neurologic deficit ( dind ) , secondary cerebral ischemia , symptomatic ischemia , vasospasm , clinical vasospasm , symptomatic vasospasm , and cerebral infarction . while dci is most purely a clinical definition , many previous studies had combined its definition with angiographic or anatomic evidence of vasospasm , making it impossible to compare many studies . vasospasm , although often associated with dci , is defined as an anatomic narrowing of the intracranial arteries from various causes such as constriction , swelling , endothelial remodeling , and fibrosis . while it is generally true that vasospasm is strongly associated with dci and cerebral infarction [ 24 ] , patients with asah can have poor clinical and functional outcomes without vasospasm . recently , the international multidisciplinary consensus conference on the critical care management of subarachnoid hemorrhage published guidelines for a uniform definition of dci for use in clinical trials and observational studies . it recommended that separately defined outcomes for angiographic vasospasm , dci , cerebral infarction , and functional outcome be utilized in future studies . there is disagreement in the literature , however , as to the relationship between age and incidence of vasospasm after ruptured asah . the definition of vasospasm used in these studies differed along with the method of diagnosing vasospasm , which makes the interpretation of these studies difficult . two of the studies performed only bivariate analyses of age as a predictor of vasospasm and were not controlled for other common confounders [ 7 , 12 ] . three studies suggesting an inverse relationship between increasing age and increased risk of vasospasm utilized threshold cerebral blood flow velocity ( cbfv ) values from transcranial doppler ( tcd ) to define vasospasm . in elderly patients , the differences in the definition of vasospasm , measurement modality , and age categories used in these studies make the interpretation of their results difficult . smoking has been suggested for decades as one of the most important risk factors for subarachnoid hemorrhage and recent evidence firmly supports this relationship [ 1921 ] . however , there is consensus in the recent literature suggesting that smoking is associated with an increased risk of dci after asah . cocaine use can also cause coronary artery vasospasm and is one established cause of prinzmetal angina . additionally , it has been associated with neurological complications such as intraparenchymal hemorrhage , intraventricular hemorrhage , and cerebral infarction . there is controversy in the literature on the effect of cocaine use in the aftermath of asah . a study in 2010 casts further doubt on the association between cocaine use and vasospasm . showed that of 600 patients with asah included in the study , 31 ( 5% ) had a history of recent cocaine use . however , no association was found between cocaine use and hunt - hess score , fisher grade , short - term outcome ( modified rankin scale > 3 ) , symptomatic vasospasm , radiologic vasospasm , stroke , or mortality . the largest study to date of 142 patients with cocaine use - associated asah by chang et al . in 2013 reported that cocaine users tended to be younger ( 49 years versus 53 years , p < 0.001 ) and had no difference in hunt - hess score , intraventricular hemorrhage , and hydrocephalus , which was consistent with previous studies . most importantly , however , cocaine use was not an independent predictor of dci when controlled for with other known independent predictors such as age , hunt - hess , wfns , and admission glasgow coma scale ( gcs ) score . overall , there seem to be few studies on the effect of cocaine use on vasospasm after asah and there is no definite consensus between them . there is a need for well - powered studies with well - defined outcomes comparing angiographic vasospasm , clinical outcome , cerebral infarction , and mortality in cocaine - associated asah . the relationship between the amount and density of blood detected on an initial computed tomography ( ct ) scan of patients admitted for a diagnosis of ruptured asah and angiographic vasospasm was first observed in the late 1970s . found that amongst 26 patients with high density hemorrhage on initial ct scans , 84.6% developed cerebral vasospasm while none of 8 patients did not develop cerebral vasospasm . found that there was a relationship between the extent of subarachnoid blood on admission ct imaging and the subsequent development of vasospasm only if the ct was performed within 24 hours of aneurysmal rupture . these data suggest that the earlier the ct scan after ruptured asah , the greater the prognostic value in predicting cerebral vasospasm . one of the best predictors of cerebral vasospasm to date is the characteristic of asah on admission ct shortly after aneurysmal rupture of the aneurysm [ 33 , 34 ] . in the well - known analysis between the amount of subarachnoid blood and development of cerebral vasospasm , fisher et al . suggested an asah grading system based on an analysis of 276 patients who had a ct scan within 72 hours of rupture , which found that the best predictors of dci were blood in both lateral ventricles ( odds ratio ( or ) 4.1 , 95% ci 1.79.8 ) and a thick clot completely filling any cistern or fissure ( odds ratio ( or ) 2.3 , 95% ci 1.59.5 ) these two characteristics were found to be independently predictive of cerebral vasospasm , and the claassen scale incorporated separate categories for cisternal blood with and without bilateral intraventricular hemorrhage . the role of cisternal blood in predicting vasospasm has been firmly established in the literature . a modified fisher ct grading scale taking into account thick cisternal and ventricular blood outperformed the original fisher scale in predicting symptomatic vasospasm in a study of 1355 asah patients in the placebo arm of a randomized controlled trial ( rct ) of tirilazad . the highest score ( grade iv sah ) does not have the highest risk of vasospasm , which can be confusing , and there is no category for patients who have both thick cisternal blood and intraventricular hemorrhage , even though these may be independent and additive predictors of vasospasm . found that in modern practice , the fisher grade only predicted symptomatic vasospasm in half of the patients . this may indicate that in the era of modern practice , where nimodipine , hypertensive therapy , and endovascular therapy have become established , the fisher grade may be outdated . while there are a variety of grading schemes for interpretation of ct scans , each possesses limitations to its validity , both internal and external , and/or ease of implementation in the clinical practice setting . various methods are currently used to detect vasospasm including tcd , ct angiography , digital subtraction angiography ( dsa ) , and ct perfusion imaging . of these , tcd is the most widely used but the standard of reference for the anatomic demonstration of cerebral vasospasm is dsa . ct perfusion imaging has shown good specificity and accuracy in detecting vasospasm with a specificity of 100% and an accuracy of 92.3% . specifically , the cerebral blood flow reduction on perfusion ct was found to significantly predict the clinical outcome of patients . a meta - analysis comparing the accuracy of ct angiography and ct perfusion for detecting vasospasm found that they had similar specificity and sensitivity , with an overall sensitivity and specificity of 79.6% and 93.1% , respectively , for ct angiography and 74.1% and 93.0% , respectively , for ct perfusion . because of the different detection characteristics for these modalities , there is controversy over the modern incidence of vasospasm and that with increased specificity of more recent techniques , the actual incidence of vasospasm may be lower than noted in previous studies . a mainstay in the prophylaxis and treatment of cerebral vasospasm in the past was hyperdynamic therapy , also known as triple - h therapy , which utilized the three approaches of hypervolemia , induced hypertension , and slight hemodilution with the aim of improving cerebral blood flow ( cbf ) . in contemporary practice , there is much disagreement on the use of hyperdynamic therapy in the prophylaxis and treatment of vasospasm after asah . a survey of 626 physicians ( anesthesiologists , internists , neurologists , and neurosurgeons ) in europe and the united states found that 39% and 52% of respondents would use hyperdynamic therapy for the prophylaxis and treatment of vasospasm after asah , respectively , . this combined treatment approach has not yet been assessed for efficacy in any large rcts , and as such , evidence for efficacy has been based mostly on case series of patients with dci attributed to vasospasm . in the early 2000s , two rcts compared the effect of prophylactic hypervolemia to normovolemia . published results from an rct of 32 patients who were randomized to prophylactic normovolemia or in addition to having no benefit , prophylactic hyperdynamic therapy was found to have greater costs and more frequent complications such as excess bleeding , congestive heart failure , and infections . a recent , comprehensive review of 11 studies in the literature on prophylactic hyperdynamic therapy concluded that the available evidence failed to show a benefit for the use of prophylactic hyperdynamic therapy and suggested harm in using overly aggressive hydration . induced arterial hypertension in 58 patients with intravascular volume expansion , blockade of the vagal depressor response , and the administration of antidiuretics and vasopressor agents , resulting in a permanent reversal of neurological deterioration in 43 patients . found that hypervolemic hemodilution therapy after symptomatic vasospasm could reverse neurological deterioration due to cerebral vasospasm . in 2003 , a systematic review of 4 prospective studies of triple - h therapy found that it reduced symptomatic vasospasm ( relative risk ( rr ) 0.45 , 95% ci 0.530.87 ) and death ( rr 0.68 , 95% ci 0.530.87 ) but had no effect on dind ( rr 0.54 , 95% ci 0.21.49 ) . a study of the recent literature indicates that hypertension may be the most important component of hyperdynamic therapy . in 1990 , otsubo published a study reporting that induced hypertension in the presence of normovolemia reduced the signs and symptoms of vasospasm in 54% of patients . reported that the administration of a saline bolus of 15 ml / kg/1 hr in patients with normovolemia at baseline with new symptoms of vasospasm produced an increase in cbf . reported that in patients with a poor - grade sah , administration of 2 ml / kg of 23.5% hypertonic saline solution decreased intracranial pressure and increased cerebral perfusion pressure , transcranial doppler velocities , and cbf [ 76 , 77 ] . the literature regarding hypervolemic therapy is therefore still inconclusive regarding the efficacy of hypervolemia . despite this , there is some evidence that hypovolemia is associated with increased risk of dci , and therefore , normovolemia should be attained via initial fluid resuscitation in patients with hypovolemia . hemodilution , particularly the lowering of hematocrit , attempts to decrease the viscosity of blood and thereby improves blood flow to ischemic regions of the brain via the poiseuille equation , which states that the resistance to flow is inversely proportional to viscosity . recommended maintaining a hematocrit of 3032% utilizing hemodilution with colloids to maximize oxygen delivery to ischemic brain in the setting of vasospasm . in humans , the role of hemodilution in treatment of cerebral vasospasm has also been scrutinized , but with no good consensus in the literature . retrospectively reviewed the management of 441 patients and found that postoperative packed rbc transfusion actually had a positive correlation with angiographic vasospasm with an odds ratio of 1.68 ( 95% ci , 1.022.75 ) additionally , intraoperative rbc transfusion was found to be correlated with worse outcome . in 2012 , neither routine hemodilution nor transfusion was recommended for routine treatment of dci or vasospasm in the guidelines for the management of aneurysmal subarachnoid hemorrhage by the american stroke association and based on the current literature ; this seems reasonable . there were conflicting results in the literature regarding the usage of albumin for intravascular volume expansion . administered 5% albumin ( 500 ml in 30 minutes ) to assess the effect of volume expansion and found a reduction in cbf following albumin administration in patients with vasospasm during the first 2 weeks after asah with no net change in the 3rd week . found that there was a beneficial effect of albumin when used as a fluid for volume expansion in patients with sah . the first randomized controlled trial of nimodipine in patients with subarachnoid hemorrhage was published by allen et al . , the authors presumed that the efficacy of nimodipine resulted from its effects on the prevention of cerebral arterial spasm . thus , it is now thought that the beneficial effect of nimodipine may be through neuroprotective factors rather than the prevention of vasospasm . the use of nimodipine in the prevention of symptomatic vasospasm is no longer a controversial topic in cerebral vasospasm management . based on 3 rcts and 158 patients found that the incidence of vasospasm ( rr = 0.73 , 95% ci 0.540.99 ) , delayed ischemic deficits ( rr = 0.38 , 95% ci 0.170.83 ) , and mortality ( rr = 0.22 , 95% ci 0.060.82 ) were reduced in the group receiving statin versus placebo . , and 2 pseudo - rcts which were published as abstracts in the literature . magnesium is a widely used , a cost - effective therapy that is well - established in the fields of obstetrics and cardiology . promisingly , in numerous earlier small pilot clinical trials , magnesium sulfate showed a trend towards improving clinical outcomes in patients with asah [ 123130 ] . however , the results of three recent phase iii , randomized , placebo - controlled , double - blind clinical trials of magnesium sulfate have shown that intravenous magnesium sulfate may not be as effective as previously hoped . reported that amongst 110 patients randomized to 10 days of intravenous magnesium sulfate to titrate serum magnesium to 2.02.5 mmol / l and control , the incidence of delayed ischemic infarction was significantly lower in the magnesium group ( or 0.28 , 95% ci 0.120.64 ) than in the control group . showed that in 1204 patients randomized to magnesium and placebo , there was no difference in the incidence of poor outcome ( rr 1.03 , 95% ci 0.851.25 ) . the authors also performed an updated meta - analysis with 2047 patients , the largest study to date , and concluded that magnesium is not superior to placebo for reduction of poor outcome after asah and that routine administration of magnesium after asah could not be recommended . based on this new evidence , the routine administration of magnesium sulfate after asah can not be recommended . in a phase iii trial of aneurysmal clipping patients by the same investigators , conscious-2 , with 1157 patients , clazosentan had no significant effect on the primary endpoint , which was defined as mortality or cerebral vasospasm related morbidity 6 weeks after sah . consistent with the results of conscious-2 , there was no effect of the lower dose of clazosentan , 5 mg / h , on the primary outcome , defined as all - cause mortality , cerebral vasospasm - related new cerebral infarcts , dind , or rescue therapy . there was also no effect of this lower dose on the secondary outcome , defined as a gos - e of at least 4 at week 12 . however , the larger dose of clazosentan , 15 mg / h , did significantly reduce cerebral vasospasm - related morbidity and mortality within 6 weeks asah . the authors theorized that the complications of clazosentan , including pulmonary complications , anemia , and hypotension , may have biased the effect of clazosentan on cerebral vasospasm towards the null . this review has focused on some risk factors for vasospasm and dci after asah , the use of ct imaging schemes to grade asah . other therapies , such as magnesium sulfates , statins , and endothelin-1a receptor antagonists showed initial promise but have shown disappointing results in the most recent literature and are thus unproven . interestingly , roughly one - third of patients develop dci without angiographic vasospasm , which suggests that vasospasm may be only part of the picture in dci development after asah . recent research efforts have been vigorously undertaken to elucidate the role of other factors such as delayed effects of global cerebral ischemia , thromboembolism , microcirculatory dysfunction , and cortical spreading depression , which likely also play a role in the outcome after asah . in the future , hypotheses based on the known pathophysiology of vasospasm are expected to lead to well - powered rcts that will help to elucidate the efficacy of these unproven management strategies for the prevention and treatment of vasospasm after asah .

wrd wielu problemw sygnalizowanych przez dorastajcych pacjentw z wrodzonymi wadami serca ( grown - up congenital heart przekonanie o bliej nieokrelonym ograniczeniu oglnej sprawnoci zwizanym z wad serca i jej wczeniejszym leczeniem . poprawie aktywnoci fizycznej pacjentw z guch moe suy odpowiednio przygotowany program kompleksowej rehabilitacji kardiologicznej ( krk - guch ) stanowicy kolejny , odlegy etap leczenia . ocena wpywu krk - guch na aktywno fizyczn pacjentw w odlegym okresie po chirurgicznej korekcji wrodzonych wad serca . do badania wczono 57 pacjentw ( 30 kobiet , 27 mczyzn ) w wieku 23,7 4,1 roku , bdcych minimum 12 miesicy po korekcji operacji ubytku w przegrodzie midzykomorowej ( ventricular septal defects vsd ) lub w przegrodzie midzyprzedsionkowej ( atrial septal defects asd ) . grupa a ( n = 31 ) , oraz nieuczestniczc w programie rehabilitacji grupa b ( n = 26 ) . u wszystkich pacjentw wykonano wstpne badania czynnociowe , po czym wdroono program krk - guch . po 30 dniach od badania wstpnego ponownie oceniono pacjentw z obu grup , stosujc takie same narzdzia badawcze jak we wstpnym badaniu . u pacjentw z grupy nierehabilitowanej ( grupa b ) stwierdzono istotnie nisz aktywno fizyczn po miesicu od badania wstpnego ni w grupie pacjentw rehabilitowanych w ramach programu krk - guch ( grupa a ) . i tym samym najprawdopodobniej jako ycia pacjentw w pnym okresie po chirurgicznej korekcji wrodzonych wad serca . zaproponowany program krk - guch moe stanowi uzupenienie holistycznej opieki w opisywanej grupie pacjentw po korekcji wrodzonych wad serca . adolescents and young adults after surgery of grown - up congenital heart ( guch ) defects constitute a growing group of patients requiring cardiac care and multifaceted support . the reduction of early mortality and the prolongation of survival among patients with congenital heart defects ( chds ) may appear as a tangible success [ 1 , 2 ] . however , apart from good short - term results and long - term survival , to fully assess the efficacy of the interventional treatment of cardiac defects , a broadly understood quality of life evaluation of adolescent patients with cardiac issues that were successfully treated during childhood is needed . among the many issues raised by adolescent guch patients , the widespread belief concerning unclearly defined restrictions in general fitness , associated with congenital heart defects and their previous treatment , appears to be fundamental . it directly impacts the patients everyday life and reduces their physical activity . undergoing cardiac surgery in childhood leads to a decrease in expectations , which naturally reduces the readiness of the patients themselves to take up various forms of physical activity . notwithstanding , the physical activity of guch patients may be improved by a properly prepared comprehensive cardiac rehabilitation ( ccr - guch ) program , serving as another stage of treatment with proven influence on exercise tolerance and quality of life . the present study evaluates the impact of comprehensive cardiac rehabilitation on the physical activity of guch patients participating in a specially developed authorial program dedicated to this group of patients . its basic premise is that the low physical activity of guch patients increases their predisposition to cardiovascular diseases proportionally more in the case of those less active , similarly to the general population . the issue appeared especially interesting due to the potential for increasing the activity of adolescent patients with congenital heart diseases , which could consequently improve their general condition and reduce the risk of additional cardiovascular problems natural for this age in long - term observation . the aim of this study was to evaluate the influence of the ccr - guch program on the long - term physical activity of adolescent patients after chd surgery . fifty - seven patients with chds ( 30 women and 27 men ) at the mean age of 23.7 4.1 years , who met the inclusion criteria and provided their free and informed consent , were invited to participate in the program of comprehensive cardiac rehabilitation designed at the chair and clinic of rehabilitation of the medical university of gdask in the years 2007 - 2009 . all the patients had undergone cardiac surgery procedures in childhood ( at the age of 6 on average ) ; these included either the correction of ventricular septal defects ( vsd ) or the repair of atrial septal defects ( asd ) . out of all patients invited to participate in the ccr - guch program , 31 patients ( 17 women and 14 men ) were ultimately included in the program . the remaining 26 patients , who did not participate in the rehabilitation program due to logistic , economic , or social reasons , were provided information about the program as well as about the expected positive results of the involved controlled physical exertion and its importance for normal functioning . the chd patients constituting the study group were divided into two subgroups : those participating in rehabilitation ( a ) and non - participants ( b ) . the control group consisted of 30 healthy students ( 15 women and 15 men ) at the mean age of 24.4 1.97 years , free of any additional comorbidities , or physical / mental limitations , who provided their free and informed consent for their participation and the anonymous use of the obtained results for the purpose of the present study ( group c ) . identical inclusion criteria were used for the examination of guch patients ( groups a and b ) : clinical diagnosis condition after the repair of shunt - related cardiac defects ( asd or vsd ) employing median sternotomy ; time from the cardiac surgery over 12 months . the inclusion criteria for all three groups ( a , b , and c ) were : age over 18 years as well as free and informed written consent for participation in the study . the exclusion criteria included : active inflammatory diseases , life - threatening cardiac dysrhythmias , impaired motor function preventing the patients from performing the tasks of the rehabilitation program , coexistence of other heart defects ( congenital or acquired ) , significant deterioration of clinical condition within the past month , acute cardiac or neurological events within the past 6 months , positive results of the cardiac diagnostic test , lack of consent , or mental illnesses precluding patient cooperation . no statistically significant differences between groups a and b were observed in terms of the distribution of sex , height , body mass , or the basic functional indicators of the circulatory system . the mean age of the patients at the time of surgery was 5.18 2.8 years . the resting heart rate ( hrrest ) was 90 bpm on average ; right bundle branch block ( rbbb ) was revealed on resting electrocardiograms of 15 patients , premature ventricular contractions ( pvc ) were diagnosed in 4 patients , and premature supraventricular contractions ( psvc ) were diagnosed in 2 patients . no statistically significant differences were noted between groups a , b , and c in terms of selected initial anthropometric indices ( table i ) . patient characteristics for the study groups ( a undergoing rehabilitation , b no rehabilitation ) and controls ( c ) bmi body mass index , sd standard deviation all patients ( groups a , b , and c ) underwent physical examination , and their medical histories were obtained . the patients underwent cardiopulmonary exercise testing on a cycloergometer using the metasoft studio software ( cortex ) ; the selected ramp protocol consisted of an initial load of 20 w increasing by 10 w per minute . limits of maximal fatigue were put in place along with the standard indications for test termination ; the following parameters were evaluated : hrrest , hrmax , exercise time , exercise load in watts , vo2 peak . the evaluation of physical activity was based on the stanford questionnaire , which served as a tool for the objective assessment of patients with different parameters . it was composed of questions concerning the patients current lifestyle and was divided into two parts , including questions about everyday exercise of low - activity ( stanford i ) and high - activity exercise ( stanford ii ) . each test item was worth one point , and the final score reflected the current physical activity of the respondents . additionally , a written questionnaire was drawn up especially for the purposes of the ccr - guch program . the subjects from both study groups ( a , b ) and the controls ( c ) were asked to answer questions about their lifestyle ( whether they considered themselves physically active and whether they were apprehensive of engaging in some forms of physical activity ) . they were also asked about their participation in physical education classes during their school years and about tobacco use . the guch patients ( a , b ) were also asked whether they had ever participated in cardiac rehabilitation and whether they saw the need for engaging in controlled physical training sessions . after 30 days following the initial examination and after completion of the ccr - guch program , the physical activity of patients in groups a and b was evaluated again , using the same methods as during the initial examination ( stanford i and ii questionnaires , own questionnaire ) . the ccr - guch program was implemented during the course of four weeks and was based on recommendations concerning comprehensive cardiac rehabilitation in adults . the kinesiatric program involved a cycle of half - hour monitored cycloergometer training sessions and general fitness training with elements of aerobics and nordic walking . at the gym , the subjects engaged in exercises with and without equipment as well as resistance exercise . breathing exercises were introduced in order to strengthen the respiratory muscles and to teach proper breathing both during exertion and in situ ations requiring the improvement of ventilation , e.g. dyspnea or fatigue . resistance training was performed 2 - 3 times per week on fitness stations using weights up to 20 kg ; the sessions consisted of no more than 4 series of 15 repetitions . the psychological influence consisted of emotional support , the stimulation of self - confidence , and the improvement of self - esteem . during the educational part of the program , which included classes conducted by a cardiologist , a psychologist , and a dietician , the patients learned methods of coping with emotional stress and changing the habits adversely affecting their predisposition to cardiovascular diseases . the patients were instructed how to measure their pulse ( monitoring the pulse and assessing potential cardiac dysrhythmias ) and arterial pressure . moreover , the participants were instructed in the proper use of everyday exercise , including proper respiration during various forms of activity ( walking , running , resistance training ) . they also received advice concerning the optimal selection , planning , and performance of exercises and were shown various forms of physical activity , including sports and recreational activities which exert favorable effects on the cardiovascular system and facilitate the achievement of good short - term and long - term results [ 8 , 9 ] . the performed analysis of the initial stanford questionnaire results demonstrated that the guch patients ( a and b ) were less physically active than their healthy peers ( mean scores : stanford i 1.9 vs. 2.9 points , p < 0.001 ; stanford ii preliminary results of the physical activity examination among the study groups guch patients ( a and b ) and the controls healthy students ( c ) before the start of the ccr - guch program guch grown - up congenital heart , ccr comprehensive cardiac rehabilitation , stanford i low - activity exercise , stanford ii high - activity exercise subsequently , the guch patients undergoing rehabilitation ( a ) were compared with the group that did not undergo the ccr - guch program ( b ) . both groups were characterized by similar baseline physical activity according to the initial stanford questionnaire ( table iii ) . results of the stanford questionnaire among the study groups ( a undergoing rehabilitation and b no rehabilitation ) before the start of the ccr - guch program ccr comprehensive cardiac rehabilitation , guch grown - up congenital heart , stanford i low - activity exercise , stanford ii high - activity exercise forty - seven guch patients ( 82% ) did not participate , while all students ( 100% ) did participate in physical education during their school years . apprehension concerning engaging in physical exertion in everyday life was reported by 35 guch respondents ( 61% ) , while all students declared that they felt no fear of exercise . twenty - six guch patients ( 45.6% ) declared themselves to be physically active in everyday life ; all the students answered this question in the same manner . all the guch patients ( 100% ) declared the need to improve their physical capacity and activity . similar answers were given to the question concerning the readiness to participate in controlled ccr - guch training sessions ; however , 26 patients ( 45.6% ) did not ultimately participate in the program . three of the guch patients ( 5.2% ) and 2 students ( 6.4% ) reported tobacco use . after the intensive rehabilitation program conducted over the course of 1 month , the subjects were again asked to fill out the stanford questionnaire . seven patients who did not undergo rehabilitation ( group b ) did not answer the questions . the analysis of the remaining 50 questionnaires demonstrated that , after the period of 1 month , the patients who were not included in the ccr - guch program ( group b ) were characterized by significantly lower physical activity in comparison to the patients participating in the training sessions ( group a ) ( mean scores : stanford i 1.8 vs. 3.2 points , p < 0.001 ; stanford ii 0.13 vs. 1.2 points , results of the stanford questionnaire among the study groups ( a undergoing rehabilitation and b no rehabilitation ) after the end of the one - month ccr - guch program ccr comprehensive cardiac rehabilitation , guch grown - up congenital heart , stanford i low - activity exercise , stanford ii high - activity exercise the analysis of both parts of the questionnaire also revealed that , after the completion of the ccr - guch program , the 31 patients from group a engaged in both low - activity ( stanford i ) and high - activity exercise ( stanford ii ) much more frequently ( p < 0.001 and p < 0.001 ) ( fig . the results of the stanford questionnaire in group a before the start and after the completion of the ccr - guch program are presented in table v. results of the stanford questionnaire among the study groups ( a undergoing rehabilitation and b no rehabilitation ) before the start and after the end of the one - month of the ccr - guch program ; a ) stanford i ; b ) stanford ii results of the stanford questionnaire in study group a ( undergoing rehabilitation ) before the start and after the end of the ccr - guch program ( n = 31 ) ccr comprehensive cardiac rehabilitation , guch grown - up congenital heart , stanford i low - activity exercise , stanford ii high - activity exercise the unequivocally positive opinion of the effects of the ccr - guch program , expressed by 24% of the guch patients , should be underscored at this point . asked about the benefits that they believed were achieved by participating in the program , 20 group a patients ( 64.5% ) responded that their subjective well - being was much better , 5 patients ( 16.1% ) wrote that they tired less during everyday activities , 2 patients ( 6.4% ) no longer feared increasing the load of physical exercise , and 3 patients ( 9.6% ) felt safer during their everyday activity . their descriptive answers to the questionnaire handed out after the completion of the rehabilitation program were more likely to include information that they tended to use the stairs more often than the elevator , that they tended to walk rather than drive for short distances , and that they would more frequently go out for walks after dinner or supper . the questionnaire score reflecting improvements in everyday physical activity corresponds with the results of the stanford i questionnaire , assessing the same type of activity : the mean stanford i score changed from 1.97 points before ccr - guch to 3.2 after completion of the program . congenital heart defects surgery is typically performed during the patient 's childhood ; the age of patients undergoing these operations is increasingly lower , and increasingly younger children undergo successful cardiac surgical treatment using state of the art technology . the dominant group among adolescents and young adults with chds is formed by those who had undergone cardiac surgery , but , unfortunately , have predominantly sedentary lifestyles or undertake exercise that is not recommended for their health condition . this often results from the lack of precise information concerning the type and intensity of exercise that they could engage in . telling these patients : you can do everything , but in moderation or the basis for the selection of physical activity for individuals with chd is constituted by the standards of sports cardiology included in the recommendations of consecutive sports conferences in bethesda . the newest recommendations concerning the use of physical exercise by individuals with chd were listed in the european society of cardiology guidelines for the management of grown - up congenital heart disease ( new version 2010 ) . regrettably , many adolescents and young adults after the surgical treatment of simple shunt - related cardiac defects avoid intense physical activity [ 13 , 14 ] . the bethesda recommendations permit any kind of exercise after the successful and uneventful repair of simple intracardiac shunts . the guidelines of the european society of cardiology ( esc ) also contain no contraindications for physical exercise in this patient group . the decided majority of patients operated on due to simple shunt - related heart defects ( atrial and ventricular septal defects ) may perform physical exercise of any kind and intensity starting from the 6th postoperative month , provided no complications , pulmonary hypertension , or dysrhythmias are observed . it should be stressed that information concerning the physical exercise recommended to this patient group is still lacking ; as a result , many patients are unsure what types of activities are truly safe . the patients are unfamiliar with their capacity or the potential risks and restrictions ; therefore , it appears necessary to create professional teams dedicated to managing patients after chd repair . physical activity may be objectively assessed using a questionnaire including questions concerning the use of various exercise types of different intensity . for the purposes of this study , i.e. determination of the current physical activity profiles of patients , the data obtained from the stanford i and ii questionnaires were immensely useful in evaluating guch patients , even though the test was originally designed for other purposes [ 7 , 16 ] . the conducted analysis of the questionnaire 's results demonstrated that , after the completion of the rehabilitation program , the everyday physical activity of our patients increased . after the program ended , the subjects tended more often to report engaging in high - activity exercise ( the average stanford ii scores increased from 0.13 before the rehabilitation to 0.63 after the rehabilitation ) . it is , therefore , to be expected that , owing to this active attitude , the patients will be able to maintain the improvement of physical capacity and exercise tolerance achieved during the comprehensive cardiac rehabilitation program for a longer period of time . it should be underscored that the ultimate examination with the stanford questionnaires demonstrated that the patients undergoing comprehensive rehabilitation had statistically better results than the patients who refused to participate in the rehabilitation program ( mean scores : stanford i a : 3.2 vs. b : 1.8 ; stanford ii a : 1.2 vs. b : 0.13 , p < 0.001 ) . this may indicate that the elements of the program were properly selected and may confirm the program 's effectiveness , including in terms of the increase in awareness concerning the need for developing basic health - oriented habits and an active lifestyle . when analyzing the obtained responses , one should pay attention to the subjective opinions of patients concerning the benefits resulting from participating in the rehabilitation program . after completion of the training cycle , the patients declared that they felt much better , tired less , felt safer during physical activity , and did not fear engaging in types of activities that used to constitute an thus , it should be expected that , in the future , the group of patients who underwent the ccr - guch program will be more aware of their capabilities and will be better able to see their limits without overexerting themselves , which , unfortunately , tends to happen in the group of young adults with chds . the final results of the young adult guch patients were closer to the results of the controls , recruited ( as in previous studies conducted by the authors ) among young , healthy students . in accordance with the study 's methodology , cardiac rehabilitation conducted in the study group significantly improved the physical activity of over half of the participants . based on the obtained data , it can be surmised that the expected positive results of training and an improvement of the cardiovascular system 's adaptation to physical exercise were achieved during the program . the results confirm previously published literature data ; however , there are still few comprehensive studies devoted to the physical capacity of guch patients despite the growing number of such patients in the population [ 17 , 18 ] . the ccr - guch rehabilitation in the study group was designed in accordance with the guidelines for conducting physical training , published by the american heart association and with the experience of the authors in providing treatment and conducting physical training sessions for cardiovascular patients [ 3 , 14 , 22 ] . one limitation of the study was the lack of randomization during group selection ; however , the authors deemed refraining to invite the patients to participate in the program unethical . the number of patients included in the program was also restricted by the financial limits of the study . the intensity of physical exercise was determined based on the initial cpet . in order to maintain safety , the patients were monitored with electrocardiography during cycloergometer training , while the intensity of general fitness training and nordic walking sessions was controlled only by pulse measurements . in accordance with the premises of the ccr - guch program , acquiring self - control reduced anxiety related to continuing the activities and physical training in home conditions , without the supervision of physical therapists or physicians . in conclusion , it should be underscored that the improvement of the initially lowered physical activity of guch patients undergoing rehabilitation documented in this study , contrasted with the results of patients who did not undergo rehabilitation , confirms the need to qualify and include guch patients in comprehensive programs of late rehabilitation [ 19 , 20 ] . engaging in the comprehensive program of late cardiac rehabilitation improves the physical activity of patients after the surgical correction of congenital heart diseases . comprehensive cardiac rehabilitation reduces the fears concerning various forms of physical activity that are often encountered in this group of patients , and , in consequence , improves the patients quality of life . it appears justified to introduce the comprehensive rehabilitation program as a supplement to holistic care in the group of adolescent patients after congenital heart diseases .

introductionthe group of grown - up patients with congenital heart defects ( grown - up congenital heart guch ) complains of a number of specific medical and non - medical problems . the presented program of comprehensive cardiac rehabilitation ( ccr - guch ) , dedicated to the above mentioned group , can potentially improve the physical activity of guch patients.aimthe aim of the study was to assess the effect of the comprehensive cardiac rehabilitation program on the physical activity of guch patients.material and methodsthe invitation to take part in the ccr - guch program was addressed to a group of 57 patients ( mean age : 23.7 4.1 years ) who had undergone the surgical correction of ventricular septal defects ( vsd ) or atrial septal defects ( asd ) at least 12 months earlier . the patients were divided into two groups : a patients undergoing rehabilitation , and b patients who did not participate in the program . the patients were initially examined using functional and stress tests , and the program of comprehensive cardiac rehabilitation was started in group a. after 30 days , the patients from both groups underwent further testing using the same methods as during the initial evaluation.resultsafter one month of rehabilitation , the physical activity parameters of patients participating in the ccr - guch program ( group a ) were significantly better than those observed among non - participants ( group b).conclusionsthe introduction of the comprehensive rehabilitation program improves the physical activity and , consequently , the quality of life of guch patients . the ccr - guch program appears to be a justified supplement to holistic care in the late rehabilitation of patients after the surgical correction of congenital heart defects .

Wstp Cel Materia i metody Wyniki Wnioski Introduction Material and methods Comprehensive cardiac rehabilitation program Results Discussion Conclusions Disclosure

wrd wielu problemw sygnalizowanych przez dorastajcych pacjentw z wrodzonymi wadami serca ( grown - up congenital heart przekonanie o bliej nieokrelonym ograniczeniu oglnej sprawnoci zwizanym z wad serca i jej wczeniejszym leczeniem . poprawie aktywnoci fizycznej pacjentw z guch moe suy odpowiednio przygotowany program kompleksowej rehabilitacji kardiologicznej ( krk - guch ) stanowicy kolejny , odlegy etap leczenia . do badania wczono 57 pacjentw ( 30 kobiet , 27 mczyzn ) w wieku 23,7 4,1 roku , bdcych minimum 12 miesicy po korekcji operacji ubytku w przegrodzie midzykomorowej ( ventricular septal defects vsd ) lub w przegrodzie midzyprzedsionkowej ( atrial septal defects asd ) . u pacjentw z grupy nierehabilitowanej ( grupa b ) stwierdzono istotnie nisz aktywno fizyczn po miesicu od badania wstpnego ni w grupie pacjentw rehabilitowanych w ramach programu krk - guch ( grupa a ) . adolescents and young adults after surgery of grown - up congenital heart ( guch ) defects constitute a growing group of patients requiring cardiac care and multifaceted support . the reduction of early mortality and the prolongation of survival among patients with congenital heart defects ( chds ) may appear as a tangible success [ 1 , 2 ] . however , apart from good short - term results and long - term survival , to fully assess the efficacy of the interventional treatment of cardiac defects , a broadly understood quality of life evaluation of adolescent patients with cardiac issues that were successfully treated during childhood is needed . among the many issues raised by adolescent guch patients , the widespread belief concerning unclearly defined restrictions in general fitness , associated with congenital heart defects and their previous treatment , appears to be fundamental . it directly impacts the patients everyday life and reduces their physical activity . undergoing cardiac surgery in childhood leads to a decrease in expectations , which naturally reduces the readiness of the patients themselves to take up various forms of physical activity . notwithstanding , the physical activity of guch patients may be improved by a properly prepared comprehensive cardiac rehabilitation ( ccr - guch ) program , serving as another stage of treatment with proven influence on exercise tolerance and quality of life . the present study evaluates the impact of comprehensive cardiac rehabilitation on the physical activity of guch patients participating in a specially developed authorial program dedicated to this group of patients . its basic premise is that the low physical activity of guch patients increases their predisposition to cardiovascular diseases proportionally more in the case of those less active , similarly to the general population . the issue appeared especially interesting due to the potential for increasing the activity of adolescent patients with congenital heart diseases , which could consequently improve their general condition and reduce the risk of additional cardiovascular problems natural for this age in long - term observation . the aim of this study was to evaluate the influence of the ccr - guch program on the long - term physical activity of adolescent patients after chd surgery . fifty - seven patients with chds ( 30 women and 27 men ) at the mean age of 23.7 4.1 years , who met the inclusion criteria and provided their free and informed consent , were invited to participate in the program of comprehensive cardiac rehabilitation designed at the chair and clinic of rehabilitation of the medical university of gdask in the years 2007 - 2009 . all the patients had undergone cardiac surgery procedures in childhood ( at the age of 6 on average ) ; these included either the correction of ventricular septal defects ( vsd ) or the repair of atrial septal defects ( asd ) . out of all patients invited to participate in the ccr - guch program , 31 patients ( 17 women and 14 men ) were ultimately included in the program . the remaining 26 patients , who did not participate in the rehabilitation program due to logistic , economic , or social reasons , were provided information about the program as well as about the expected positive results of the involved controlled physical exertion and its importance for normal functioning . the chd patients constituting the study group were divided into two subgroups : those participating in rehabilitation ( a ) and non - participants ( b ) . the control group consisted of 30 healthy students ( 15 women and 15 men ) at the mean age of 24.4 1.97 years , free of any additional comorbidities , or physical / mental limitations , who provided their free and informed consent for their participation and the anonymous use of the obtained results for the purpose of the present study ( group c ) . identical inclusion criteria were used for the examination of guch patients ( groups a and b ) : clinical diagnosis condition after the repair of shunt - related cardiac defects ( asd or vsd ) employing median sternotomy ; time from the cardiac surgery over 12 months . the inclusion criteria for all three groups ( a , b , and c ) were : age over 18 years as well as free and informed written consent for participation in the study . the exclusion criteria included : active inflammatory diseases , life - threatening cardiac dysrhythmias , impaired motor function preventing the patients from performing the tasks of the rehabilitation program , coexistence of other heart defects ( congenital or acquired ) , significant deterioration of clinical condition within the past month , acute cardiac or neurological events within the past 6 months , positive results of the cardiac diagnostic test , lack of consent , or mental illnesses precluding patient cooperation . the mean age of the patients at the time of surgery was 5.18 2.8 years . the resting heart rate ( hrrest ) was 90 bpm on average ; right bundle branch block ( rbbb ) was revealed on resting electrocardiograms of 15 patients , premature ventricular contractions ( pvc ) were diagnosed in 4 patients , and premature supraventricular contractions ( psvc ) were diagnosed in 2 patients . patient characteristics for the study groups ( a undergoing rehabilitation , b no rehabilitation ) and controls ( c ) bmi body mass index , sd standard deviation all patients ( groups a , b , and c ) underwent physical examination , and their medical histories were obtained . the patients underwent cardiopulmonary exercise testing on a cycloergometer using the metasoft studio software ( cortex ) ; the selected ramp protocol consisted of an initial load of 20 w increasing by 10 w per minute . the evaluation of physical activity was based on the stanford questionnaire , which served as a tool for the objective assessment of patients with different parameters . it was composed of questions concerning the patients current lifestyle and was divided into two parts , including questions about everyday exercise of low - activity ( stanford i ) and high - activity exercise ( stanford ii ) . each test item was worth one point , and the final score reflected the current physical activity of the respondents . additionally , a written questionnaire was drawn up especially for the purposes of the ccr - guch program . the subjects from both study groups ( a , b ) and the controls ( c ) were asked to answer questions about their lifestyle ( whether they considered themselves physically active and whether they were apprehensive of engaging in some forms of physical activity ) . the guch patients ( a , b ) were also asked whether they had ever participated in cardiac rehabilitation and whether they saw the need for engaging in controlled physical training sessions . after 30 days following the initial examination and after completion of the ccr - guch program , the physical activity of patients in groups a and b was evaluated again , using the same methods as during the initial examination ( stanford i and ii questionnaires , own questionnaire ) . the ccr - guch program was implemented during the course of four weeks and was based on recommendations concerning comprehensive cardiac rehabilitation in adults . the psychological influence consisted of emotional support , the stimulation of self - confidence , and the improvement of self - esteem . during the educational part of the program , which included classes conducted by a cardiologist , a psychologist , and a dietician , the patients learned methods of coping with emotional stress and changing the habits adversely affecting their predisposition to cardiovascular diseases . the patients were instructed how to measure their pulse ( monitoring the pulse and assessing potential cardiac dysrhythmias ) and arterial pressure . moreover , the participants were instructed in the proper use of everyday exercise , including proper respiration during various forms of activity ( walking , running , resistance training ) . they also received advice concerning the optimal selection , planning , and performance of exercises and were shown various forms of physical activity , including sports and recreational activities which exert favorable effects on the cardiovascular system and facilitate the achievement of good short - term and long - term results [ 8 , 9 ] . the performed analysis of the initial stanford questionnaire results demonstrated that the guch patients ( a and b ) were less physically active than their healthy peers ( mean scores : stanford i 1.9 vs. 2.9 points , p < 0.001 ; stanford ii preliminary results of the physical activity examination among the study groups guch patients ( a and b ) and the controls healthy students ( c ) before the start of the ccr - guch program guch grown - up congenital heart , ccr comprehensive cardiac rehabilitation , stanford i low - activity exercise , stanford ii high - activity exercise subsequently , the guch patients undergoing rehabilitation ( a ) were compared with the group that did not undergo the ccr - guch program ( b ) . both groups were characterized by similar baseline physical activity according to the initial stanford questionnaire ( table iii ) . results of the stanford questionnaire among the study groups ( a undergoing rehabilitation and b no rehabilitation ) before the start of the ccr - guch program ccr comprehensive cardiac rehabilitation , guch grown - up congenital heart , stanford i low - activity exercise , stanford ii high - activity exercise forty - seven guch patients ( 82% ) did not participate , while all students ( 100% ) did participate in physical education during their school years . twenty - six guch patients ( 45.6% ) declared themselves to be physically active in everyday life ; all the students answered this question in the same manner . all the guch patients ( 100% ) declared the need to improve their physical capacity and activity . similar answers were given to the question concerning the readiness to participate in controlled ccr - guch training sessions ; however , 26 patients ( 45.6% ) did not ultimately participate in the program . three of the guch patients ( 5.2% ) and 2 students ( 6.4% ) reported tobacco use . after the intensive rehabilitation program conducted over the course of 1 month , the subjects were again asked to fill out the stanford questionnaire . seven patients who did not undergo rehabilitation ( group b ) did not answer the questions . the analysis of the remaining 50 questionnaires demonstrated that , after the period of 1 month , the patients who were not included in the ccr - guch program ( group b ) were characterized by significantly lower physical activity in comparison to the patients participating in the training sessions ( group a ) ( mean scores : stanford i 1.8 vs. 3.2 points , p < 0.001 ; stanford ii 0.13 vs. 1.2 points , results of the stanford questionnaire among the study groups ( a undergoing rehabilitation and b no rehabilitation ) after the end of the one - month ccr - guch program ccr comprehensive cardiac rehabilitation , guch grown - up congenital heart , stanford i low - activity exercise , stanford ii high - activity exercise the analysis of both parts of the questionnaire also revealed that , after the completion of the ccr - guch program , the 31 patients from group a engaged in both low - activity ( stanford i ) and high - activity exercise ( stanford ii ) much more frequently ( p < 0.001 and p < 0.001 ) ( fig . the results of the stanford questionnaire in group a before the start and after the completion of the ccr - guch program are presented in table v. results of the stanford questionnaire among the study groups ( a undergoing rehabilitation and b no rehabilitation ) before the start and after the end of the one - month of the ccr - guch program ; a ) stanford i ; b ) stanford ii results of the stanford questionnaire in study group a ( undergoing rehabilitation ) before the start and after the end of the ccr - guch program ( n = 31 ) ccr comprehensive cardiac rehabilitation , guch grown - up congenital heart , stanford i low - activity exercise , stanford ii high - activity exercise the unequivocally positive opinion of the effects of the ccr - guch program , expressed by 24% of the guch patients , should be underscored at this point . asked about the benefits that they believed were achieved by participating in the program , 20 group a patients ( 64.5% ) responded that their subjective well - being was much better , 5 patients ( 16.1% ) wrote that they tired less during everyday activities , 2 patients ( 6.4% ) no longer feared increasing the load of physical exercise , and 3 patients ( 9.6% ) felt safer during their everyday activity . their descriptive answers to the questionnaire handed out after the completion of the rehabilitation program were more likely to include information that they tended to use the stairs more often than the elevator , that they tended to walk rather than drive for short distances , and that they would more frequently go out for walks after dinner or supper . the questionnaire score reflecting improvements in everyday physical activity corresponds with the results of the stanford i questionnaire , assessing the same type of activity : the mean stanford i score changed from 1.97 points before ccr - guch to 3.2 after completion of the program . congenital heart defects surgery is typically performed during the patient 's childhood ; the age of patients undergoing these operations is increasingly lower , and increasingly younger children undergo successful cardiac surgical treatment using state of the art technology . the newest recommendations concerning the use of physical exercise by individuals with chd were listed in the european society of cardiology guidelines for the management of grown - up congenital heart disease ( new version 2010 ) . regrettably , many adolescents and young adults after the surgical treatment of simple shunt - related cardiac defects avoid intense physical activity [ 13 , 14 ] . the decided majority of patients operated on due to simple shunt - related heart defects ( atrial and ventricular septal defects ) may perform physical exercise of any kind and intensity starting from the 6th postoperative month , provided no complications , pulmonary hypertension , or dysrhythmias are observed . the patients are unfamiliar with their capacity or the potential risks and restrictions ; therefore , it appears necessary to create professional teams dedicated to managing patients after chd repair . determination of the current physical activity profiles of patients , the data obtained from the stanford i and ii questionnaires were immensely useful in evaluating guch patients , even though the test was originally designed for other purposes [ 7 , 16 ] . the conducted analysis of the questionnaire 's results demonstrated that , after the completion of the rehabilitation program , the everyday physical activity of our patients increased . after the program ended , the subjects tended more often to report engaging in high - activity exercise ( the average stanford ii scores increased from 0.13 before the rehabilitation to 0.63 after the rehabilitation ) . it is , therefore , to be expected that , owing to this active attitude , the patients will be able to maintain the improvement of physical capacity and exercise tolerance achieved during the comprehensive cardiac rehabilitation program for a longer period of time . it should be underscored that the ultimate examination with the stanford questionnaires demonstrated that the patients undergoing comprehensive rehabilitation had statistically better results than the patients who refused to participate in the rehabilitation program ( mean scores : stanford i a : 3.2 vs. b : 1.8 ; stanford ii a : 1.2 vs. b : 0.13 , p < 0.001 ) . this may indicate that the elements of the program were properly selected and may confirm the program 's effectiveness , including in terms of the increase in awareness concerning the need for developing basic health - oriented habits and an active lifestyle . when analyzing the obtained responses , one should pay attention to the subjective opinions of patients concerning the benefits resulting from participating in the rehabilitation program . after completion of the training cycle , the patients declared that they felt much better , tired less , felt safer during physical activity , and did not fear engaging in types of activities that used to constitute an thus , it should be expected that , in the future , the group of patients who underwent the ccr - guch program will be more aware of their capabilities and will be better able to see their limits without overexerting themselves , which , unfortunately , tends to happen in the group of young adults with chds . the final results of the young adult guch patients were closer to the results of the controls , recruited ( as in previous studies conducted by the authors ) among young , healthy students . in accordance with the study 's methodology , cardiac rehabilitation conducted in the study group significantly improved the physical activity of over half of the participants . based on the obtained data , it can be surmised that the expected positive results of training and an improvement of the cardiovascular system 's adaptation to physical exercise were achieved during the program . the results confirm previously published literature data ; however , there are still few comprehensive studies devoted to the physical capacity of guch patients despite the growing number of such patients in the population [ 17 , 18 ] . the ccr - guch rehabilitation in the study group was designed in accordance with the guidelines for conducting physical training , published by the american heart association and with the experience of the authors in providing treatment and conducting physical training sessions for cardiovascular patients [ 3 , 14 , 22 ] . one limitation of the study was the lack of randomization during group selection ; however , the authors deemed refraining to invite the patients to participate in the program unethical . the number of patients included in the program was also restricted by the financial limits of the study . the intensity of physical exercise was determined based on the initial cpet . in order to maintain safety , the patients were monitored with electrocardiography during cycloergometer training , while the intensity of general fitness training and nordic walking sessions was controlled only by pulse measurements . in accordance with the premises of the ccr - guch program , acquiring self - control reduced anxiety related to continuing the activities and physical training in home conditions , without the supervision of physical therapists or physicians . in conclusion , it should be underscored that the improvement of the initially lowered physical activity of guch patients undergoing rehabilitation documented in this study , contrasted with the results of patients who did not undergo rehabilitation , confirms the need to qualify and include guch patients in comprehensive programs of late rehabilitation [ 19 , 20 ] . engaging in the comprehensive program of late cardiac rehabilitation improves the physical activity of patients after the surgical correction of congenital heart diseases . comprehensive cardiac rehabilitation reduces the fears concerning various forms of physical activity that are often encountered in this group of patients , and , in consequence , improves the patients quality of life . it appears justified to introduce the comprehensive rehabilitation program as a supplement to holistic care in the group of adolescent patients after congenital heart diseases .